suPAR在宫颈癌中的临床意义及其基因靶向治疗机制的研究
|
摘要
宫颈癌是危害女性健康的重要疾病之一,流行病学调查研究表示,近年来呈现逐年增加、发病年轻化的趋势。由于筛查宫颈癌的工作不完善,我国的发病率仍是发达国家的6倍,80%确诊时已是浸润癌。手术是目前早期宫颈癌最主要的治疗手段,然而仍有10%-15%的患者出现复发和转移。如何筛选出有复发和转移高危因素的患者,并采取个体化的综合治疗以提高生存率和生活质量,是妇科肿瘤领域值得探讨的课题。因而,寻找有效的方法抑制肿瘤的侵袭及转移至关重要。
目前,国内外学者普遍认为:肿瘤的发生、发展、浸润、转移是一种多因素共同参与的复杂过程。uPA/uPAR系统在肿瘤的生长、侵袭、转移中起着重要作用,血液中的suPAR可能是体内反映uPA系统活性的更方便、更可靠的有效指标。本研究进一步探讨uPAR与宫颈癌细胞侵袭、转移及血管形成的关系,以期寻找癌转移更有效的分子生物学指标;进一步探讨suPAR早期诊断的临床作用和意义;并通过携带suPAR基因的质粒pcDNA3.1-suPAR转染宫颈癌Hela细胞及裸鼠皮下移植瘤的构建过程中,深入研究suPAR基因对宫颈癌裸鼠皮下移植瘤的抑制作用,进一步阐明suPAR基因表达与宫颈癌侵袭、转移的分子生物学变化,探讨suPAR靶向基因治疗在抗肿瘤治疗中的价值,为肿瘤的靶点治疗寻找新的途径。
第一部分uPAR、VEGF-C在宫颈鳞状细胞癌的表达及临床意义
目的:本部分通过检测uPAR、VEGF-C在宫颈鳞状细胞癌组织中的免疫组化表达情况,分析与宫颈癌细胞侵袭、转移及血管形成的关系,以期寻找癌转移更敏感的分子生物学指标。
方法:本研究通过回顾性分析40例Ⅰ-Ⅱ期宫颈癌患者的临床资料,免疫组化方法检测uPAR和VEGF-C在宫颈鳞状细胞癌中的表达,以进一步探讨uPAR和VEGF-C在宫颈鳞状细胞癌中的浸润、转移作用,为判断宫颈癌的预后和正确选择治疗方案提供新的依据。
结果:
1 uPAR、VEGF-C宫颈鳞癌组织的免疫组化表达结果
uPAR在Ⅰ、Ⅱ期宫颈癌组织阳性表达率均85%,VEGF-C在Ⅰ、Ⅱ期宫颈癌组织阳性表达率分别为80%、90%。uPAR、VEGF-C在Ⅰ、Ⅱ期宫颈癌组织的表达明显高于CIN及正常组织(P<0.05),而Ⅰ和Ⅱ期宫颈癌组织之间无显著性差异(P>0.05)。uPAR和VEGF-C的表达在宫颈癌各级分化中,G1与G2,G1与G3均有显著性差异(P<0.05),而G2和G3无显著性差异(P>0.05)。
2 uPAR、VEGF-C在宫颈鳞癌复发、转移的随访结果
40例患者至随访终止时,uPAR阳性表达34例中23例复发,复发率57.50% (23/40),11例局部复发27.50% (11/40),3例远处转移7.50% (3/40),9例死亡22.50% (9/40),死于宫颈癌;VEGF-C阳性34例17例复发,复发率42.50%(17/40),7例局部复发17.50% (7/40),1例远处转移2.50% (1/40),9例死亡22.50% (9/40),两组间复发率、转移率、死亡率无统计学意义(P>0.05),但两组相关性分析(r=0.998,P=0.000<0.05),有显著相关。
结论:
1宫颈鳞癌患者癌组织uPAR、VEGF-C呈异常高表达,且随着级别的增加而增高,表明了uPAR、VEGF-C在宫颈癌侵袭、转移的过程中发挥着重要的作用;
2高表达的uPAR、VEGF-C提示宫颈癌的侵袭、转移性更强,预后较差;
3 uPAR、VEGF-C的检测对于早期宫颈鳞癌有实际临床意义,有助于更准确的评估患者的预后及指导治疗。
第二部分suPAR在宫颈癌血浆的定量检测及早期诊断的意义
目的:检测正常宫颈、宫颈上皮内瘤变、宫颈癌及晚期癌放化疗前后血浆suPAR水平,以进一步探讨suPAR、SccAg在宫颈癌发生发展中的作用和早期诊断的临床意义。
方法:采用ELISA方法测定60例宫颈鳞癌患者血浆中suPAR、SccAg值,以38例健康妇女的正常宫颈做对照。测定suPAR、SccAg在正常宫颈、宫颈上皮内瘤变、宫颈癌及晚期癌放化疗前后中的水平,比较、分析。
结果:
1健康妇女血浆suPAR含量水平的测定结果
38例健康妇女血浆suPAR水平为0.8967±0.0411log10(ng/ml),参考值范围:0.8297~0.9581 log10(ng/ml),不同年龄对照组的suPAR水平的关系。提示对照组妇女各年龄之间suPAR水平比较,差别无统计学意义(P=0.969>0.05)。
2宫颈癌及CIN患者血浆suPAR含量的测定结果
各期宫颈癌及CIN患者的血浆suPAR含量比对照组明显升高,均有统计学意义(P<0.01);SNK-q两两比较得出:Ⅰa~Ⅱa宫颈癌患者较癌前病变患者血浆suPAR升高,Ⅱb~Ⅳ宫颈癌患者较癌前病变及Ⅰa ~Ⅱa的患者suPAR水平均升高,均有统计学意义(P值均<0.05);显示宫颈癌患者血浆suPAR含量与分期有关,其期别越高血浆suPAR含量越高。
3宫颈癌患者Ⅱb ~Ⅳ放化疗前后血浆suPAR含量的测定结果
Ⅱb ~Ⅳ期宫颈癌患者放化疗后血浆suPAR含量较放化疗前明显降低,具有统计学意义(P<0.05)。
4 suPAR、SccAg在宫颈癌患者血浆中含量测定值的结果比较
suPAR在宫颈癌患者血浆中的灵敏度、特异度、阳性预测值、阴性预测值分别为98.43%、66.67%、91.30%、92.30%,SccAg的灵敏度、特异度、阳性预测值、阴性预测值分别为57.81%、94.44%、97.40%、38.60%。ROC曲线下面积suPAR为0.858,标准误0.064,可信区间(0.733,0.983)。SccAg为0.805,标准误0.050,可信区间(0.707,0.904)。suPAR优于SccAg。
结论:
1血浆suPAR随着宫颈癌分化程度、临床期别的增加而升高,suPAR与其有明显相关。
2 suPAR的灵敏度、特异度、阳性预测值、阴性预测值分别为98.43%、66.67%、91.30%、92.30%,SccAg的灵敏度、特异度、阳性预测值、阴性预测值分别为57.81%、94.44%、97.40%、38.60%。suPAR诊断准确率高。可做为宫颈癌早期诊断、监测病情进展、判断预后的有价值指标。
第三部分稳定转染supAR基因的宫颈癌裸鼠皮下移植瘤的构建及其基因靶向治疗机制的研究
目的:通过构建携带suPAR基因的质粒pcDNA-suPAR转染宫颈癌Hela细胞及裸鼠皮下移植瘤的构建过程中,深入研究suPAR基因转染对宫颈癌裸鼠皮下移植瘤的作用,进一步阐明suPAR基因表达与宫颈癌侵袭、转移的分子生物学变化,为宫颈癌的有效靶点治疗提供理论依据。
方法:本部分研究对suPAR基因的转录调控进行了以下研究:通过用RT-PCR的方法从人肝癌细胞株HepG2中克隆到全长为849bp的suPAR基因片段,ELISA法检测suPAR基因的表达水平与细胞的侵袭转移能力存在正相关;真核表达载体pc-suPAR的构建与酶切鉴定;用ELISA法检测suPAR在真核细胞Hela中的有效表达;稳定转染suPAR基因的Hela细胞;肿瘤接种于裸鼠,观察肿瘤的生长曲线。通过研究suPAR基因在转录水平的调控机制,分析该基因的转录调控与细胞侵袭、转移的关系。
结果:
1 suPAR基因的获得
用RT-PCR的方法成功地从人肝癌细胞株HepG2中克隆到全长为849bp的suPAR基因片段。
2携带suPAR基因的真核表达载体的获得
2.1 psuPAR酶切鉴定
suPAR基因的RT-PCR产物与质粒pcDNA3.1/V5-His用EocRI和XbaI双酶切割后,经Ligation连接,转化TOPO10受体菌,然后用上述双酶酶切鉴定,电泳结果可见在大约849bp和5500bp处出现2条带,与预期结果相同。
2.2 psuPAR测序鉴定
将重组载体psuPAR进行测序,结果也显示,重组表达载体psuPAR携带的suPAR核苷酸序列及其编码的氨基酸序列也与GeneBank人suPAR基因序列(1-849bp )完全一致。
3 suPAR在真核细胞Hela中的有效表达
为检测suPAR在真核细胞的表达情况,将psuPAR转染Hela细胞,72 h后,收集转染细胞上清经ELISA方法检测suPAR表达。转染质粒psuPAR后,Hela细胞培养上清中suPAR的表达明显高于未转染的Hela细胞(p<0.05),说明psuPAR质粒可在Hela细胞中良好表达。
4稳定转染supAR基因的Hela细胞的获得
将suPAR基因插入含有新霉素(neo)抗性基因的真核表达载体pcDNA3.1-V5/His,得到重组表达质粒pcsuPAR。将该表达质粒转染Hela细胞,经G418(800μg/ml)完全培养基进行加压筛选,得到阳性克隆。并改用浓度为400μg/ml的G418维持。稳定表达suPAR基因的Hela细胞克隆,用ELISA方法进行鉴定。将ELISA测得的各个克隆的OD值按照标准曲线y=0.197x-0.291换算出各个克隆ng/ml值。
5 suPAR基因对荷宫颈癌裸鼠皮下移植瘤的体内生长抑制作用结果
将稳定表达suPAR基因的Hela细胞与野生型Hela细胞分别皮下移植到裸鼠,发现野生型Hela宫颈癌裸鼠移植瘤在第10d可明显触摸到肿瘤块,而稳定转染suPAR的宫颈癌裸鼠移植瘤则生长缓慢。在荷宫颈癌裸鼠皮下移植瘤生长的第30d,处死小鼠,剥离肿瘤块称重,结果显示Hela/ pcDNA3.1-suPAR组重量明显低于Hela组(P<0.01)。Hela/ pcDNA3.1- suPAR组比Hela组石蜡HE切片显示核固缩、核浓染、核分裂明显减少。
结论:
1 suPAR基因的表达水平与肿瘤的侵袭转移能力密切相关,高水平预示着肿瘤的侵袭转移能力强,预后差;
2成功获得suPAR基因;
3成功获得携带suPAR基因的真核表达载体;
4成功获得稳定转染suPAR基因的Hela细胞株(pcDNA3.1-suPAR);
5成功获得pcDNA3.1-suPAR转移至宫颈癌裸鼠移植瘤动物模型;
6 pcDNA3.1-suPAR基因裸鼠移植瘤接种显示suPAR基因有明显抑瘤作用,表明suPAR基因是基因靶向治疗宫颈癌的有效靶点。
Cervical cancer is one of the most important diseases threatening women’s health. Epidemiological investigation has revealed that the incidence of cervical cancer is getting higher yearly and the age of the patient affected tends to be younger. Since the screening system has not been well developed, the incidence of cervical cancer in China was still six times as much that in the developed countries, in which 80% of cases has developed to the invasive cancer when they were diagnosed. At present operation is the major and effective therapeutic measure for early cases, while 10~15% of patients eventually developed to recurrent or metastasis diseases. It has become an important strategy to screen the patients with high risk of recurrence and metastasis and set up individual therapeutic regime to improve the survival rate and life quality in gynecological oncology field. It is also very important to find an effective approach to prevent the cervical cancer from invasion and metastasis.
It has been considered that the tumor development, growth, invasion and metastasis are a complicated process, and UPA/uPAR system plays an important role in all these tumor bioactivities. suPAR in blood may be a more convenient and reliable index to reflect the actions of uPA system. The current study intended to investigate the relationship between uPAR and the invasion, metastasis and vascular formation of cervical cancer, and explore the clinical significance of detecting suPAR in patients with earlier cervical cancer, as well as its possibility to be a biological biomarker in cancer micrometastasis. To further detect the function of suPAR in inhibiting the growth of cervical cancer, suPAR plasmid (pcDNA3.1-suPAR) was constructed and transfered into Hela cells, and the expression of suPRA and tumor growth in nude mice were examined. Our study will provide effective theoretical basis for the treatment of cervical cancer by using suPAR gene target strategy.
PartⅠThe expression and clinical significance of uPAR and VEGF-C in cervical squamous cancer
Objectives: To analyze the expression of uPAR in cervical carcinoma, the relationship between VEGF-C expression and cancer cell invasion, cell metastasis and angiogenesis, and to find a more sensitive biological indicator for cancer micrometastasis.
Methods: The expressions of uPAR and VEGF-C in 40 cases of early cervical squamous cancer tissues (stage I and stage II) were examined by using immunohistochemistry method and the roles of uPAR and VEGF-C in the infiltration and metastasis of early cervical squamous cancer were detected by retrospectively analyzing the clinical data.
Results: 1 Immunohistochemical results of uPAR and VEGF-C in cervical carcinoma
The positive staining rates of uPAR were 85% in both stage I and stage II cervical carcinoma tissues, and the expression rates of VEGF-C in stage I and stage II cervical carcinoma tissues were 80% and 90%, respectively. The expressions of uPAR and VEGF-C in cervical carcinoma of both stageⅠandⅡwere higher than those in CIN and normal tissues (P<0.05), while no statistical difference (P>0.05) was showed between stage I and stage II tissues. The expressions of uPAR and VEGF-C were Significant between G1 and G2, as well as G1 and G3, but no significant difference between G2 and G3 groups (P>0.05).
2 uPAR, VEGF-C and prognosis of cervical carcinoma
40 cases of cervical carcinoma were followed -up. In uPAR positive group, 23 out of 34 patients recurred (57.50% of recurrence rate), 11 cases had recurrent disease (27.50%), 3 cases developed distant metastasis (7.50%) and 9 cases died (22.50%). In VEGF-C positive group, 17 out of 34 patients recurred (42.50% of recurrence rate), 7 cases had recurrent disease (17.50%), 1 case developed distant metastasis (2.50%) and 9 cases died (22.50%).There was no significant difference between the recurrent/metastatic/dead rates of the two groups (P >0.05), while the relativity analysis was significant between two groups.
Conclusions: 1 The expressions of uPAR and VEGF-C were tremendously increased in patients with cervical cancer, and were correlated with increased grade change, indicating that uPAR and VEGF-C play an important role in the processes of tumor growth and invasion in patients with cervical cancer.
2 The group with higher expressions of uPAR and VEGF-C showed stronger invasiveness and poorer prognosis in patients with cervical carcinoma,
3 The detection of uPAR and VEGF-C showed clinical significance for early stage of cervical carcinoma, which might help to evaluate the prognosis and guide treatment, reduce the distant metastasis and improve survival.
Part II Clinical value of detecting plasma suPAR in patients with early cervical squamous cancer
Objectives: To detect the expressions of suPAR in tissues of normal cervix, cervical intraepithelial neoplasia and cervical carcinoma in order to explore the related clinical significance.
Methods: ELISA was used to analyze suPAR, SccAg values in 60 patients with cervical squamous carcinoma compared with normal cervical tissues from 38 healthy women as the control group. SuPAR and SccAg in normal cervix, cervical intraepithelial neoplasia, cervical cancer and advanced carcinoma were detected and compared. The clinical significance of suPAR and SccAg was discussed.
Results: 1 The Plasma suPAR level in healthy women
suPAR level was 0.8967±0.0411 Log10(ng/ml) in 38 cases of healthy women, and the reference range was 0.8297~0.9581 Log10(ng/ml)
2 The plasma level of suPAR in patients with cervical carcinoma and CIN
SuPAR levels in cervical carcinoma and CIN were statistically different from that in control group (P<0.01). Furthermore, the suPAR level increased with the grade, the higher the grade, the higher the suPAR level.
3 Detection of plasma suPAR in patients with stageⅡb～Ⅳcervical carcinoma before and after chemotherapy and radio therapy.
The suPAR level in patients with invasive cervical carcinoma was significantly decreased after chemotherapy and radio therapy (P<0.05).
4 Comparison of sensitivity, specificity of uPAR and SccAg
The sensitivity, specificity, positive predictive value and negative predictive value of SuPAR were 98.43%, 66.67%, 91.30% and 92.30%, respectively, while those for SccAg were 57.81%, 94.44%, 97.40% and 38.60% respectively. The area of ROC of suPAR and SccAg were 0.858 and 0.805, respectively. SuPAR was superior to SccAg in diagnosing accuracy.
Conclusions: 1 Plasma suPAR and SccAg increased with the increased degree of cervical lesions, the depth of myometrial invasion and clinical stage. suPAR were obviously associated with loading dose of turmor.
2 Since SuPAR had higher sensitivity, specificity and accuracy, it was an useful indicator for monitoring tumor progress and evaluating prognosis in patients with cervical cancer.
PartⅢThe construction of subcutaneously implanted tumor with uPAR suPAR gene stablely expressed Hela cells and study on its mechanism of gene targeting therapy
Objectives: To construct a plasmid carrying suPAR gene and transfect Hela cells with pcDNA-suPAR and to carry out an in-depth study of the effect of suPAR gene on Hela cells and to elucidate suPAR gene expression, cancer invasion and metastasis changes, providing effective theoretical basis for the treatment of cervical cancer.
Methods: RT-PCR was used to obtain 849bp gene fragment from HepG2 hepatoma cell line. ELISA was used to detect the expression of suPAR gene and its relationship with cell infiltration and metastasis. Ukaryotic expression pc-suPAR vector was constructed and restriction enzyme digestion was confirmed. ELISA was used to detect the effective expression of suPAR in eukaryotic cell Hela. A stable transfection of suPAR was also carried out in Hela cells. Tumor was inoculated in nude mice to observe tumor growth curve. The relationship of gene transcriptional regulation and cell invasion and metastasis was obtained by analyzing the level of suPAR gene transcription.
Results: 1 SuPAR gene was obtained successfully
RT-PCR was successfully used to clone a full-length gene for the 849bp fragment of suPAR from human hepatoma cell line HepG2.
2 suPAR carrying eukaryotic expression vector was obtained successfully
2.1 psuPAR enzyme digestion
suPAR gene RT-PCR product and plasmid with pcDNA3.1/V5 were digested by XbaI and EocRI enzyme and ligated, then transformed to TOPO10 bacteria. The above-mentioned dual-enzyme digestion was used to verify. Electrophoresis results revealed two bands with approximately 849bp and 5500bp, which correlated with expectation.
2.2 psuPAR sequencing
The recombinant psuPAR vector was sequenced. Results also showed that the recombinant psuPAR vector carried suPAR nucleotide sequence and its encoding amino acid sequence is fully consistent with GeneBank human suPAR sequences (1-849bp) .
3 The effective expression of suPAR in eukaryotic cells of Hela
For the detection of the expression of suPAR in eukaryotic cells, psuPAR was transfected into Hela cells. 72h later, the expression of suPAR was examined by the ELISA from the supernatant of transfected cells. Expression of suPAR from the supernatant of transfected cell was mucher higher than that from not transfected (p<0.05). This result suggests that psuPAR plasmid was well expressed in Hela cells.
4 Hela cells with stable transfected suPAR gene was obtained successfully
Insert SuPAR gene into the eukaryotic expression vector pcDNA3.1-V5/His containing the neomycin gene (neo) resistance gene. Then the recombinant expression plasmid pcsuPAR was obtained. The expression plasmid was transfected into Hela cells by G418 (800μg/ml) complete medium pressure screening to obtain positive clones. A concentration of 400μg/ml of G418 was used to maintain screening. SuPAR stable gene expression in Hela cell clones was identified by ELISA methods. OD value of each clone from ElISA was converted to all cloning ng/ml value in accordance with the standard curve y = 0.197x-0.291.
5 In vivo results for suPAR gene inhibition of tumor growth in subcutaneously transplanted cervical cancer nude mice
Hela cells stably expressing SuPAR gene and wild type Hela cells were transplanted subcutaneously into nude mice respectively. On the 10th day, a touchable tumor block was found in nude mice transplanted with wild-type Hela cells but not in Hela cells stably expressing SuPAR gene. Mice were sacrificed on the 30th day after subcutaneous transplanting. Tumor block was weighted, and results showed that the weight of Hela/ pcDNA3.1-suPAR group was significantly lower than that of Hela group. Hela/pcDNA3.1-suPAR Group showed condensed nuclear staining, nuclear dense staining, decreased nuclear mitotic and significantly reduced cancer infiltration by Paraffin HE.
Conclusions:
1 suPAR gene expression level was closely related to tumor invasion and metastasis ability. Higher level of suPAR was related to stronger invasion and metastasis as well as poorer prognosis;
2 suPAR gene was obtained successfully;
3 Eukaryotic expression vector pc-suPAR construction and restriction enzyme digestion were successfully obtained;
4 Hela cell line stably transfected with suPAR gene was obtained;
5 Successfully transferred to the pcDNA3.1-suPAR cervical cancer animal model of nude mice;
6 pcDNA3.1-suPAR gene vaccination nude suPAR gene showed significant anti-tumor effect, indicating that suPAR gene transfer was an effective strategy for the treatment of cervical cancer.
目前,国内外学者普遍认为:肿瘤的发生、发展、浸润、转移是一种多因素共同参与的复杂过程。uPA/uPAR系统在肿瘤的生长、侵袭、转移中起着重要作用,血液中的suPAR可能是体内反映uPA系统活性的更方便、更可靠的有效指标。本研究进一步探讨uPAR与宫颈癌细胞侵袭、转移及血管形成的关系,以期寻找癌转移更有效的分子生物学指标;进一步探讨suPAR早期诊断的临床作用和意义;并通过携带suPAR基因的质粒pcDNA3.1-suPAR转染宫颈癌Hela细胞及裸鼠皮下移植瘤的构建过程中,深入研究suPAR基因对宫颈癌裸鼠皮下移植瘤的抑制作用,进一步阐明suPAR基因表达与宫颈癌侵袭、转移的分子生物学变化,探讨suPAR靶向基因治疗在抗肿瘤治疗中的价值,为肿瘤的靶点治疗寻找新的途径。
第一部分uPAR、VEGF-C在宫颈鳞状细胞癌的表达及临床意义
目的:本部分通过检测uPAR、VEGF-C在宫颈鳞状细胞癌组织中的免疫组化表达情况,分析与宫颈癌细胞侵袭、转移及血管形成的关系,以期寻找癌转移更敏感的分子生物学指标。
方法:本研究通过回顾性分析40例Ⅰ-Ⅱ期宫颈癌患者的临床资料,免疫组化方法检测uPAR和VEGF-C在宫颈鳞状细胞癌中的表达,以进一步探讨uPAR和VEGF-C在宫颈鳞状细胞癌中的浸润、转移作用,为判断宫颈癌的预后和正确选择治疗方案提供新的依据。
结果:
1 uPAR、VEGF-C宫颈鳞癌组织的免疫组化表达结果
uPAR在Ⅰ、Ⅱ期宫颈癌组织阳性表达率均85%,VEGF-C在Ⅰ、Ⅱ期宫颈癌组织阳性表达率分别为80%、90%。uPAR、VEGF-C在Ⅰ、Ⅱ期宫颈癌组织的表达明显高于CIN及正常组织(P<0.05),而Ⅰ和Ⅱ期宫颈癌组织之间无显著性差异(P>0.05)。uPAR和VEGF-C的表达在宫颈癌各级分化中,G1与G2,G1与G3均有显著性差异(P<0.05),而G2和G3无显著性差异(P>0.05)。
2 uPAR、VEGF-C在宫颈鳞癌复发、转移的随访结果
40例患者至随访终止时,uPAR阳性表达34例中23例复发,复发率57.50% (23/40),11例局部复发27.50% (11/40),3例远处转移7.50% (3/40),9例死亡22.50% (9/40),死于宫颈癌;VEGF-C阳性34例17例复发,复发率42.50%(17/40),7例局部复发17.50% (7/40),1例远处转移2.50% (1/40),9例死亡22.50% (9/40),两组间复发率、转移率、死亡率无统计学意义(P>0.05),但两组相关性分析(r=0.998,P=0.000<0.05),有显著相关。
结论:
1宫颈鳞癌患者癌组织uPAR、VEGF-C呈异常高表达,且随着级别的增加而增高,表明了uPAR、VEGF-C在宫颈癌侵袭、转移的过程中发挥着重要的作用;
2高表达的uPAR、VEGF-C提示宫颈癌的侵袭、转移性更强,预后较差;
3 uPAR、VEGF-C的检测对于早期宫颈鳞癌有实际临床意义,有助于更准确的评估患者的预后及指导治疗。
第二部分suPAR在宫颈癌血浆的定量检测及早期诊断的意义
目的:检测正常宫颈、宫颈上皮内瘤变、宫颈癌及晚期癌放化疗前后血浆suPAR水平,以进一步探讨suPAR、SccAg在宫颈癌发生发展中的作用和早期诊断的临床意义。
方法:采用ELISA方法测定60例宫颈鳞癌患者血浆中suPAR、SccAg值,以38例健康妇女的正常宫颈做对照。测定suPAR、SccAg在正常宫颈、宫颈上皮内瘤变、宫颈癌及晚期癌放化疗前后中的水平,比较、分析。
结果:
1健康妇女血浆suPAR含量水平的测定结果
38例健康妇女血浆suPAR水平为0.8967±0.0411log10(ng/ml),参考值范围:0.8297~0.9581 log10(ng/ml),不同年龄对照组的suPAR水平的关系。提示对照组妇女各年龄之间suPAR水平比较,差别无统计学意义(P=0.969>0.05)。
2宫颈癌及CIN患者血浆suPAR含量的测定结果
各期宫颈癌及CIN患者的血浆suPAR含量比对照组明显升高,均有统计学意义(P<0.01);SNK-q两两比较得出:Ⅰa~Ⅱa宫颈癌患者较癌前病变患者血浆suPAR升高,Ⅱb~Ⅳ宫颈癌患者较癌前病变及Ⅰa ~Ⅱa的患者suPAR水平均升高,均有统计学意义(P值均<0.05);显示宫颈癌患者血浆suPAR含量与分期有关,其期别越高血浆suPAR含量越高。
3宫颈癌患者Ⅱb ~Ⅳ放化疗前后血浆suPAR含量的测定结果
Ⅱb ~Ⅳ期宫颈癌患者放化疗后血浆suPAR含量较放化疗前明显降低,具有统计学意义(P<0.05)。
4 suPAR、SccAg在宫颈癌患者血浆中含量测定值的结果比较
suPAR在宫颈癌患者血浆中的灵敏度、特异度、阳性预测值、阴性预测值分别为98.43%、66.67%、91.30%、92.30%,SccAg的灵敏度、特异度、阳性预测值、阴性预测值分别为57.81%、94.44%、97.40%、38.60%。ROC曲线下面积suPAR为0.858,标准误0.064,可信区间(0.733,0.983)。SccAg为0.805,标准误0.050,可信区间(0.707,0.904)。suPAR优于SccAg。
结论:
1血浆suPAR随着宫颈癌分化程度、临床期别的增加而升高,suPAR与其有明显相关。
2 suPAR的灵敏度、特异度、阳性预测值、阴性预测值分别为98.43%、66.67%、91.30%、92.30%,SccAg的灵敏度、特异度、阳性预测值、阴性预测值分别为57.81%、94.44%、97.40%、38.60%。suPAR诊断准确率高。可做为宫颈癌早期诊断、监测病情进展、判断预后的有价值指标。
第三部分稳定转染supAR基因的宫颈癌裸鼠皮下移植瘤的构建及其基因靶向治疗机制的研究
目的:通过构建携带suPAR基因的质粒pcDNA-suPAR转染宫颈癌Hela细胞及裸鼠皮下移植瘤的构建过程中,深入研究suPAR基因转染对宫颈癌裸鼠皮下移植瘤的作用,进一步阐明suPAR基因表达与宫颈癌侵袭、转移的分子生物学变化,为宫颈癌的有效靶点治疗提供理论依据。
方法:本部分研究对suPAR基因的转录调控进行了以下研究:通过用RT-PCR的方法从人肝癌细胞株HepG2中克隆到全长为849bp的suPAR基因片段,ELISA法检测suPAR基因的表达水平与细胞的侵袭转移能力存在正相关;真核表达载体pc-suPAR的构建与酶切鉴定;用ELISA法检测suPAR在真核细胞Hela中的有效表达;稳定转染suPAR基因的Hela细胞;肿瘤接种于裸鼠,观察肿瘤的生长曲线。通过研究suPAR基因在转录水平的调控机制,分析该基因的转录调控与细胞侵袭、转移的关系。
结果:
1 suPAR基因的获得
用RT-PCR的方法成功地从人肝癌细胞株HepG2中克隆到全长为849bp的suPAR基因片段。
2携带suPAR基因的真核表达载体的获得
2.1 psuPAR酶切鉴定
suPAR基因的RT-PCR产物与质粒pcDNA3.1/V5-His用EocRI和XbaI双酶切割后,经Ligation连接,转化TOPO10受体菌,然后用上述双酶酶切鉴定,电泳结果可见在大约849bp和5500bp处出现2条带,与预期结果相同。
2.2 psuPAR测序鉴定
将重组载体psuPAR进行测序,结果也显示,重组表达载体psuPAR携带的suPAR核苷酸序列及其编码的氨基酸序列也与GeneBank人suPAR基因序列(1-849bp )完全一致。
3 suPAR在真核细胞Hela中的有效表达
为检测suPAR在真核细胞的表达情况,将psuPAR转染Hela细胞,72 h后,收集转染细胞上清经ELISA方法检测suPAR表达。转染质粒psuPAR后,Hela细胞培养上清中suPAR的表达明显高于未转染的Hela细胞(p<0.05),说明psuPAR质粒可在Hela细胞中良好表达。
4稳定转染supAR基因的Hela细胞的获得
将suPAR基因插入含有新霉素(neo)抗性基因的真核表达载体pcDNA3.1-V5/His,得到重组表达质粒pcsuPAR。将该表达质粒转染Hela细胞,经G418(800μg/ml)完全培养基进行加压筛选,得到阳性克隆。并改用浓度为400μg/ml的G418维持。稳定表达suPAR基因的Hela细胞克隆,用ELISA方法进行鉴定。将ELISA测得的各个克隆的OD值按照标准曲线y=0.197x-0.291换算出各个克隆ng/ml值。
5 suPAR基因对荷宫颈癌裸鼠皮下移植瘤的体内生长抑制作用结果
将稳定表达suPAR基因的Hela细胞与野生型Hela细胞分别皮下移植到裸鼠,发现野生型Hela宫颈癌裸鼠移植瘤在第10d可明显触摸到肿瘤块,而稳定转染suPAR的宫颈癌裸鼠移植瘤则生长缓慢。在荷宫颈癌裸鼠皮下移植瘤生长的第30d,处死小鼠,剥离肿瘤块称重,结果显示Hela/ pcDNA3.1-suPAR组重量明显低于Hela组(P<0.01)。Hela/ pcDNA3.1- suPAR组比Hela组石蜡HE切片显示核固缩、核浓染、核分裂明显减少。
结论:
1 suPAR基因的表达水平与肿瘤的侵袭转移能力密切相关,高水平预示着肿瘤的侵袭转移能力强,预后差;
2成功获得suPAR基因;
3成功获得携带suPAR基因的真核表达载体;
4成功获得稳定转染suPAR基因的Hela细胞株(pcDNA3.1-suPAR);
5成功获得pcDNA3.1-suPAR转移至宫颈癌裸鼠移植瘤动物模型;
6 pcDNA3.1-suPAR基因裸鼠移植瘤接种显示suPAR基因有明显抑瘤作用,表明suPAR基因是基因靶向治疗宫颈癌的有效靶点。
Cervical cancer is one of the most important diseases threatening women’s health. Epidemiological investigation has revealed that the incidence of cervical cancer is getting higher yearly and the age of the patient affected tends to be younger. Since the screening system has not been well developed, the incidence of cervical cancer in China was still six times as much that in the developed countries, in which 80% of cases has developed to the invasive cancer when they were diagnosed. At present operation is the major and effective therapeutic measure for early cases, while 10~15% of patients eventually developed to recurrent or metastasis diseases. It has become an important strategy to screen the patients with high risk of recurrence and metastasis and set up individual therapeutic regime to improve the survival rate and life quality in gynecological oncology field. It is also very important to find an effective approach to prevent the cervical cancer from invasion and metastasis.
It has been considered that the tumor development, growth, invasion and metastasis are a complicated process, and UPA/uPAR system plays an important role in all these tumor bioactivities. suPAR in blood may be a more convenient and reliable index to reflect the actions of uPA system. The current study intended to investigate the relationship between uPAR and the invasion, metastasis and vascular formation of cervical cancer, and explore the clinical significance of detecting suPAR in patients with earlier cervical cancer, as well as its possibility to be a biological biomarker in cancer micrometastasis. To further detect the function of suPAR in inhibiting the growth of cervical cancer, suPAR plasmid (pcDNA3.1-suPAR) was constructed and transfered into Hela cells, and the expression of suPRA and tumor growth in nude mice were examined. Our study will provide effective theoretical basis for the treatment of cervical cancer by using suPAR gene target strategy.
PartⅠThe expression and clinical significance of uPAR and VEGF-C in cervical squamous cancer
Objectives: To analyze the expression of uPAR in cervical carcinoma, the relationship between VEGF-C expression and cancer cell invasion, cell metastasis and angiogenesis, and to find a more sensitive biological indicator for cancer micrometastasis.
Methods: The expressions of uPAR and VEGF-C in 40 cases of early cervical squamous cancer tissues (stage I and stage II) were examined by using immunohistochemistry method and the roles of uPAR and VEGF-C in the infiltration and metastasis of early cervical squamous cancer were detected by retrospectively analyzing the clinical data.
Results: 1 Immunohistochemical results of uPAR and VEGF-C in cervical carcinoma
The positive staining rates of uPAR were 85% in both stage I and stage II cervical carcinoma tissues, and the expression rates of VEGF-C in stage I and stage II cervical carcinoma tissues were 80% and 90%, respectively. The expressions of uPAR and VEGF-C in cervical carcinoma of both stageⅠandⅡwere higher than those in CIN and normal tissues (P<0.05), while no statistical difference (P>0.05) was showed between stage I and stage II tissues. The expressions of uPAR and VEGF-C were Significant between G1 and G2, as well as G1 and G3, but no significant difference between G2 and G3 groups (P>0.05).
2 uPAR, VEGF-C and prognosis of cervical carcinoma
40 cases of cervical carcinoma were followed -up. In uPAR positive group, 23 out of 34 patients recurred (57.50% of recurrence rate), 11 cases had recurrent disease (27.50%), 3 cases developed distant metastasis (7.50%) and 9 cases died (22.50%). In VEGF-C positive group, 17 out of 34 patients recurred (42.50% of recurrence rate), 7 cases had recurrent disease (17.50%), 1 case developed distant metastasis (2.50%) and 9 cases died (22.50%).There was no significant difference between the recurrent/metastatic/dead rates of the two groups (P >0.05), while the relativity analysis was significant between two groups.
Conclusions: 1 The expressions of uPAR and VEGF-C were tremendously increased in patients with cervical cancer, and were correlated with increased grade change, indicating that uPAR and VEGF-C play an important role in the processes of tumor growth and invasion in patients with cervical cancer.
2 The group with higher expressions of uPAR and VEGF-C showed stronger invasiveness and poorer prognosis in patients with cervical carcinoma,
3 The detection of uPAR and VEGF-C showed clinical significance for early stage of cervical carcinoma, which might help to evaluate the prognosis and guide treatment, reduce the distant metastasis and improve survival.
Part II Clinical value of detecting plasma suPAR in patients with early cervical squamous cancer
Objectives: To detect the expressions of suPAR in tissues of normal cervix, cervical intraepithelial neoplasia and cervical carcinoma in order to explore the related clinical significance.
Methods: ELISA was used to analyze suPAR, SccAg values in 60 patients with cervical squamous carcinoma compared with normal cervical tissues from 38 healthy women as the control group. SuPAR and SccAg in normal cervix, cervical intraepithelial neoplasia, cervical cancer and advanced carcinoma were detected and compared. The clinical significance of suPAR and SccAg was discussed.
Results: 1 The Plasma suPAR level in healthy women
suPAR level was 0.8967±0.0411 Log10(ng/ml) in 38 cases of healthy women, and the reference range was 0.8297~0.9581 Log10(ng/ml)
2 The plasma level of suPAR in patients with cervical carcinoma and CIN
SuPAR levels in cervical carcinoma and CIN were statistically different from that in control group (P<0.01). Furthermore, the suPAR level increased with the grade, the higher the grade, the higher the suPAR level.
3 Detection of plasma suPAR in patients with stageⅡb～Ⅳcervical carcinoma before and after chemotherapy and radio therapy.
The suPAR level in patients with invasive cervical carcinoma was significantly decreased after chemotherapy and radio therapy (P<0.05).
4 Comparison of sensitivity, specificity of uPAR and SccAg
The sensitivity, specificity, positive predictive value and negative predictive value of SuPAR were 98.43%, 66.67%, 91.30% and 92.30%, respectively, while those for SccAg were 57.81%, 94.44%, 97.40% and 38.60% respectively. The area of ROC of suPAR and SccAg were 0.858 and 0.805, respectively. SuPAR was superior to SccAg in diagnosing accuracy.
Conclusions: 1 Plasma suPAR and SccAg increased with the increased degree of cervical lesions, the depth of myometrial invasion and clinical stage. suPAR were obviously associated with loading dose of turmor.
2 Since SuPAR had higher sensitivity, specificity and accuracy, it was an useful indicator for monitoring tumor progress and evaluating prognosis in patients with cervical cancer.
PartⅢThe construction of subcutaneously implanted tumor with uPAR suPAR gene stablely expressed Hela cells and study on its mechanism of gene targeting therapy
Objectives: To construct a plasmid carrying suPAR gene and transfect Hela cells with pcDNA-suPAR and to carry out an in-depth study of the effect of suPAR gene on Hela cells and to elucidate suPAR gene expression, cancer invasion and metastasis changes, providing effective theoretical basis for the treatment of cervical cancer.
Methods: RT-PCR was used to obtain 849bp gene fragment from HepG2 hepatoma cell line. ELISA was used to detect the expression of suPAR gene and its relationship with cell infiltration and metastasis. Ukaryotic expression pc-suPAR vector was constructed and restriction enzyme digestion was confirmed. ELISA was used to detect the effective expression of suPAR in eukaryotic cell Hela. A stable transfection of suPAR was also carried out in Hela cells. Tumor was inoculated in nude mice to observe tumor growth curve. The relationship of gene transcriptional regulation and cell invasion and metastasis was obtained by analyzing the level of suPAR gene transcription.
Results: 1 SuPAR gene was obtained successfully
RT-PCR was successfully used to clone a full-length gene for the 849bp fragment of suPAR from human hepatoma cell line HepG2.
2 suPAR carrying eukaryotic expression vector was obtained successfully
2.1 psuPAR enzyme digestion
suPAR gene RT-PCR product and plasmid with pcDNA3.1/V5 were digested by XbaI and EocRI enzyme and ligated, then transformed to TOPO10 bacteria. The above-mentioned dual-enzyme digestion was used to verify. Electrophoresis results revealed two bands with approximately 849bp and 5500bp, which correlated with expectation.
2.2 psuPAR sequencing
The recombinant psuPAR vector was sequenced. Results also showed that the recombinant psuPAR vector carried suPAR nucleotide sequence and its encoding amino acid sequence is fully consistent with GeneBank human suPAR sequences (1-849bp) .
3 The effective expression of suPAR in eukaryotic cells of Hela
For the detection of the expression of suPAR in eukaryotic cells, psuPAR was transfected into Hela cells. 72h later, the expression of suPAR was examined by the ELISA from the supernatant of transfected cells. Expression of suPAR from the supernatant of transfected cell was mucher higher than that from not transfected (p<0.05). This result suggests that psuPAR plasmid was well expressed in Hela cells.
4 Hela cells with stable transfected suPAR gene was obtained successfully
Insert SuPAR gene into the eukaryotic expression vector pcDNA3.1-V5/His containing the neomycin gene (neo) resistance gene. Then the recombinant expression plasmid pcsuPAR was obtained. The expression plasmid was transfected into Hela cells by G418 (800μg/ml) complete medium pressure screening to obtain positive clones. A concentration of 400μg/ml of G418 was used to maintain screening. SuPAR stable gene expression in Hela cell clones was identified by ELISA methods. OD value of each clone from ElISA was converted to all cloning ng/ml value in accordance with the standard curve y = 0.197x-0.291.
5 In vivo results for suPAR gene inhibition of tumor growth in subcutaneously transplanted cervical cancer nude mice
Hela cells stably expressing SuPAR gene and wild type Hela cells were transplanted subcutaneously into nude mice respectively. On the 10th day, a touchable tumor block was found in nude mice transplanted with wild-type Hela cells but not in Hela cells stably expressing SuPAR gene. Mice were sacrificed on the 30th day after subcutaneous transplanting. Tumor block was weighted, and results showed that the weight of Hela/ pcDNA3.1-suPAR group was significantly lower than that of Hela group. Hela/pcDNA3.1-suPAR Group showed condensed nuclear staining, nuclear dense staining, decreased nuclear mitotic and significantly reduced cancer infiltration by Paraffin HE.
Conclusions:
1 suPAR gene expression level was closely related to tumor invasion and metastasis ability. Higher level of suPAR was related to stronger invasion and metastasis as well as poorer prognosis;
2 suPAR gene was obtained successfully;
3 Eukaryotic expression vector pc-suPAR construction and restriction enzyme digestion were successfully obtained;
4 Hela cell line stably transfected with suPAR gene was obtained;
5 Successfully transferred to the pcDNA3.1-suPAR cervical cancer animal model of nude mice;
6 pcDNA3.1-suPAR gene vaccination nude suPAR gene showed significant anti-tumor effect, indicating that suPAR gene transfer was an effective strategy for the treatment of cervical cancer.
引文
1 Parkin DM , Pisani P , Feflay J. Global cancer statistics CA Cancer J Clin , 1999 ,49 (1):33-64
2 Lentz SE,Muderspach LI, Felix JC,et al.Indentification of micrometastases in histologically negative lymph nodes of early-stage cervical cancer patients.Obstet Gynecol,2004,103(6):1204-1210
3 AokiY,Sasaki M,Watanabe M,etal.High-risk group in node-positive patients with stage IB,IIA,and IIB cervical carcinoma:after radical hysterctomy and postoperative pelvic irradiation. Gynecol Oncol, 2000, 77(2):305-309
4 Van Trappen PO, GyselmanVG, Lowe DG,etal. Molecular quantification and mapping of lymph node micrometastases in cervical cancer. Lancet, 2001,357(9249):15-20
5 Juretzka MM,Jensen KC,Longacre TA,etal.Detection of Pelvic lymph node micrometastasis in stage IA2-IB2 cervical cancer by immunohistochemical analysis.Gynecol Oncol,2004,93(l):107-111
6 Marchiole P,Buenerd A, Benehaib M,etal.Clinical significance of lympho vascular space involvement and lymph node micrometastases in early-stage cervical cancer:a retrospective case-control surgicopatholo- gical study.Gynecol Oncol,2005,97(3):727-732
7 Van Trappen PO, Gyselman VG, Lowe DG, etal.Molecular quantification and mapping of lymph-node micrometastases in cervical cancer. Lancet, 2001, 357(9249):15-20
8 Galizia G, Lieto E, Ferraraccio F, etal. Determination of molecular marker expression can predict clinical outcome in colon carcinomas.Clin Cancer Res, 2004,10(10):3490-3499
9 Gonzalez Bosquet J,Keeney GL, Mariani A,etal.Cytokeratin staining ofresected lymph nodes may improve the sensitivity of surgical staging for endometrial cancer.Gynecol Oncol,2003, 91(3):518-525
10 Juretzka MM,Jensen KC,Longacre TA,et al.Detection of pelvic lymph node micrometastasis in stage IA2-IB2 cervical cancer by immunohis- tochemical analysis.Gynecol Oncol,2004,93(1):107-111
11 Marchiole P,Buenerd A,Scoazec JY,et al.Sentinel lymph node biopsy is not accurate in predicting lymph node status for patients with cervical carcinoma.Cancer,2004,100(10):2154-2159
12 Lentz SE,Muders Paeh Ll,Felix JC,etal.Indentification of micrometastases in histologically negative lymph nodes of early-stage cervical cancer patients. Obstet Gynecol,2004,103(6):1204-1210
13 Van Trappen PO, GyselmanVG, Lowe D G, etal. Molecular quantification and mapping of lymph-node micrometastases in cervical cancer. Lancet, 2001,357(9249):15-20
14 Malur S,Krause N,Kohler C,et al.Sentinel lymph node detection in Patients with cervical cancer.Gynecol Oncol,2001,80(2):254-257
15 Nezos A, Pissimisis N , Lembessis P.Detection of circulating tumor cells in bladder cancer patients. Cancer Treatment Reviews, 2009, 35(3): 272-279
16 Voulgari A, Pintzas A.Epithelial–mesenchymal transition in cancer metastasis: Mechanisms, markers and strategies to overcome drug resistance in the clinic.Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, Available online 21 March 2009
17 Angenete E, Langenski?ld M, Palmgren I.UPA and PAI-1 in Rectal Cancer-Relationship to Radiotherapy and Clinical Outcome.Journal of Surgical Research, 2009,153,1,(1): 46-53
18 Kalliom?ki T, McCallum G, Wells PG,et al. Progression and metastasis in a transgenic mouse breast cancer model: Effects of exposure to in vivo hypoxiaCancer Letters,, Available online 7 April 2009
19朱世能,陆世伦.肿瘤基础理论(第二版):上海医科大学出版社,264-279
20 YuanC,WangP,LaiC,et al.Recurrence and survival analyses of 1,115cervicalcancer patients treated with radically hysterectomy.Gynecol Obstet Invest,1999,47(2):127-132
21 Takeda N, Sakuragi N,Takeda M,etal.Multivariate analysis of histopathologic prognostic faetors for invasive cervicale cancer treated with radical hysterectomy and systematic retroperitoneal lymphadenec- tomy. Acta Obstet Gynecol Scand,2002,81(12):1144-1151
22 Marchiole P, Buenerd A, Benchaib M,etal. Clinical significance of lympho vasculars space involvement and lymph node micrometastases in early- stage cervicalcancer:a retrospective case-control surgico-pathological study. Gynecol Oncol,2005,97(3):727-732
23 Xuenong Zou, Lijin Zou, Ying He,et al. Molecular treatment strategies and surgical reconstruction for metastatic bone diseases.Cancer Treatment Reviews, 2008,34,6(10): 527-538
24 Brankin B,Campbell M,Canning P, et al.Endostatin modulates VEGF-mediated barrier dysfunction in the retinal microvascular endothelium.Experimental Eye Research, 2005, 81,1(5): 22-31
25 Gaur P, Bose D, Samuel S,et al.Targeting Tumor Angiogenesis.Seminars in Oncology, 2009,36,1(3):S12-S19
26 Vaheri A, Enzerink A , R?s?nen K,et al.Nemosis, a novel way of fibroblast activation, in inflammation and cancer.Experimental Cell Research, Available online 17 March 2009
27 Yu J, Bian D, Mahanivong C, et al.p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression. J Biol Chem, 2004, 279 ( 48 ): 50446 -50454
1 Riisbro R, Stephens R.W, Bru¨nner N, et al. Soluble urokinase plasminogen activator receptor in preoperatively obtained plasma from patients with gynecological cancer or benign gynecological diseases. Gynecologic Oncology ,2001,82:523-531
2 Dano K,Romer J,Nielsen BS,et al.Cancer invasion and tissue remodeling-cooperation of protease systems and cell types. APMIS,1999,107(1):120- 127
3 Rabbani SA,Mazar AP.The role of the plasminogen activation system inangiogenesis and metastasis.Surg Oncol Clin NAm,2001,10(2): 393
4 Nguyen DH,Hussaini M,Gonias SL.Binding of urokinase type plasminogen activator to its receptor in MCF-7 cells activates extracellular signal-regulated kinase 1 and 2 which is required for increasedcellular motility.J Biol Chem,1998,273:8502-8507
5 Angenete E, Langenski?ld M, Palmgren I.uPA and PAI-1 in Rectal Cancer-Relationship to Radiotherapy and Clinical Outcome.Journal of Surgical Research, 2009,153,1,(1): 46-53
6 Premkumar VG, Yuvaraj S, Sathish S. Anti-angiogenic potential of CoenzymeQ10, ribo?avin and niacin in breast cancer patients undergoing tamoxifen therapy. Vascular Pharmacology,2008,48:191-201
7 RabbaniSA,MazarAP.The role of the plasminogen activation systeminangiogenesis and metastasis.Surg Oncol Clin N Am,2001,10(2):393
8 Resnati M,Pallavicini,Iserhan CN,et al.The fibrinolytic receptor for urokinase Activates the G protein-coupled chemotactic receptor.Proc Natl Acad Sci USA,2002,99(3):1359-1364
9 Mizukami I, Garni-Wagner B, DeAngelo L.M, et al.Immunologic detection of the cellular receptor for urokinase plasminogen activator.Clinical Immunology and Immunopathology, 1994, 71(1): 96-104
10 Ellis V,Behrendt N,Dano K.Plasminogen activation by receptor-bound urokinase.Akinetic study with both cell-associated and isolated receptor.J Biol Chem,1991,266(19):12752-12758
11 Kruger A,Soeltl R,Lutz V,et al.Reduction of breast carcinoma tumor growth and lung colonization by overexpression of the soluble urokinase-type plasminogen activator receptor(CD87). Cancer Gene Ther,2000,7(2):292-299
12 Lutz R,Sabine R,Sonja P,et al.Urokinase gene expression indicates early invasive growth in souamous cell lesions of the uterine cervix.Pathol 1999,189(2):245-250
13 Riethdorf S, Neffen E.F, Cviko A,et al.p16INK4A expression as biomarker for HPV 16-related vulvar neoplasias. Human Pathology, 2004, 35(12): 1477-1483
14 Susan Kasper ,et al.Identification, characterization, and biological relevance of prostate cancer stem cells from clinical specimens.Urologic Oncology: Seminars and Original Investigations, 2009, 27,3(5): 301-303
15 Masafumi M, Furukawa J,Takenaka A,et al. Expression of urokinase-type plasminogen activator system in prostate cancer: Correlation with clinicopathological outcomes in patients undergoing radical prostatectomy. Urologic Oncology: Seminars and Original Investigations, 2009, 27,2(3): 180-186
16 Kathleen D,Aamir A,Asfar S et al.Evolving role of uPA/uPAR system in human cancers.Cancer Treatment Reviews,2008,34(2):122-136
17 Chao A, Kung-Chu H, Wang C,et al. Positron emission tomography in evaluating the feasibility of curative intent in cervical cancer patients with limited distant lymph node metastases. Gynecologic Oncology,2008, 8,110(2): 172-178
18 Rombach G,Scharl A,Vierbuchen M,et al.Detection of squamous cell carcinoma antigen in normal squamous epithelia and in squamous cell carcinoma of the uterine cervix.Cancer,1989,63(7):1337-1342
19 Takeshima N.The value of squamous cell carcinoma antigen as a predictor of nodal metastasis in cervical cancer.Gynecol Oncol,1998,68(3):263-266
1 Kathleen Dass, Aamir Ahmad, Asfar S,et al. Evolving role of uPA/uPAR system in human cancers. Cancer Treatment Reviews ,2008,34:122-136
2 Voorzanger-Rousselot N, Garnero P, et al. Biochemical markers in oncology. Part I: Molecular basis. Part II: Clinical uses. Cancer Treatment Reviews ,2007,33: 230- 283
3 Schmitt M,Wilhelm OG,Reuning U,et al.The urokinase plasminogen activator system as a novel target for tumour therapy.Fibrinolysis Proteolysis,2000,14z;114-132
4 Giulio M, Dellabella M, Fazioli F,et al. Increased urokinase-type plasminogen activator receptor and epidermal growth factor receptor in serum of patients with prostate cancer. American urological association,2009, 181(3), 1393-1400
5 Pepper MS.Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis.Arterioscler Thromb Vase Biol,2001,21:1104-1117
6 Tang H,Kerins DM,Hao Q,et al.The urokinase-type plasminogen activator receptor mediates tyrosine phosphorylation of focal adhesion proteins and activation of mitogen-activated protein kinase in cultured endothelial cells.J Biol Chem,1998,273:18268-18272
7 Foekens JA, Peters HA,Look MP,et al.The urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients.Cancer Res,2000,60:636-643
8 Ribatti D, Leali D, Vacca A,et al. In vivo angiogenic activity of urokinase:role of endogenous fibroblast growth factor-2.J Cell Sci,1999, 112: 4213-4221
9 Paolo Mignatti,Danie B.Biology and biochemistry of proteins in tumor invasion.Phyisol Rev 1993,73:161
10 Ellis V,Behrendt N,Dano K.Plasminogen activation by receptor-bound urokinase.Akinetic study with both cell-associated and isolated receptor.JBiol Chem,1991,266(19):12752-12758
11 Pedersen N,Schmitt M,Ronne E,Nicoletti M.I,et al.A ligand-free,soluble urokinase receptor is present in the ascites fluid from patients with ovarian cancer J.Clin Invert.1993;92,2160-2167
12 Resnati M ,Guttinger M ,Valcamonica S,et a1.Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect.1996 15(7): 1572-1582
13 Chavakis T, Willouweit A, Lupu F. The soluble urokinase receptor: mechanisms of release and functional activities. Atherosclerosis, 1999, 144, 1(5): 15-17
14 Wilhelm O,Weidle.V,Hohl S,et al.Recombinant soluble urokinase receptor as a scavenger for urokinase-type plasminogen activator(uPA).Inhibition of proliferation and invasion of human ovarian cancer cells.FEBS Lett,1994,Jan 10;337(2):131-134
15 Kruger A,Soeltl R,Lutz V,et a1.Reduction of breast carcinoma tumor growth and lung colonization by overexpression of the soluble urokinase-type plasminogen activator receptor(CD87).Cancer Gene Ther,2000,7(2): 292-299
16 Ohno1 Y, Ohori M, Akimoto S. Identification of bone metastasis markers in prostate cancer. European Journal of Cancer Supplements, 2008, 6, 9(7): 68
17 Milanese G, Dellabella M, Fazioli F. Increased urokinase-type plasminogen activator receptor and epidermal growth factor receptor in serum of patients with prostate cancer. The Journal of Urology, 2009, 181,3(3): 1393-1400
18 Verena Lutz,Ute reuning,Achim Kruger,Thomas Luther,et al.High Level Synthesis of Recombinant Soluble Urokinase Receptor(CD87) by Ovarian Cancer Cells Reduces Intraperitoneal Tumor Growth and Spread in Nude Mice.Biol.Chem.2001,3(382):789-798
19 Gardsvoll H,Werner F, Sondergaard L,et al. Characte-rization of low-glycosylated forms of soluble human urokinase receptor rxpressed inDrosophila Schneider 2 cells after deletion of glycosylation-sites.Protein Expression and Purification ,2004, 34: 284- 295
20 Chau I, Rigg A, Cunningham D,et al. Matrix metalloproteinase inhibitors/an emphasis on gastrointestinal malignancies.Critical reviews in Oncology/Hematology ,2003 ,45:151-176
21 Henrik G?rdsvoll, Hansen L, J?rgensen T, et al. A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor.Protein Expression and Purification, 2007, 52(2):384-394
22 Fevang A, Eugen-Olsen J, Yndestad A. Enhanced levels of urokinase plasminogen activator and its soluble receptor in common variable immunodeficiency. Clinical Immunology, In Press, Corrected Proof,2009, 15(2)
23 Lutz V,Reuning U,Kruger A,et a1.High level synthesis of recombinant soluble urokinase receptor(CD87) by ovarian cancer cells reduces intraperitoneal tumor growth and spread in nude mice.Biol Chem, 2001, 382(5): 789-798
24 Resnati M.Pallavicini I,Wang J M,et a1.The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/I XA4R.Proc Natl Acad Sci USA, 2002,99(3):1359-l364
25 Bernd R. Binder, Judit Mihaly,et al. The plasminogen activator inhibitor“paradox”in cancer. Immunology Letters, 2008, 118(2):116-124
26 Chau I, Rigg A, Cunningham D,et al. Matrix metalloproteinase inhibitors/ an emphasis on gastrointestinal malignancies.Critical reviews in Oncology/Hematology,2003,45:151-176
1 Dano K,Andreasen PA,Grohndahl-Hansen J et al.Plasminogen activators, tissue degradation and cancer,Adv Cancer Res 1985,44:139-266
2 Strickland S.Richards WG.Invasion of the trophoblasts.Cell 1992,71:355-357
3 Klift C.Tissue type-plasminogen activator.Boca Raton CRC press Inc, 1988
4 Malur S,Krause N,Kohler C,et al.Sentinel lymph node detection in Patients with cervical cancer.Gynecol Oncol,2001,80(2):254-257
5 Nezos A, Pissimisis N , Lembessis P.Detection of circulating tumor cells in bladder cancer patients. Cancer Treatment Reviews, 2009, 35(3):272-279
6 Voulgari A, Pintzas A.Epithelial–mesenchymal transition in cancer metastasis: Mechanisms, markers and strategies to overcome drug resistance in the clinic.Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, Available online 21 March 2009
7 Angenete E, Langenski?ld M, Palmgren I.uPA and PAI-1 in RectalCancer-Relationship to Radiotherapy and Clinical Outcome.Journal of Surgical Research, 2009,153,1,(1):46-53
8 Dear AE & Medcalf RL.The urokinase-type plasminogen activator (CD87)is a pleiotropic molecule Eur J Biochem1998,252:185-193
9 Takeda N,Sakuragi N,Takeda M,etal.Multivariate analysis of histopathologic prognostic faetors for invasive cervicale cancer treated with radical hysterectomy and systematic retroperitoneal lymphadenec- tomy.Acta Obstet Gynecol Scand, 2002, 81(12):1144-1151
10 Slavica Kvolik, Marko Jukic, Marko Matijevic,et al.An overview of coagulation disorders in cancer patients.Surgical Oncology,Available online 25 April 2009
11 Y. Li, P.J. Cozzi .Targeting uPA/uPAR in prostate cancer.Cancer Treatment Reviews, 2007,33,6(10):521-527
12 Chloe Milsom, Joanne Yu, Linda May,et al.The role of tumor-and host-related tissue factor pools in oncogene-driven tumor progression. Thrombosis Research, 2007,120,2:S82-S91
13 Ricard V. Solé, Carlos Rodríguez-Caso, Thomas S,et al.Cancer stem cells as the engine of unstable tumor progression.Journal of Theoretical Biology, 2008,253,4(21):629-637
14 Carroll VA,Binder BR.The role of the plasminogen activation system in cancer.Semin Thromb Hemost 1999,25(2):183-197
15 Duffy MJ.Proteases as prognostic markers in cancer.Clin Cancer Res.1996,2(4):613-618
16 Oberholster A.J, Heyns P.S, et al.Online condition monitoring of axial-flow turbomachinery blades using rotor-axial Eulerian laser Doppler vibrometry.Mechanical Systems and Signal Processing, 2009, 23,5(6): 1634-1643
17 Keller S, Anne-Kathleen K, MarméF,et al.Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes Cancer Letters, 2009, 278(1):73-81
18 Ossowski L,Russo-Payne H,Wilson EL.Inhibition of urokinase typeplasminogen activators by antibodies:The effect on dissemination of a human tumor in the nude mouse.Cancer Res 1991,51:274-281
19 Achbarou A,Kaiser S,Tremblay G,et al.Urokinase overproduction results in increased skeletal metastasis by prostate cancer cells in vivo.Cancer Res 1994,54:2372-2377
20 Jae Young Cho,M.D.Hyun Cheol Chung,M.D.Sung Hoon Noh,M.D.et al.High level of urokanise-type plasminogen activator is a new prosnostic marker in patients with gastric carcinoma. Cancer, 1997,79(5):878
21 Xuenong Zou, Lijin Zou, Ying He,et al. Molecular treatment strategies and surgical reconstruction for metastatic bone diseases.Cancer Treatment Reviews, 2008,34,6(10):527-538
22 Daneri-Navarro A,Del Toro-Arreola S,Sanchez-Hernandez PE,et al. Immunosuppressive activity of proteases in cervical carcinoma.Gynecol Oncol,2005,98(1):111-117
23 Stepanova V,Mukhina S,Kohier E,et al.Urokinase plasminogen activator induces human smooth muscle cell migration and proliferation via distinct receptor-depentent and proteolysis–dependent mechanisms.Mol Cell Biochem. 1999, 195(1-2):199-206
24 Nguyen DH,Webb DJ,Catling AD,et al.Urokinase-type Plasminogen Activator Stimulates the Ras/ERK Signaling Pathway and MCF-7 Cell Migration by a Mechanism that Requires FAK,Src,and Shc:Rapid dissociation of Grb2/Sos from Shc is associated with the transient phosphorylation of ERK in urokinase-treated cells.J Biol Chem.2000
25 Dumler I,Stepanova V,Jerke U,et al.Urokinase-induced mitogenesis is mediated by casein kinase 2 and nucleolin.Curr Biol.1999, 16-30: 9(24):1468-1476
26 Planus E,Barlovatz-Meimon G,Rogers RA,et al.Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading.J Cell Sci.1997,110(Pt9):1091-1098
27 Hildenbrand R,Dilger I,Horlin A,Stutte HJ.Urokinase plasminogen activator induces angiogenesis and tumor vessel invasion in breastcancer .Pathol Res Pract.1995,191(5):403-409
28 RabbaniSA,MazarAP.The role of the plasminogen activation system inangiogenesis and metastasis.Surg Oncol Clin NAm,2001,10(2):393
29 Nguyen DH,Hussaini M,Gonias SL.Binding of urokinase type plasminogen activator to its receptor in MCF-7 cells activates extracellular signal-regulated kinase 1 and 2 which is required for increasedcellular motility.J Biol Chem,1998,273:8502-8507
30 Ikuko F Mizukami, Beth Ann Garni-Wagner, L. Michael DeAngelo,et al. Immunologic Detection of the Cellular Receptor for Urokinase Plasminogen Activator.Clinical Immunology and Immunopathology, 1994, 71(1):96-104
31 Susan Kasper ,et al.Identification, characterization, and biological relevance of prostate cancer stem cells from clinical specimens.Urologic Oncology: Seminars and Original Investigations, 2009, 27,3(5): 301-303
32 Masafumi Kumano, Hideaki Miyake, Mototsugu Muramaki, Junya Furukawa, Atsushi Takenaka, Masato Fujisawa. Expression of urokinase-type plasminogen activator system in prostate cancer: Correlation with clinicopathological outcomes in patients undergoing radical prostatectomy. Urologic Oncology: Seminars and Original Investigations, 2009, 27(2):180-186
33 Sabine Riethdorf, Eduardo F. Neffen, Aida Cviko, Thomas L?ning, Christopher P. Crum, Lutz Riethdorf.p16INK4A expression as biomarker for HPV 16-related vulvar neoplasias. Human Pathology, 2004,35(12):1477-1483
34 Motoi Sugimura, Hiroshi Kobayashi, Naohiro Kanayama, Toshihiko Terao. Clinical significance of urokinase-type plasminogen activator (uPA) in invasive cervical cancer of the uterus .Gynecologic Oncology, 1992,46(3): 330-336
35 Wilhelm O,Weidle.V,Hohl S,et al.Recombinant soluble urokinase receptor as a scavenger for urokinase-type plasminogen activator(uPA).Inhibition of proliferation and invasion of human ovarian cancer cells.FEBSLett,1994,Jan 10;337(2):131-134
36 Resnati M,Guttinger M,Valcamonica S,et al.Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactice- ffect.EMBOJ,1996,15(7):1572-1582
37 Minji J,Keena S,Thoma S,et al.Soluble urohinase-type polasminogen activator receptor inhibits cancer cell growth and invasion by direct urohinase-independent effects on cell singnaling.Biol Chem, 2003, 287:46692- 46698
38 Resnati M,Fazioli F, Sidenius N,et al.A urokinase-sensitive region of the human urokinase receptor is responsible for it schemotactic activity. EMBOJ,1997,16:7279-7286
39 Warren Schlecht,Genesio Murano and DouglasBoyd.Examination of the role of the urokanise receptor in human colon cancer.Cancer Res,1989,49:6064
40 Schmitt M,Harbeck N,Thomssen C,et al.Clinical impact of the plasminogen activation system in tumor invasion and metastasis: prognostic relevance and target for therapy.Thromb Haemost. 1997, 78(1):285-296
41 Kobayashi H,Suzuki M,Kanayama N,et al.Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade. European Journal of Biochemistry, 2002;269(16):3945-3957
42 Ossowski L,Aguirre-Ghiso JA.Urokinase receptor and integrin partnership:coordination of signaling for cell adhesion,migration and growth.Curr Opin Cell Biol,2000;12(5):613-620
43 Aguirre Ghiso JA,Alonso DF,Farias EF,et al.Deregulation of the signaling pathways controlling urokinase production.Its relationship with the invasive phenotype. Biochem.1999,263(2):295-304
1 Tsoumpou I, M. Arbyn, M. Kyrgiou,et al.p16INK4a immunostaining in cytological and histological specimens from the uterine cervix: A systematic review and meta-analysis.Cancer Treatment Reviews, 2009, 35,3(5): 210-220
2 Wentzensen N, Sherman M, Schiffman M,et al.Utility of methylation markers in cervical cancer early detection: Appraisal of the state-of-the-science.Gynecologic Oncology, 2009,112,2(5) :293-299
3 Piepoli, R. Cotugno, M. Quitadamo, et al.Methylation study of CDH1, P16,APC and MLH1 genes promoter along multistep gastric carcinogenesis. Digestive and Liver Disease, 2006,38,1(3):S110-S111
4 Neller M, J. Alejandro López, Christopher W,et al.Antigens for cancer immunotherapy.Seminars in Immunology, 2008,20,5(10):286-295
5 Oliveira-Cunha M, Siriwardena A, Byers R,et al.Molecular diagnosis in pancreatic cancer.Diagnostic Histopathology, 2008, 14,5(5):214-222
6 Tang-Yuan Chu, et al.Risk Factors and Genetic Markers of Human Papillomavirus-induced Cervical Carcinogenesis: A Focus on Chinese Populations in Southeast Asia and Southern China.Tzu Chi Medical Journal, 2008,20,2(6): 91-100
7 Sindos M, Ndisang D, Pisal N,et al.Measurement of Brn-3a levels in Pap smears provides a novel diagnostic marker for the detection of cervical neoplasia.Gynecologic Oncology, 2003, 90,2(8):366-371
8 Malur S,Krause N,Kohler C,et al.Sentinel lymph node detection in Patients with cervical cancer.Gynecol Oncol,2001,80(2):254-257
9 Nezos A, Pissimisis N , Lembessis P.Detection of circulating tumor cells in bladder cancer patients. Cancer Treatment Reviews, 2009, 35(3):272-279
10 Manavi M, Hudelist G, Fink-Retter A,et al.Gene profiling in Pap-cell smears of high-risk human papillomavirus-positive squamous cervical carcinoma.Gynecologic Oncology, 2007, 105,2(5): 418-426
11 Dano K,Romer J,Nielsen BS,et al.Cancer invasion and tissue remo deling-cooperation of protease systems and cell types. APMIS, 1999, 107(1):120-127
12 Resnati M,Pallavicini,Iserhan CN,et al.The fibrinolytic receptor for urokinase Activates the G protein-coupled chemotactic receptor.Proc Natl Acad Sci USA,2002,99(3):1359-1364
13 Richter C, Leo C, Schütz A,et al.Apaf-1 is regulated by hypoxia in cervical cancer.Pathology - Research and Practice, 2004,200,4: 329-330
14 Bergers G,Benjamin LE.Tumorigenesis and the angiogenic switch .Nat ure Rev Cancer,2003,3l:401-410
15 Keyt BA, Berleau LT, Nguyen HV, et al.The carboxyl-terminal domain (111–165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem , 1996,271:7788-7795
16 Ribatti D, Leali D, Vacca A, et al: In vivo angiogenic activity of urokinase: Role of endogenous fibroblast growth factor-2. J Cell Sci,1999, 112:4213–4221
17 Lansink M, Koolwijk P, van Hinsbergh V, Kooistra T: Effect of steroid hormones and retinoids on the formation of capillary- like tubular structures of human microvascular endothelial cells in fibrin matrices is related to urokinase expression. Blood 92:927-938, 1998
18 Chao A, Wang T, Chyong-Huey Lai,et al. Overview of Microarray Analysis of Gene Expression and its Applications to Cervical Cancer Investigation.Taiwanese Journal of Obstetrics and Gynecology, 2007, 46,4(12):363-373
19 Saldivar J.S, Lopez D, Rebecca,et al.COX-2 overexpression as a biomarker of early cervical carcinogenesis: A pilot study.Gynecologic Oncology, Supplement 1, October 2007, 107,1:S155-S162
2 Lentz SE,Muderspach LI, Felix JC,et al.Indentification of micrometastases in histologically negative lymph nodes of early-stage cervical cancer patients.Obstet Gynecol,2004,103(6):1204-1210
3 AokiY,Sasaki M,Watanabe M,etal.High-risk group in node-positive patients with stage IB,IIA,and IIB cervical carcinoma:after radical hysterctomy and postoperative pelvic irradiation. Gynecol Oncol, 2000, 77(2):305-309
4 Van Trappen PO, GyselmanVG, Lowe DG,etal. Molecular quantification and mapping of lymph node micrometastases in cervical cancer. Lancet, 2001,357(9249):15-20
5 Juretzka MM,Jensen KC,Longacre TA,etal.Detection of Pelvic lymph node micrometastasis in stage IA2-IB2 cervical cancer by immunohistochemical analysis.Gynecol Oncol,2004,93(l):107-111
6 Marchiole P,Buenerd A, Benehaib M,etal.Clinical significance of lympho vascular space involvement and lymph node micrometastases in early-stage cervical cancer:a retrospective case-control surgicopatholo- gical study.Gynecol Oncol,2005,97(3):727-732
7 Van Trappen PO, Gyselman VG, Lowe DG, etal.Molecular quantification and mapping of lymph-node micrometastases in cervical cancer. Lancet, 2001, 357(9249):15-20
8 Galizia G, Lieto E, Ferraraccio F, etal. Determination of molecular marker expression can predict clinical outcome in colon carcinomas.Clin Cancer Res, 2004,10(10):3490-3499
9 Gonzalez Bosquet J,Keeney GL, Mariani A,etal.Cytokeratin staining ofresected lymph nodes may improve the sensitivity of surgical staging for endometrial cancer.Gynecol Oncol,2003, 91(3):518-525
10 Juretzka MM,Jensen KC,Longacre TA,et al.Detection of pelvic lymph node micrometastasis in stage IA2-IB2 cervical cancer by immunohis- tochemical analysis.Gynecol Oncol,2004,93(1):107-111
11 Marchiole P,Buenerd A,Scoazec JY,et al.Sentinel lymph node biopsy is not accurate in predicting lymph node status for patients with cervical carcinoma.Cancer,2004,100(10):2154-2159
12 Lentz SE,Muders Paeh Ll,Felix JC,etal.Indentification of micrometastases in histologically negative lymph nodes of early-stage cervical cancer patients. Obstet Gynecol,2004,103(6):1204-1210
13 Van Trappen PO, GyselmanVG, Lowe D G, etal. Molecular quantification and mapping of lymph-node micrometastases in cervical cancer. Lancet, 2001,357(9249):15-20
14 Malur S,Krause N,Kohler C,et al.Sentinel lymph node detection in Patients with cervical cancer.Gynecol Oncol,2001,80(2):254-257
15 Nezos A, Pissimisis N , Lembessis P.Detection of circulating tumor cells in bladder cancer patients. Cancer Treatment Reviews, 2009, 35(3): 272-279
16 Voulgari A, Pintzas A.Epithelial–mesenchymal transition in cancer metastasis: Mechanisms, markers and strategies to overcome drug resistance in the clinic.Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, Available online 21 March 2009
17 Angenete E, Langenski?ld M, Palmgren I.UPA and PAI-1 in Rectal Cancer-Relationship to Radiotherapy and Clinical Outcome.Journal of Surgical Research, 2009,153,1,(1): 46-53
18 Kalliom?ki T, McCallum G, Wells PG,et al. Progression and metastasis in a transgenic mouse breast cancer model: Effects of exposure to in vivo hypoxiaCancer Letters,, Available online 7 April 2009
19朱世能,陆世伦.肿瘤基础理论(第二版):上海医科大学出版社,264-279
20 YuanC,WangP,LaiC,et al.Recurrence and survival analyses of 1,115cervicalcancer patients treated with radically hysterectomy.Gynecol Obstet Invest,1999,47(2):127-132
21 Takeda N, Sakuragi N,Takeda M,etal.Multivariate analysis of histopathologic prognostic faetors for invasive cervicale cancer treated with radical hysterectomy and systematic retroperitoneal lymphadenec- tomy. Acta Obstet Gynecol Scand,2002,81(12):1144-1151
22 Marchiole P, Buenerd A, Benchaib M,etal. Clinical significance of lympho vasculars space involvement and lymph node micrometastases in early- stage cervicalcancer:a retrospective case-control surgico-pathological study. Gynecol Oncol,2005,97(3):727-732
23 Xuenong Zou, Lijin Zou, Ying He,et al. Molecular treatment strategies and surgical reconstruction for metastatic bone diseases.Cancer Treatment Reviews, 2008,34,6(10): 527-538
24 Brankin B,Campbell M,Canning P, et al.Endostatin modulates VEGF-mediated barrier dysfunction in the retinal microvascular endothelium.Experimental Eye Research, 2005, 81,1(5): 22-31
25 Gaur P, Bose D, Samuel S,et al.Targeting Tumor Angiogenesis.Seminars in Oncology, 2009,36,1(3):S12-S19
26 Vaheri A, Enzerink A , R?s?nen K,et al.Nemosis, a novel way of fibroblast activation, in inflammation and cancer.Experimental Cell Research, Available online 17 March 2009
27 Yu J, Bian D, Mahanivong C, et al.p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on urokinase plasminogen activator expression. J Biol Chem, 2004, 279 ( 48 ): 50446 -50454
1 Riisbro R, Stephens R.W, Bru¨nner N, et al. Soluble urokinase plasminogen activator receptor in preoperatively obtained plasma from patients with gynecological cancer or benign gynecological diseases. Gynecologic Oncology ,2001,82:523-531
2 Dano K,Romer J,Nielsen BS,et al.Cancer invasion and tissue remodeling-cooperation of protease systems and cell types. APMIS,1999,107(1):120- 127
3 Rabbani SA,Mazar AP.The role of the plasminogen activation system inangiogenesis and metastasis.Surg Oncol Clin NAm,2001,10(2): 393
4 Nguyen DH,Hussaini M,Gonias SL.Binding of urokinase type plasminogen activator to its receptor in MCF-7 cells activates extracellular signal-regulated kinase 1 and 2 which is required for increasedcellular motility.J Biol Chem,1998,273:8502-8507
5 Angenete E, Langenski?ld M, Palmgren I.uPA and PAI-1 in Rectal Cancer-Relationship to Radiotherapy and Clinical Outcome.Journal of Surgical Research, 2009,153,1,(1): 46-53
6 Premkumar VG, Yuvaraj S, Sathish S. Anti-angiogenic potential of CoenzymeQ10, ribo?avin and niacin in breast cancer patients undergoing tamoxifen therapy. Vascular Pharmacology,2008,48:191-201
7 RabbaniSA,MazarAP.The role of the plasminogen activation systeminangiogenesis and metastasis.Surg Oncol Clin N Am,2001,10(2):393
8 Resnati M,Pallavicini,Iserhan CN,et al.The fibrinolytic receptor for urokinase Activates the G protein-coupled chemotactic receptor.Proc Natl Acad Sci USA,2002,99(3):1359-1364
9 Mizukami I, Garni-Wagner B, DeAngelo L.M, et al.Immunologic detection of the cellular receptor for urokinase plasminogen activator.Clinical Immunology and Immunopathology, 1994, 71(1): 96-104
10 Ellis V,Behrendt N,Dano K.Plasminogen activation by receptor-bound urokinase.Akinetic study with both cell-associated and isolated receptor.J Biol Chem,1991,266(19):12752-12758
11 Kruger A,Soeltl R,Lutz V,et al.Reduction of breast carcinoma tumor growth and lung colonization by overexpression of the soluble urokinase-type plasminogen activator receptor(CD87). Cancer Gene Ther,2000,7(2):292-299
12 Lutz R,Sabine R,Sonja P,et al.Urokinase gene expression indicates early invasive growth in souamous cell lesions of the uterine cervix.Pathol 1999,189(2):245-250
13 Riethdorf S, Neffen E.F, Cviko A,et al.p16INK4A expression as biomarker for HPV 16-related vulvar neoplasias. Human Pathology, 2004, 35(12): 1477-1483
14 Susan Kasper ,et al.Identification, characterization, and biological relevance of prostate cancer stem cells from clinical specimens.Urologic Oncology: Seminars and Original Investigations, 2009, 27,3(5): 301-303
15 Masafumi M, Furukawa J,Takenaka A,et al. Expression of urokinase-type plasminogen activator system in prostate cancer: Correlation with clinicopathological outcomes in patients undergoing radical prostatectomy. Urologic Oncology: Seminars and Original Investigations, 2009, 27,2(3): 180-186
16 Kathleen D,Aamir A,Asfar S et al.Evolving role of uPA/uPAR system in human cancers.Cancer Treatment Reviews,2008,34(2):122-136
17 Chao A, Kung-Chu H, Wang C,et al. Positron emission tomography in evaluating the feasibility of curative intent in cervical cancer patients with limited distant lymph node metastases. Gynecologic Oncology,2008, 8,110(2): 172-178
18 Rombach G,Scharl A,Vierbuchen M,et al.Detection of squamous cell carcinoma antigen in normal squamous epithelia and in squamous cell carcinoma of the uterine cervix.Cancer,1989,63(7):1337-1342
19 Takeshima N.The value of squamous cell carcinoma antigen as a predictor of nodal metastasis in cervical cancer.Gynecol Oncol,1998,68(3):263-266
1 Kathleen Dass, Aamir Ahmad, Asfar S,et al. Evolving role of uPA/uPAR system in human cancers. Cancer Treatment Reviews ,2008,34:122-136
2 Voorzanger-Rousselot N, Garnero P, et al. Biochemical markers in oncology. Part I: Molecular basis. Part II: Clinical uses. Cancer Treatment Reviews ,2007,33: 230- 283
3 Schmitt M,Wilhelm OG,Reuning U,et al.The urokinase plasminogen activator system as a novel target for tumour therapy.Fibrinolysis Proteolysis,2000,14z;114-132
4 Giulio M, Dellabella M, Fazioli F,et al. Increased urokinase-type plasminogen activator receptor and epidermal growth factor receptor in serum of patients with prostate cancer. American urological association,2009, 181(3), 1393-1400
5 Pepper MS.Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis.Arterioscler Thromb Vase Biol,2001,21:1104-1117
6 Tang H,Kerins DM,Hao Q,et al.The urokinase-type plasminogen activator receptor mediates tyrosine phosphorylation of focal adhesion proteins and activation of mitogen-activated protein kinase in cultured endothelial cells.J Biol Chem,1998,273:18268-18272
7 Foekens JA, Peters HA,Look MP,et al.The urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients.Cancer Res,2000,60:636-643
8 Ribatti D, Leali D, Vacca A,et al. In vivo angiogenic activity of urokinase:role of endogenous fibroblast growth factor-2.J Cell Sci,1999, 112: 4213-4221
9 Paolo Mignatti,Danie B.Biology and biochemistry of proteins in tumor invasion.Phyisol Rev 1993,73:161
10 Ellis V,Behrendt N,Dano K.Plasminogen activation by receptor-bound urokinase.Akinetic study with both cell-associated and isolated receptor.JBiol Chem,1991,266(19):12752-12758
11 Pedersen N,Schmitt M,Ronne E,Nicoletti M.I,et al.A ligand-free,soluble urokinase receptor is present in the ascites fluid from patients with ovarian cancer J.Clin Invert.1993;92,2160-2167
12 Resnati M ,Guttinger M ,Valcamonica S,et a1.Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect.1996 15(7): 1572-1582
13 Chavakis T, Willouweit A, Lupu F. The soluble urokinase receptor: mechanisms of release and functional activities. Atherosclerosis, 1999, 144, 1(5): 15-17
14 Wilhelm O,Weidle.V,Hohl S,et al.Recombinant soluble urokinase receptor as a scavenger for urokinase-type plasminogen activator(uPA).Inhibition of proliferation and invasion of human ovarian cancer cells.FEBS Lett,1994,Jan 10;337(2):131-134
15 Kruger A,Soeltl R,Lutz V,et a1.Reduction of breast carcinoma tumor growth and lung colonization by overexpression of the soluble urokinase-type plasminogen activator receptor(CD87).Cancer Gene Ther,2000,7(2): 292-299
16 Ohno1 Y, Ohori M, Akimoto S. Identification of bone metastasis markers in prostate cancer. European Journal of Cancer Supplements, 2008, 6, 9(7): 68
17 Milanese G, Dellabella M, Fazioli F. Increased urokinase-type plasminogen activator receptor and epidermal growth factor receptor in serum of patients with prostate cancer. The Journal of Urology, 2009, 181,3(3): 1393-1400
18 Verena Lutz,Ute reuning,Achim Kruger,Thomas Luther,et al.High Level Synthesis of Recombinant Soluble Urokinase Receptor(CD87) by Ovarian Cancer Cells Reduces Intraperitoneal Tumor Growth and Spread in Nude Mice.Biol.Chem.2001,3(382):789-798
19 Gardsvoll H,Werner F, Sondergaard L,et al. Characte-rization of low-glycosylated forms of soluble human urokinase receptor rxpressed inDrosophila Schneider 2 cells after deletion of glycosylation-sites.Protein Expression and Purification ,2004, 34: 284- 295
20 Chau I, Rigg A, Cunningham D,et al. Matrix metalloproteinase inhibitors/an emphasis on gastrointestinal malignancies.Critical reviews in Oncology/Hematology ,2003 ,45:151-176
21 Henrik G?rdsvoll, Hansen L, J?rgensen T, et al. A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor.Protein Expression and Purification, 2007, 52(2):384-394
22 Fevang A, Eugen-Olsen J, Yndestad A. Enhanced levels of urokinase plasminogen activator and its soluble receptor in common variable immunodeficiency. Clinical Immunology, In Press, Corrected Proof,2009, 15(2)
23 Lutz V,Reuning U,Kruger A,et a1.High level synthesis of recombinant soluble urokinase receptor(CD87) by ovarian cancer cells reduces intraperitoneal tumor growth and spread in nude mice.Biol Chem, 2001, 382(5): 789-798
24 Resnati M.Pallavicini I,Wang J M,et a1.The fibrinolytic receptor for urokinase activates the G protein-coupled chemotactic receptor FPRL1/I XA4R.Proc Natl Acad Sci USA, 2002,99(3):1359-l364
25 Bernd R. Binder, Judit Mihaly,et al. The plasminogen activator inhibitor“paradox”in cancer. Immunology Letters, 2008, 118(2):116-124
26 Chau I, Rigg A, Cunningham D,et al. Matrix metalloproteinase inhibitors/ an emphasis on gastrointestinal malignancies.Critical reviews in Oncology/Hematology,2003,45:151-176
1 Dano K,Andreasen PA,Grohndahl-Hansen J et al.Plasminogen activators, tissue degradation and cancer,Adv Cancer Res 1985,44:139-266
2 Strickland S.Richards WG.Invasion of the trophoblasts.Cell 1992,71:355-357
3 Klift C.Tissue type-plasminogen activator.Boca Raton CRC press Inc, 1988
4 Malur S,Krause N,Kohler C,et al.Sentinel lymph node detection in Patients with cervical cancer.Gynecol Oncol,2001,80(2):254-257
5 Nezos A, Pissimisis N , Lembessis P.Detection of circulating tumor cells in bladder cancer patients. Cancer Treatment Reviews, 2009, 35(3):272-279
6 Voulgari A, Pintzas A.Epithelial–mesenchymal transition in cancer metastasis: Mechanisms, markers and strategies to overcome drug resistance in the clinic.Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, Available online 21 March 2009
7 Angenete E, Langenski?ld M, Palmgren I.uPA and PAI-1 in RectalCancer-Relationship to Radiotherapy and Clinical Outcome.Journal of Surgical Research, 2009,153,1,(1):46-53
8 Dear AE & Medcalf RL.The urokinase-type plasminogen activator (CD87)is a pleiotropic molecule Eur J Biochem1998,252:185-193
9 Takeda N,Sakuragi N,Takeda M,etal.Multivariate analysis of histopathologic prognostic faetors for invasive cervicale cancer treated with radical hysterectomy and systematic retroperitoneal lymphadenec- tomy.Acta Obstet Gynecol Scand, 2002, 81(12):1144-1151
10 Slavica Kvolik, Marko Jukic, Marko Matijevic,et al.An overview of coagulation disorders in cancer patients.Surgical Oncology,Available online 25 April 2009
11 Y. Li, P.J. Cozzi .Targeting uPA/uPAR in prostate cancer.Cancer Treatment Reviews, 2007,33,6(10):521-527
12 Chloe Milsom, Joanne Yu, Linda May,et al.The role of tumor-and host-related tissue factor pools in oncogene-driven tumor progression. Thrombosis Research, 2007,120,2:S82-S91
13 Ricard V. Solé, Carlos Rodríguez-Caso, Thomas S,et al.Cancer stem cells as the engine of unstable tumor progression.Journal of Theoretical Biology, 2008,253,4(21):629-637
14 Carroll VA,Binder BR.The role of the plasminogen activation system in cancer.Semin Thromb Hemost 1999,25(2):183-197
15 Duffy MJ.Proteases as prognostic markers in cancer.Clin Cancer Res.1996,2(4):613-618
16 Oberholster A.J, Heyns P.S, et al.Online condition monitoring of axial-flow turbomachinery blades using rotor-axial Eulerian laser Doppler vibrometry.Mechanical Systems and Signal Processing, 2009, 23,5(6): 1634-1643
17 Keller S, Anne-Kathleen K, MarméF,et al.Systemic presence and tumor-growth promoting effect of ovarian carcinoma released exosomes Cancer Letters, 2009, 278(1):73-81
18 Ossowski L,Russo-Payne H,Wilson EL.Inhibition of urokinase typeplasminogen activators by antibodies:The effect on dissemination of a human tumor in the nude mouse.Cancer Res 1991,51:274-281
19 Achbarou A,Kaiser S,Tremblay G,et al.Urokinase overproduction results in increased skeletal metastasis by prostate cancer cells in vivo.Cancer Res 1994,54:2372-2377
20 Jae Young Cho,M.D.Hyun Cheol Chung,M.D.Sung Hoon Noh,M.D.et al.High level of urokanise-type plasminogen activator is a new prosnostic marker in patients with gastric carcinoma. Cancer, 1997,79(5):878
21 Xuenong Zou, Lijin Zou, Ying He,et al. Molecular treatment strategies and surgical reconstruction for metastatic bone diseases.Cancer Treatment Reviews, 2008,34,6(10):527-538
22 Daneri-Navarro A,Del Toro-Arreola S,Sanchez-Hernandez PE,et al. Immunosuppressive activity of proteases in cervical carcinoma.Gynecol Oncol,2005,98(1):111-117
23 Stepanova V,Mukhina S,Kohier E,et al.Urokinase plasminogen activator induces human smooth muscle cell migration and proliferation via distinct receptor-depentent and proteolysis–dependent mechanisms.Mol Cell Biochem. 1999, 195(1-2):199-206
24 Nguyen DH,Webb DJ,Catling AD,et al.Urokinase-type Plasminogen Activator Stimulates the Ras/ERK Signaling Pathway and MCF-7 Cell Migration by a Mechanism that Requires FAK,Src,and Shc:Rapid dissociation of Grb2/Sos from Shc is associated with the transient phosphorylation of ERK in urokinase-treated cells.J Biol Chem.2000
25 Dumler I,Stepanova V,Jerke U,et al.Urokinase-induced mitogenesis is mediated by casein kinase 2 and nucleolin.Curr Biol.1999, 16-30: 9(24):1468-1476
26 Planus E,Barlovatz-Meimon G,Rogers RA,et al.Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading.J Cell Sci.1997,110(Pt9):1091-1098
27 Hildenbrand R,Dilger I,Horlin A,Stutte HJ.Urokinase plasminogen activator induces angiogenesis and tumor vessel invasion in breastcancer .Pathol Res Pract.1995,191(5):403-409
28 RabbaniSA,MazarAP.The role of the plasminogen activation system inangiogenesis and metastasis.Surg Oncol Clin NAm,2001,10(2):393
29 Nguyen DH,Hussaini M,Gonias SL.Binding of urokinase type plasminogen activator to its receptor in MCF-7 cells activates extracellular signal-regulated kinase 1 and 2 which is required for increasedcellular motility.J Biol Chem,1998,273:8502-8507
30 Ikuko F Mizukami, Beth Ann Garni-Wagner, L. Michael DeAngelo,et al. Immunologic Detection of the Cellular Receptor for Urokinase Plasminogen Activator.Clinical Immunology and Immunopathology, 1994, 71(1):96-104
31 Susan Kasper ,et al.Identification, characterization, and biological relevance of prostate cancer stem cells from clinical specimens.Urologic Oncology: Seminars and Original Investigations, 2009, 27,3(5): 301-303
32 Masafumi Kumano, Hideaki Miyake, Mototsugu Muramaki, Junya Furukawa, Atsushi Takenaka, Masato Fujisawa. Expression of urokinase-type plasminogen activator system in prostate cancer: Correlation with clinicopathological outcomes in patients undergoing radical prostatectomy. Urologic Oncology: Seminars and Original Investigations, 2009, 27(2):180-186
33 Sabine Riethdorf, Eduardo F. Neffen, Aida Cviko, Thomas L?ning, Christopher P. Crum, Lutz Riethdorf.p16INK4A expression as biomarker for HPV 16-related vulvar neoplasias. Human Pathology, 2004,35(12):1477-1483
34 Motoi Sugimura, Hiroshi Kobayashi, Naohiro Kanayama, Toshihiko Terao. Clinical significance of urokinase-type plasminogen activator (uPA) in invasive cervical cancer of the uterus .Gynecologic Oncology, 1992,46(3): 330-336
35 Wilhelm O,Weidle.V,Hohl S,et al.Recombinant soluble urokinase receptor as a scavenger for urokinase-type plasminogen activator(uPA).Inhibition of proliferation and invasion of human ovarian cancer cells.FEBSLett,1994,Jan 10;337(2):131-134
36 Resnati M,Guttinger M,Valcamonica S,et al.Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactice- ffect.EMBOJ,1996,15(7):1572-1582
37 Minji J,Keena S,Thoma S,et al.Soluble urohinase-type polasminogen activator receptor inhibits cancer cell growth and invasion by direct urohinase-independent effects on cell singnaling.Biol Chem, 2003, 287:46692- 46698
38 Resnati M,Fazioli F, Sidenius N,et al.A urokinase-sensitive region of the human urokinase receptor is responsible for it schemotactic activity. EMBOJ,1997,16:7279-7286
39 Warren Schlecht,Genesio Murano and DouglasBoyd.Examination of the role of the urokanise receptor in human colon cancer.Cancer Res,1989,49:6064
40 Schmitt M,Harbeck N,Thomssen C,et al.Clinical impact of the plasminogen activation system in tumor invasion and metastasis: prognostic relevance and target for therapy.Thromb Haemost. 1997, 78(1):285-296
41 Kobayashi H,Suzuki M,Kanayama N,et al.Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal-regulated kinase-dependent cascade. European Journal of Biochemistry, 2002;269(16):3945-3957
42 Ossowski L,Aguirre-Ghiso JA.Urokinase receptor and integrin partnership:coordination of signaling for cell adhesion,migration and growth.Curr Opin Cell Biol,2000;12(5):613-620
43 Aguirre Ghiso JA,Alonso DF,Farias EF,et al.Deregulation of the signaling pathways controlling urokinase production.Its relationship with the invasive phenotype. Biochem.1999,263(2):295-304
1 Tsoumpou I, M. Arbyn, M. Kyrgiou,et al.p16INK4a immunostaining in cytological and histological specimens from the uterine cervix: A systematic review and meta-analysis.Cancer Treatment Reviews, 2009, 35,3(5): 210-220
2 Wentzensen N, Sherman M, Schiffman M,et al.Utility of methylation markers in cervical cancer early detection: Appraisal of the state-of-the-science.Gynecologic Oncology, 2009,112,2(5) :293-299
3 Piepoli, R. Cotugno, M. Quitadamo, et al.Methylation study of CDH1, P16,APC and MLH1 genes promoter along multistep gastric carcinogenesis. Digestive and Liver Disease, 2006,38,1(3):S110-S111
4 Neller M, J. Alejandro López, Christopher W,et al.Antigens for cancer immunotherapy.Seminars in Immunology, 2008,20,5(10):286-295
5 Oliveira-Cunha M, Siriwardena A, Byers R,et al.Molecular diagnosis in pancreatic cancer.Diagnostic Histopathology, 2008, 14,5(5):214-222
6 Tang-Yuan Chu, et al.Risk Factors and Genetic Markers of Human Papillomavirus-induced Cervical Carcinogenesis: A Focus on Chinese Populations in Southeast Asia and Southern China.Tzu Chi Medical Journal, 2008,20,2(6): 91-100
7 Sindos M, Ndisang D, Pisal N,et al.Measurement of Brn-3a levels in Pap smears provides a novel diagnostic marker for the detection of cervical neoplasia.Gynecologic Oncology, 2003, 90,2(8):366-371
8 Malur S,Krause N,Kohler C,et al.Sentinel lymph node detection in Patients with cervical cancer.Gynecol Oncol,2001,80(2):254-257
9 Nezos A, Pissimisis N , Lembessis P.Detection of circulating tumor cells in bladder cancer patients. Cancer Treatment Reviews, 2009, 35(3):272-279
10 Manavi M, Hudelist G, Fink-Retter A,et al.Gene profiling in Pap-cell smears of high-risk human papillomavirus-positive squamous cervical carcinoma.Gynecologic Oncology, 2007, 105,2(5): 418-426
11 Dano K,Romer J,Nielsen BS,et al.Cancer invasion and tissue remo deling-cooperation of protease systems and cell types. APMIS, 1999, 107(1):120-127
12 Resnati M,Pallavicini,Iserhan CN,et al.The fibrinolytic receptor for urokinase Activates the G protein-coupled chemotactic receptor.Proc Natl Acad Sci USA,2002,99(3):1359-1364
13 Richter C, Leo C, Schütz A,et al.Apaf-1 is regulated by hypoxia in cervical cancer.Pathology - Research and Practice, 2004,200,4: 329-330
14 Bergers G,Benjamin LE.Tumorigenesis and the angiogenic switch .Nat ure Rev Cancer,2003,3l:401-410
15 Keyt BA, Berleau LT, Nguyen HV, et al.The carboxyl-terminal domain (111–165) of vascular endothelial growth factor is critical for its mitogenic potency. J Biol Chem , 1996,271:7788-7795
16 Ribatti D, Leali D, Vacca A, et al: In vivo angiogenic activity of urokinase: Role of endogenous fibroblast growth factor-2. J Cell Sci,1999, 112:4213–4221
17 Lansink M, Koolwijk P, van Hinsbergh V, Kooistra T: Effect of steroid hormones and retinoids on the formation of capillary- like tubular structures of human microvascular endothelial cells in fibrin matrices is related to urokinase expression. Blood 92:927-938, 1998
18 Chao A, Wang T, Chyong-Huey Lai,et al. Overview of Microarray Analysis of Gene Expression and its Applications to Cervical Cancer Investigation.Taiwanese Journal of Obstetrics and Gynecology, 2007, 46,4(12):363-373
19 Saldivar J.S, Lopez D, Rebecca,et al.COX-2 overexpression as a biomarker of early cervical carcinogenesis: A pilot study.Gynecologic Oncology, Supplement 1, October 2007, 107,1:S155-S162