卵巢粘液性癌表面粘液的分解及其对泰素作用的影响
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摘要
晚期卵巢粘液性癌使用经典TP(泰素类+铂类)方案化疗的有效率明显低于其它类型卵巢上皮性癌,5年存活率仅为30～40%。分泌粘液是这一病理类型卵巢癌的特点,这些粘液是否会影响化疗药物的作用,粘液分解酶的使用能否提高化疗疗效,对这些问题,目前尚无相关研究。本研究通过查阅文献分析筛选,以Endo-α-N-acetylgalactosaminidase(Endo-α-N-乙酰半乳糖转移酶)作为粘液分解酶,结合体内外实验评价粘液分解前后泰素化疗作用的差异,以期探索卵巢粘液性癌的新的联合治疗方法。课题分为以下三个部分进行研究:
第一部分人卵巢粘液性癌粘液的分布及其所含粘蛋白类型的鉴定
目的:研究卵巢粘液性癌细胞株OMC685所分泌粘液在细胞和组织的分布,并鉴定该粘液中所含粘蛋白(mucin)的类型。
方法:1.培养卵巢粘液性癌细胞株OMC685,待细胞密度长至70%从形态学(光镜、扫描电镜、透射电镜)观察卵巢粘液性癌细胞及其表面粘液分布;OMC685细胞贴壁后培养48 h、72 h,密度分别长至30%、70%,行阿尔辛蓝—过碘酸雪夫(AB-PAS)染色,光镜下观察细胞粘液分布;2.用OMC685制成裸小鼠皮下瘤,行HE与AB-PAS粘液特殊染色,光镜下观察卵巢粘液性癌组织表面粘液分布;3.将OMC685制成细胞爬片,使用分泌性粘蛋白mucin 2、mucin 5AC、mucin 5B及mucin 6单克隆抗体行免疫细胞化学检测,鉴定该分泌性粘蛋白的类型;4.培养至对数生长期的OMC685重悬于培养液,以30%的密度接种,贴壁后换无血清无酚红培养液,培养24、48、72h分别提取培养液,ELISA法检测其中mucin2含量。
结果:1.光学显微镜下见卵巢粘液性癌细胞株OMC685细胞大小不一,以多边形为主,少数瘤巨细胞及多核细胞点缀其中,分裂相多见。透射电子显微镜下见瘤细胞为卵圆形或多边形,细胞间细胞连接多为紧密连接;细胞表面见微绒毛;核大,核质比高,染色质分布不均;较多细胞内可见的核糖体板层复合体,具有腺上皮恶性肿瘤细胞的特征;扫描电镜见细胞表面多微绒毛,表面分布网状物质;OMC685表面分布蓝染(酸性)或紫红染(中性)粘液,随培养时间延长,细胞周围粘液增多,同法处理卵巢浆液性癌细胞株SKOV3,表面无特异染色粘液分布;2.OMC685裸鼠皮下瘤HE染色见细胞形态学特点同培养细胞,AB-PAS染色见瘤结节周围环绕蓝染合并紫红染粘液层,细胞间隙可见小片状紫红染中性粘液池,提示体内卵巢粘液性癌组织表面覆盖有粘液层:3.OMC685免疫细胞化学染色显示粘蛋白mucin 2阳性,mugin 5AC,mucin 5B及mucin 6阴性,与之对照卵巢浆液性癌细胞株CaoV3上述四种粘蛋白均为阴性,提示卵巢粘液性癌较为特异地分泌mumin 2;4.与空白无血清无酚红培养液对比,24、48、72h培养液均为阳性,其中48h时(细胞长至对数生长期)mucin 2含量达峰值。
结论:证实卵巢粘液性癌细胞OMC685表面分布有粘液层,裸鼠皮下移植瘤组织表面亦覆盖有粘液层,通过免疫细胞化学、ELISA法确认该粘液成分主要为mucin2,并且在细胞培养48 h时(细胞长至对数生长期)分泌达峰值。
第二部分筛选卵巢粘液性癌粘液分解酶并观察其分解肿瘤细胞周围粘液的作用
目的:筛选出较为特异的卵巢粘液性癌粘液分解酶,并确认该酶的分解效果。
方法:1.粘液的主要成分为粘蛋白,是一种大分子量糖蛋白,以较高特异性、适宜细胞生长环境使用的原则,通过查阅文献筛选出适宜的粘液分解酶;2.培养细胞长至对数生长期,吸净培养液,取不稀释、1:10、1:100、1:1000、1:10000稀释五个浓度的粘液分解酶Endo-α-N-acetylgalactosaminidase覆盖细胞,5%CO_2 37℃细胞培养箱中孵育3 h后倒置显微镜下观察细胞生长状态;在前述实验结果的基础上再取1:200、1:400、1:800三个稀释倍数的酶反应液加入细胞培养板,方法同上,37℃细胞培养箱中孵育3 h后倒置显微镜下观察细胞生长状态;3.培养至对数生长期的OMC685重悬于培养液,以1×10~5个/ml的密度接种,贴壁后换无血清无酚红培养液,培养24、48、72 h分别提取培养液,其中取部分48 h培养液以200:1的比例加入Endo-α-N-acetylgalactosaminidase,37℃培养箱中孵3 h,ELISA检测细胞培养液中mucin 2的含量;4.OMC685细胞爬片长至30%,70%,用1:200稀释的Endo-α-N-acetylgalactosaminidase反应液在37℃培养箱中孵育3 h,AB-PAS染色光镜下观察细胞表面粘液的分解,扫描电镜观察细胞表面结构的变化。
结果:1.根据文献,Endo-α-N-acetylgalactosaminidase能够较特异地作用于粘蛋白的糖苷键,分解粘液;2.Endo-α-N-acetylgalactosaminidase能够作用于细胞生长环境,以1:200稀释度用于体外实验可达到作用于细胞的有效工作浓度:3.ELISA法检测结果提示,使用Endo-α-N-acety]galactosaminidase后48 h培养液中mucin 2含量降低,与使用该酶前mucin 2的含量相比差异有显著性,证明Endo-α-N-acetylgalactosaminidase对粘液的分解是有效的;4.OMC685细胞爬片AB-PAS染色提示细胞长至70%表面粘液多于细胞密度30%,Endo-α-N-acetylgalactosaminidase作用后细胞表面粘液层基本消失,细胞突起回缩。扫描电镜观察到细胞表面网状结构减少,细胞表面微绒毛粗而短,细胞贴壁状态未改变。
结论:Endo-α-N-acetylgalactosaminidase可以较有效地分解细胞表面粘液层,孵育后培养液中mucin 2含量降低,可以用于进一步实验,借以观察对泰素化疗的影响。
第三部分体内外实验验证粘液分解对泰素作用于卵巢粘液性癌的影响
目的:研究粘液分解对泰素治疗卵巢粘液性癌效果的影响。
方法:1.在体外实验中,以MTT法检测不同浓度泰素对OMC685的杀伤作用,选取泰素作用于OMC685的有效工作浓度;2.取不同浓度泰素分别作用于SKOV3与CaoV3,选择本部分体外实验的对照细胞系;3.消化对数生长期OMC685制成细胞悬液,以0.5×10~5/ml接种于96孔板,分对照组、加酶组、加泰素组、酶加泰素组共四组,每组取3复孔,待细胞贴壁后继续培养24 h,加酶组与酶加泰素组吸净细胞培养液,每孔加入1:200稀释的酶反应液,37℃培养箱孵育3 h后吸除酶反应液,加泰素组与酶加泰素组再加入0.1μmol/L含泰素培养液,24、48、72 h后MTT法检测活细胞量,Brdu法检测OMC685的增殖程度;4.消化对数生长期SKOV3制成细胞悬液,以1×10~6/ml接种于96孔板,分为对照组、加粘液组、加泰素组、粘液加泰素组共四组,细胞贴壁后同时提取培养OMC685达48h的含粘液培养液覆盖于加粘液组和粘液加泰素组SKOV3表面,加泰素组和粘液加泰素组再予含泰素培养液覆盖于其上,各组细胞培养液中泰素终浓度均为0.1μmol/L,24、48、72h后MTT法检测活细胞量,Brdu法检测细胞增殖;5.在体内实验中,首先需初步探索体内使用Endo-α-N-acetylgalactosaminidase辅助泰素治疗的工作浓度,裸小鼠皮下接种OMC685,四周后皮下瘤长至直径约1cm取出,修剪成直径3mm的近圆形小瘤块缝于裸小鼠大网膜表面,分为泰素组、1:200稀释酶加泰素组、1:40稀释酶加泰素组,生理盐水组作为对照组,每组3只。接种3天后隔天给药,生理盐水组腹腔注射0.2ml生理盐水,泰素组每次腹腔注射0.2 ml 1mg/ml泰素,酶加泰素组先予相应稀释浓度酶每只0.2ml腹腔注射,3 h后再予1mg/ml泰素0.2ml腹腔注射,共给药4次。停药3天后检测肿瘤大小及各脏器的肿瘤转移情况;6.根据前述实验结果选择1:200稀释酶反应液作为体内实验分解酶的工作浓度,同前法制作裸小鼠网膜移植瘤模型,分为三组:生理盐水组、泰素组、1:200稀释酶加泰素组,每组10只,给药方法同前,停药一周后牺牲裸小鼠,检测肿瘤大小、重量,观察腹腔及各脏器肿瘤转移情况。
结果:1.在体外作用于0MC685 0.1μmol/L泰素能达到有效治疗效果;2.不同泰素浓度组与单细胞对照组相比对CaoV3的抑制率差异无显著性,说明该细胞株可能为泰素耐药株;泰素作用于SKOV3在浓度达0.1、1μmol/L时活细胞量与对照组相比差异有显著性,该两组问差异无显著性,故选择SKOV3作为体外实验对照细胞系,0.1μmol/L作为其有效工作浓度;3.Endo-α-N-acetylgalactosaminidase孵育3h后使用泰素,24、48、72h行MTT法及Brdu法检测,酶加泰素组与单用泰素组相比对OMC685的抑制率均明显增加,并随时间增加差异显著性增加;4.SKOV3覆盖粘液培养液后使用泰素治疗,MTT及Brdu检测均提示对SKOV3的抑制率弱于单用泰素,48、72 h组差异有显著性;5.筛选酶浓度的体内实验中,网膜移植的裸小鼠93.3%成瘤,生理盐水对照组网膜移植瘤体积46.09±5.9 mm~3,泰素组网膜移植瘤体积13.57±1.26 mm~3,1:200稀释度酶加泰素组网膜移植瘤体积9.21±1.2 mm~3,1:40稀释度酶加泰素组网膜移植瘤体积7.7±0.66 mm~3,从结果中可以看出1:200稀释酶加泰素组与1:40稀释酶加泰素组相差不大,故选择1:200稀释度作为体内实验的工作浓度;6.选择1:200稀释度作为分解酶有效工作浓度,给药方法同前,给药两周后牺牲裸小鼠,单用生理盐水组网膜移植瘤体积111.62±31.05mm~3,单用泰素组网膜瘤体积61.04±12.59mm~3,1:200酶加泰素组网膜瘤体积34.76±5.46mm~3;生理盐水组与泰素组差异有显著性(P＜0.01),泰素组与酶加泰素组差异有显著性(P＜0.01):泰素组抑瘤率达37.38%,1:200稀释度酶加泰素组抑瘤率达53.46%;说明1:200稀释度酶辅助泰素用药对OMC685的抑瘤率高于单用泰素。各组均未见其它器官有瘤结节形成。
结论:体外体内实验均提示分解酶辅助泰素化疗可以增强泰素作用的效果。
总结:
人卵巢粘液性癌表面分布有粘液层,Endo-α-N-acetylgalactosaminidase可对其有效分解,这一分解能够辅助提高泰素治疗的抑瘤率。
The classical chemotherapy which uses TP scenario (Taxol and platinum) is obviously less effective in the treatment of advanced stage mucinous ovarian carcinoma than in that of other epithelial ovarian cancer. For patients with advanced mucinous ovarian cancer, the 5 year survival rate is just 30～40%. The typical character of mucinous ovarian cancer is its ability to secret mucus. Then whether mucous layer substantially decreases the effect of anti-cancer agents on mucinous ovarian cancer remains unknown, and whether the chemotherapy effect will be elevated by degrading the mucous layer need to be investigated. There has been few report elucidating this problem so far. In this study, the proper methods to degrade the mucous layer are been compared and the anti-cancer effect of Taxol before and after degradation of mucous layer is estimated. The whole study is divided into 3 parts as follows:
PartⅠIdentification of mucin type in the human mucinous ovarian cancer and the characteristic distribution of the mucous layer on the cellular and tissue level
Objective: To study the distribution of mucous layer secreted by mucinous ovarian cancer cell line 0MC685 on the cellular and tissue level, and identify the type of mucin.
Methods: 1. The mucous ovarian cancer cell line OMC685 were cultured to 70% convergence, and then the distribution of its surface mucus were observed morphologically with optic microscope, scanning electronic microscope, and transmission electronic microscope. The 0MC685 cell grew to 30% and 70% convergence at 24、48 hour after cells adhered to the dish' s bottom in normal culture condition. Stained with AB-PAS method, the distribution of mucous layer can be observed under optic microscope. The serous ovarian cancer cell line SKOV3 were compared; 2. Use 0MC685 cell line to obtain the subcutaneous cancer model, and the tumor specimen were stained by HE and AB-PAS methods. The distribution of surface mucus in the artificial mucinous ovarian cancer were observed under optic microscope; 3. The OMC685 cells were cultured and let to climb on a glass slice. The slice were stained by immunohistochemistry method to detect the detailed type of the mucin, where the Mucin 2、Mucin 5AC、Mucin 5B, and Mucin 6 monoclonal antibodies were utilized. The results were compared with that of serous ovarian cancer cell line CaoV3; 4. OMC685 cell were re-suspended in culture medium when grew to the logarithmic stage. Cells were added in the dish at the level of 30% convergence. At 24、48、and 72 hours after replenished with serum free medium, the concentration of mucin 2 within the medium were measured with ELISA method.
Result: 1. The OMC685 cells were different in size under optic microscope and the main morphological appearance was multi-angular type. There were some giant cancer cells and multi-nuclear cells within the cancer tissue, and the cell division image could be found often. Under transmission electronic microscope, the cancer cells were olival and multi-angular, of which the tight junction is the major type of inter-cellular junction. Micro-villi were abundant on the surface of cancer cell. Their nuclear was large, and the nuclear-plasma ratio was high. The chromatin distributed unevenly and in most cells the ribosome layer compound, which represented the typical character of the glandular epithelial cancer malignant cells, could be found. Under scanning electronic microscope, the surface of the cancer cell is plenty of micro-villi, and some reticular substances were found on it. Blue (acidic) and purple-staining (neutral) mucus also existed. With the extension of culture time, the amount of mucus increased. There was no specific mucus to be found on the surface of SKOV3; 2. Cells with the same morphological appearance as in vitro status could be observed in the subcutaneous cancer nude mouse model, the blue and purple mucous layer surrounding the cancer nodules could be stained with AB-PAS method. Between the cells, the purple mucous pool has been observed, which prompt the mucous layer also covered the mucinous ovarian cancer tissue; 3. The immuno-histochemical stain showed: mucin 2 were positive; mucin 5AC、mucin 5B and mucin 6 were negative in the staining, compared with the staining of CaoV3; 4. Compared with the serum-free and phenol-red free blank control, medium obtained at 24, 48, 72 hours after in vitro culture were all positive, and the concentration of mucin 2 reached its summit value at the 48 hours point.
Conclusion: The mucous layer outside of mucinous ovarian cell OMC685 was certified, and the surface of cancer tissue transplanted in the subcutaneous space of nude mouse also covered by mucous layer. The main component of mucus was mucin 2, which has been demonstrated by immunohisto-chemistry and ELISA. The summit value of mucus secretion was achieved at 48 hours after cancer cells being cultured in vitro (when cell grew to the logarithmic growth stage).
PartⅡScreening of mucus degradation enzyme and its detailed function for the mucous layer of mucinous ovarian cancer
Objective: To screen out an enzyme which could specifically degrade the mucous layer of mucinous ovarian cancer, and then demonstrate the selected enzyme' s effect on the mucus.
Method: 1. The main component within the mucus is called mucin, a glycoprotein with a large molecular mass. After a thorough review of literature, several relatively proper candidates of enzyme that can specifically degrade mucus have been selected; 2. When OMC685 cells were cultured to logarithmic growth stage, take out the mediun, and no diluted, 1: 10、1: 100、1: 1000、1: 10000 diluted Endo-α-N-acetylgalactosaminidase was covered on OMC685. After cultured for 3 hours in 5%C0_2 37℃, observed the configuration of omc685; further more, 1:200、1: 400、1: 800 diluted Endo-α-N-acetylgalactosaminidase were used; 3. OMC685 cells were cultured to logarithmic growth stage, and re-suspended in the medium at the level of 1×10~5/ml. After cells adhered to the bottom of dishes, serum and phenol-red free medium was used, and the original culture medium were all changed. The culture medium was then taken out at the time point of 24、48 and 72 hours. The 48 hours culture medium was further co-incubated with Endo-α-N-acetylgalactosaminidase by the ratio of 1:200 at 37℃for 3 hours. The concentration of mucin 2 within such an enzyme-treated medium was measured by ELISA method; 4. After OMC685 cells climbed to the glass slice, and met the 30% and 70% convergence, the Endo-α-N-acetylgalactosaminidase were added into the culture medium by the ratio of 1:200. The AB-PAS stained slices were observed under optic microscope, to see whether the mucous layer had been degraded. The structure changes on the cellular surface were known by transmission electronic microscope.
Results: 1. Endo-α-N-acetylgalactosaminidase could specifically break down the mucus layer; 2. 1: 200 diluted Endo-α-N-acetylgalactosaminidase can be used in the test; 3. According to the results of ELISA test, the concentration of mucin 2 within the 48 hours culture medium was lowered significantly after the Endo-α-N-acetylgalactosaminidase had been added. This result demonstrated that Endo-α-N-acetylgalactosaminidase would be effective in the degradation of mucous layer; 4. The AB-PAS staining showed that the mucous layer was thicker on the slice where cancer cells climbed and grew to the 70% convergence, comparing to that on the slice where the cancer cells just reached 30% convergence. After treated with Endo-α-N-acetylgalactosaminidase, the mucous layer on the surface of 0MC685 cells decreased, and the cellular protuberances also drew back. Under scanning electronic mircrospcope, the reticular structures significant decreased, the cellular protuberances drew back. However, the wall-adherence status of cancer cells kept unchanged.
Conclusion: After cultured by Endo-α-N-acetylgalactosaminidase, the mucus layer on mucinous ovarian carcinoma cell was decomposed, and the mucin 2 level in the medium was depressed. So Endo-α-N-acetylgalactosaminidase can be used in further test.
PartⅢThe effect of mucus degradation on the anti-cancer function of a chemical agent: Taxol when it is used to treat the mucinous ovarian cancer Objective: To study the effect of mucous degradation on the anti-cancer function of Taxol in the treatment of mucinous ovarian cancer.
Methods: 1. In vitro experiments the cyto-toxicity on 0MC685 of Taxol was measured using MTT method and choosed the adaptive concentration of taxol; 2. Taxol was used to treat SKOV3 and CaoV3, and selected the control cell line; 3. The anti-cancer effects of Taxol were compared after 24, 48 and 72 hours' co-incubation together with Endo-α-N-acetylgalactosaminidase at 1:200 concentrations. Under the same experimental processes, the proliferation ability of OMC685 was measured with BrdU method; 4. The 48 hours culture medium of 0MC685 were added into the medium of SKOV3, and the proliferation ability of these cells were tested with MTT and BrdU method respectively, after 24、48、and 72 hours' treatment of Taxol; 5. In vivo experiments: when the subcutaneous cancer grew to 1 cm in diameter, which was about 4 weeks after 0MC685 cells had been inoculated into the nude mouse, the cancer tissue was taken out. It was cut into 3mm little cancer blocks, which were subsequently inoculated onto the surface of epiploon. Model mice were divided into four groups: Taxol group, Taxol plus 1:200 enzyme group, Taxol plus 1:40 enzyme group, and control group. The control group was used as the blank control. From the third day after inoculation, above agents (Taxol or enzyme) were given on every two days for 4 times. On the third day after the last time of anti-cancer treatment, mice were killed to examine the bulk of cancer tissues and their metastasis situations; 6. Selected 1:200 ratio as work concentration of Endo-α-N-acetylgalactosaminidase. Model mice were divided into three groups: Taxol group, Taxol plus 1:200 enzyme group, and control group, and were treated as above. On the eighth day after the last time of anti-cancer treatment, mice were killed to examine the bulk of cancer tissues and their metastasis situations.
Results: 1. Taxol can reach its effective therapeutic purpose at a concentration of 0.1 umol/L in vitro; 2. There were no significant distinctness among different concentration taxol treatment groups of CAOV3, and 0. 1、1μmol/L taxol can achieve effective therapeutic purpose. So SK0V3 was selected as control cell line, and concentration 0. 1μmol/L was used; 3. Comparing to the Taxol alone group, the proliferation-inhabiting rate of Taxol increased significantly after cancer cells had been co-incubated with Endo-α-N-acetylgalactosaminidase for 3 hours in Taxol plus enzyme group. The results kept unchanged whenever the proliferation ability were measured with MTT or BrdU method at several time points of 24、48、and 72 hours. Moreover, the statistical significance would increase with the extension of the observation interval; 4. On the contrary, after the 48 hours culture medium of OMC685 cells had been added into culture dishes of SKOV3 cells, the MTT and BrdU test could both demonstrate that the Taxol' s cyto-toxicity effect was weakened on SKOV3 cancer cells. Such a difference became more significant at the time point of 48 and 72 hours; 5. In 93.3% nude mice, the transplanted cancer cells could grow up to the visible cancer masses. In the control group, the volume of transplanted cancer mass was 46. 09±5.9 mm~3; in the Taxol alone group, the volume of transplanted cancer mass was 13. 57±1. 26 mm~3; in the Taxol plus 1:200 enzyme group, the volume of transplanted cancer mass was 9. 21±1. 2 mm~3; and in the Taxol plus 1:40 enzyme group, the volume of transplanted cancer mass was 7.7±0.66 mm~3. The differences between control group and Taxol group, as well as between Taxol group and Taxol plus enzyme group were significant. The difference between Taxol plus 1:200 enzyme group and Taxol plus 1:40 enzyme group was not significant. Therefore, 1:200 concentration was selected to use in vivo test; 6. Use 1:200 Endo-α-N-acetylgalactosaminidase as above, and kill the mice after had treated for 2 weeks. In the control group, the volume of transplanted cancer mass was 111.62±31.05 mm; in the Taxol group, the volume of transplanted cancer mass was 61.04±12.59 mm; and in the Taxol plus enzyme group, the volume of transplanted cancer mass was 34.76±5.46 mm. The differences between control group and Taxol group, as well as between Taxol group and Taxol plus enzyme group were significant(P<0.01). the cancer cell proliferation-inhibiting rate of Taxol group was 37.38%, compared with 53.46% of Taxol plus 1:200 enzyme group. The result showed that the anti-cancer effect is more obvious in Taxol plus enzyme group. No metastatic nodule of any other organ except the peritoneum cavity was found in all three experimental groups.
Conclusion: In vitro test and in vivo test both approve that Endo-α-N-acetylgalactosaminidase can effectively elevate the cancer cell proliferation-inhibiting rate of Taxol.
In summary, the mucous layer exists on the surface of human mucinous ovarian cancer, and Endo-α-N- acetylgalactosaminidase can effectively discompose it, which can be helpful to elevate the cancer cell proliferation- inhibiting rate of therapeutic remedies containing Taxol.
第一部分人卵巢粘液性癌粘液的分布及其所含粘蛋白类型的鉴定
目的:研究卵巢粘液性癌细胞株OMC685所分泌粘液在细胞和组织的分布,并鉴定该粘液中所含粘蛋白(mucin)的类型。
方法:1.培养卵巢粘液性癌细胞株OMC685,待细胞密度长至70%从形态学(光镜、扫描电镜、透射电镜)观察卵巢粘液性癌细胞及其表面粘液分布;OMC685细胞贴壁后培养48 h、72 h,密度分别长至30%、70%,行阿尔辛蓝—过碘酸雪夫(AB-PAS)染色,光镜下观察细胞粘液分布;2.用OMC685制成裸小鼠皮下瘤,行HE与AB-PAS粘液特殊染色,光镜下观察卵巢粘液性癌组织表面粘液分布;3.将OMC685制成细胞爬片,使用分泌性粘蛋白mucin 2、mucin 5AC、mucin 5B及mucin 6单克隆抗体行免疫细胞化学检测,鉴定该分泌性粘蛋白的类型;4.培养至对数生长期的OMC685重悬于培养液,以30%的密度接种,贴壁后换无血清无酚红培养液,培养24、48、72h分别提取培养液,ELISA法检测其中mucin2含量。
结果:1.光学显微镜下见卵巢粘液性癌细胞株OMC685细胞大小不一,以多边形为主,少数瘤巨细胞及多核细胞点缀其中,分裂相多见。透射电子显微镜下见瘤细胞为卵圆形或多边形,细胞间细胞连接多为紧密连接;细胞表面见微绒毛;核大,核质比高,染色质分布不均;较多细胞内可见的核糖体板层复合体,具有腺上皮恶性肿瘤细胞的特征;扫描电镜见细胞表面多微绒毛,表面分布网状物质;OMC685表面分布蓝染(酸性)或紫红染(中性)粘液,随培养时间延长,细胞周围粘液增多,同法处理卵巢浆液性癌细胞株SKOV3,表面无特异染色粘液分布;2.OMC685裸鼠皮下瘤HE染色见细胞形态学特点同培养细胞,AB-PAS染色见瘤结节周围环绕蓝染合并紫红染粘液层,细胞间隙可见小片状紫红染中性粘液池,提示体内卵巢粘液性癌组织表面覆盖有粘液层:3.OMC685免疫细胞化学染色显示粘蛋白mucin 2阳性,mugin 5AC,mucin 5B及mucin 6阴性,与之对照卵巢浆液性癌细胞株CaoV3上述四种粘蛋白均为阴性,提示卵巢粘液性癌较为特异地分泌mumin 2;4.与空白无血清无酚红培养液对比,24、48、72h培养液均为阳性,其中48h时(细胞长至对数生长期)mucin 2含量达峰值。
结论:证实卵巢粘液性癌细胞OMC685表面分布有粘液层,裸鼠皮下移植瘤组织表面亦覆盖有粘液层,通过免疫细胞化学、ELISA法确认该粘液成分主要为mucin2,并且在细胞培养48 h时(细胞长至对数生长期)分泌达峰值。
第二部分筛选卵巢粘液性癌粘液分解酶并观察其分解肿瘤细胞周围粘液的作用
目的:筛选出较为特异的卵巢粘液性癌粘液分解酶,并确认该酶的分解效果。
方法:1.粘液的主要成分为粘蛋白,是一种大分子量糖蛋白,以较高特异性、适宜细胞生长环境使用的原则,通过查阅文献筛选出适宜的粘液分解酶;2.培养细胞长至对数生长期,吸净培养液,取不稀释、1:10、1:100、1:1000、1:10000稀释五个浓度的粘液分解酶Endo-α-N-acetylgalactosaminidase覆盖细胞,5%CO_2 37℃细胞培养箱中孵育3 h后倒置显微镜下观察细胞生长状态;在前述实验结果的基础上再取1:200、1:400、1:800三个稀释倍数的酶反应液加入细胞培养板,方法同上,37℃细胞培养箱中孵育3 h后倒置显微镜下观察细胞生长状态;3.培养至对数生长期的OMC685重悬于培养液,以1×10~5个/ml的密度接种,贴壁后换无血清无酚红培养液,培养24、48、72 h分别提取培养液,其中取部分48 h培养液以200:1的比例加入Endo-α-N-acetylgalactosaminidase,37℃培养箱中孵3 h,ELISA检测细胞培养液中mucin 2的含量;4.OMC685细胞爬片长至30%,70%,用1:200稀释的Endo-α-N-acetylgalactosaminidase反应液在37℃培养箱中孵育3 h,AB-PAS染色光镜下观察细胞表面粘液的分解,扫描电镜观察细胞表面结构的变化。
结果:1.根据文献,Endo-α-N-acetylgalactosaminidase能够较特异地作用于粘蛋白的糖苷键,分解粘液;2.Endo-α-N-acetylgalactosaminidase能够作用于细胞生长环境,以1:200稀释度用于体外实验可达到作用于细胞的有效工作浓度:3.ELISA法检测结果提示,使用Endo-α-N-acety]galactosaminidase后48 h培养液中mucin 2含量降低,与使用该酶前mucin 2的含量相比差异有显著性,证明Endo-α-N-acetylgalactosaminidase对粘液的分解是有效的;4.OMC685细胞爬片AB-PAS染色提示细胞长至70%表面粘液多于细胞密度30%,Endo-α-N-acetylgalactosaminidase作用后细胞表面粘液层基本消失,细胞突起回缩。扫描电镜观察到细胞表面网状结构减少,细胞表面微绒毛粗而短,细胞贴壁状态未改变。
结论:Endo-α-N-acetylgalactosaminidase可以较有效地分解细胞表面粘液层,孵育后培养液中mucin 2含量降低,可以用于进一步实验,借以观察对泰素化疗的影响。
第三部分体内外实验验证粘液分解对泰素作用于卵巢粘液性癌的影响
目的:研究粘液分解对泰素治疗卵巢粘液性癌效果的影响。
方法:1.在体外实验中,以MTT法检测不同浓度泰素对OMC685的杀伤作用,选取泰素作用于OMC685的有效工作浓度;2.取不同浓度泰素分别作用于SKOV3与CaoV3,选择本部分体外实验的对照细胞系;3.消化对数生长期OMC685制成细胞悬液,以0.5×10~5/ml接种于96孔板,分对照组、加酶组、加泰素组、酶加泰素组共四组,每组取3复孔,待细胞贴壁后继续培养24 h,加酶组与酶加泰素组吸净细胞培养液,每孔加入1:200稀释的酶反应液,37℃培养箱孵育3 h后吸除酶反应液,加泰素组与酶加泰素组再加入0.1μmol/L含泰素培养液,24、48、72 h后MTT法检测活细胞量,Brdu法检测OMC685的增殖程度;4.消化对数生长期SKOV3制成细胞悬液,以1×10~6/ml接种于96孔板,分为对照组、加粘液组、加泰素组、粘液加泰素组共四组,细胞贴壁后同时提取培养OMC685达48h的含粘液培养液覆盖于加粘液组和粘液加泰素组SKOV3表面,加泰素组和粘液加泰素组再予含泰素培养液覆盖于其上,各组细胞培养液中泰素终浓度均为0.1μmol/L,24、48、72h后MTT法检测活细胞量,Brdu法检测细胞增殖;5.在体内实验中,首先需初步探索体内使用Endo-α-N-acetylgalactosaminidase辅助泰素治疗的工作浓度,裸小鼠皮下接种OMC685,四周后皮下瘤长至直径约1cm取出,修剪成直径3mm的近圆形小瘤块缝于裸小鼠大网膜表面,分为泰素组、1:200稀释酶加泰素组、1:40稀释酶加泰素组,生理盐水组作为对照组,每组3只。接种3天后隔天给药,生理盐水组腹腔注射0.2ml生理盐水,泰素组每次腹腔注射0.2 ml 1mg/ml泰素,酶加泰素组先予相应稀释浓度酶每只0.2ml腹腔注射,3 h后再予1mg/ml泰素0.2ml腹腔注射,共给药4次。停药3天后检测肿瘤大小及各脏器的肿瘤转移情况;6.根据前述实验结果选择1:200稀释酶反应液作为体内实验分解酶的工作浓度,同前法制作裸小鼠网膜移植瘤模型,分为三组:生理盐水组、泰素组、1:200稀释酶加泰素组,每组10只,给药方法同前,停药一周后牺牲裸小鼠,检测肿瘤大小、重量,观察腹腔及各脏器肿瘤转移情况。
结果:1.在体外作用于0MC685 0.1μmol/L泰素能达到有效治疗效果;2.不同泰素浓度组与单细胞对照组相比对CaoV3的抑制率差异无显著性,说明该细胞株可能为泰素耐药株;泰素作用于SKOV3在浓度达0.1、1μmol/L时活细胞量与对照组相比差异有显著性,该两组问差异无显著性,故选择SKOV3作为体外实验对照细胞系,0.1μmol/L作为其有效工作浓度;3.Endo-α-N-acetylgalactosaminidase孵育3h后使用泰素,24、48、72h行MTT法及Brdu法检测,酶加泰素组与单用泰素组相比对OMC685的抑制率均明显增加,并随时间增加差异显著性增加;4.SKOV3覆盖粘液培养液后使用泰素治疗,MTT及Brdu检测均提示对SKOV3的抑制率弱于单用泰素,48、72 h组差异有显著性;5.筛选酶浓度的体内实验中,网膜移植的裸小鼠93.3%成瘤,生理盐水对照组网膜移植瘤体积46.09±5.9 mm~3,泰素组网膜移植瘤体积13.57±1.26 mm~3,1:200稀释度酶加泰素组网膜移植瘤体积9.21±1.2 mm~3,1:40稀释度酶加泰素组网膜移植瘤体积7.7±0.66 mm~3,从结果中可以看出1:200稀释酶加泰素组与1:40稀释酶加泰素组相差不大,故选择1:200稀释度作为体内实验的工作浓度;6.选择1:200稀释度作为分解酶有效工作浓度,给药方法同前,给药两周后牺牲裸小鼠,单用生理盐水组网膜移植瘤体积111.62±31.05mm~3,单用泰素组网膜瘤体积61.04±12.59mm~3,1:200酶加泰素组网膜瘤体积34.76±5.46mm~3;生理盐水组与泰素组差异有显著性(P＜0.01),泰素组与酶加泰素组差异有显著性(P＜0.01):泰素组抑瘤率达37.38%,1:200稀释度酶加泰素组抑瘤率达53.46%;说明1:200稀释度酶辅助泰素用药对OMC685的抑瘤率高于单用泰素。各组均未见其它器官有瘤结节形成。
结论:体外体内实验均提示分解酶辅助泰素化疗可以增强泰素作用的效果。
总结:
人卵巢粘液性癌表面分布有粘液层,Endo-α-N-acetylgalactosaminidase可对其有效分解,这一分解能够辅助提高泰素治疗的抑瘤率。
The classical chemotherapy which uses TP scenario (Taxol and platinum) is obviously less effective in the treatment of advanced stage mucinous ovarian carcinoma than in that of other epithelial ovarian cancer. For patients with advanced mucinous ovarian cancer, the 5 year survival rate is just 30～40%. The typical character of mucinous ovarian cancer is its ability to secret mucus. Then whether mucous layer substantially decreases the effect of anti-cancer agents on mucinous ovarian cancer remains unknown, and whether the chemotherapy effect will be elevated by degrading the mucous layer need to be investigated. There has been few report elucidating this problem so far. In this study, the proper methods to degrade the mucous layer are been compared and the anti-cancer effect of Taxol before and after degradation of mucous layer is estimated. The whole study is divided into 3 parts as follows:
PartⅠIdentification of mucin type in the human mucinous ovarian cancer and the characteristic distribution of the mucous layer on the cellular and tissue level
Objective: To study the distribution of mucous layer secreted by mucinous ovarian cancer cell line 0MC685 on the cellular and tissue level, and identify the type of mucin.
Methods: 1. The mucous ovarian cancer cell line OMC685 were cultured to 70% convergence, and then the distribution of its surface mucus were observed morphologically with optic microscope, scanning electronic microscope, and transmission electronic microscope. The 0MC685 cell grew to 30% and 70% convergence at 24、48 hour after cells adhered to the dish' s bottom in normal culture condition. Stained with AB-PAS method, the distribution of mucous layer can be observed under optic microscope. The serous ovarian cancer cell line SKOV3 were compared; 2. Use 0MC685 cell line to obtain the subcutaneous cancer model, and the tumor specimen were stained by HE and AB-PAS methods. The distribution of surface mucus in the artificial mucinous ovarian cancer were observed under optic microscope; 3. The OMC685 cells were cultured and let to climb on a glass slice. The slice were stained by immunohistochemistry method to detect the detailed type of the mucin, where the Mucin 2、Mucin 5AC、Mucin 5B, and Mucin 6 monoclonal antibodies were utilized. The results were compared with that of serous ovarian cancer cell line CaoV3; 4. OMC685 cell were re-suspended in culture medium when grew to the logarithmic stage. Cells were added in the dish at the level of 30% convergence. At 24、48、and 72 hours after replenished with serum free medium, the concentration of mucin 2 within the medium were measured with ELISA method.
Result: 1. The OMC685 cells were different in size under optic microscope and the main morphological appearance was multi-angular type. There were some giant cancer cells and multi-nuclear cells within the cancer tissue, and the cell division image could be found often. Under transmission electronic microscope, the cancer cells were olival and multi-angular, of which the tight junction is the major type of inter-cellular junction. Micro-villi were abundant on the surface of cancer cell. Their nuclear was large, and the nuclear-plasma ratio was high. The chromatin distributed unevenly and in most cells the ribosome layer compound, which represented the typical character of the glandular epithelial cancer malignant cells, could be found. Under scanning electronic microscope, the surface of the cancer cell is plenty of micro-villi, and some reticular substances were found on it. Blue (acidic) and purple-staining (neutral) mucus also existed. With the extension of culture time, the amount of mucus increased. There was no specific mucus to be found on the surface of SKOV3; 2. Cells with the same morphological appearance as in vitro status could be observed in the subcutaneous cancer nude mouse model, the blue and purple mucous layer surrounding the cancer nodules could be stained with AB-PAS method. Between the cells, the purple mucous pool has been observed, which prompt the mucous layer also covered the mucinous ovarian cancer tissue; 3. The immuno-histochemical stain showed: mucin 2 were positive; mucin 5AC、mucin 5B and mucin 6 were negative in the staining, compared with the staining of CaoV3; 4. Compared with the serum-free and phenol-red free blank control, medium obtained at 24, 48, 72 hours after in vitro culture were all positive, and the concentration of mucin 2 reached its summit value at the 48 hours point.
Conclusion: The mucous layer outside of mucinous ovarian cell OMC685 was certified, and the surface of cancer tissue transplanted in the subcutaneous space of nude mouse also covered by mucous layer. The main component of mucus was mucin 2, which has been demonstrated by immunohisto-chemistry and ELISA. The summit value of mucus secretion was achieved at 48 hours after cancer cells being cultured in vitro (when cell grew to the logarithmic growth stage).
PartⅡScreening of mucus degradation enzyme and its detailed function for the mucous layer of mucinous ovarian cancer
Objective: To screen out an enzyme which could specifically degrade the mucous layer of mucinous ovarian cancer, and then demonstrate the selected enzyme' s effect on the mucus.
Method: 1. The main component within the mucus is called mucin, a glycoprotein with a large molecular mass. After a thorough review of literature, several relatively proper candidates of enzyme that can specifically degrade mucus have been selected; 2. When OMC685 cells were cultured to logarithmic growth stage, take out the mediun, and no diluted, 1: 10、1: 100、1: 1000、1: 10000 diluted Endo-α-N-acetylgalactosaminidase was covered on OMC685. After cultured for 3 hours in 5%C0_2 37℃, observed the configuration of omc685; further more, 1:200、1: 400、1: 800 diluted Endo-α-N-acetylgalactosaminidase were used; 3. OMC685 cells were cultured to logarithmic growth stage, and re-suspended in the medium at the level of 1×10~5/ml. After cells adhered to the bottom of dishes, serum and phenol-red free medium was used, and the original culture medium were all changed. The culture medium was then taken out at the time point of 24、48 and 72 hours. The 48 hours culture medium was further co-incubated with Endo-α-N-acetylgalactosaminidase by the ratio of 1:200 at 37℃for 3 hours. The concentration of mucin 2 within such an enzyme-treated medium was measured by ELISA method; 4. After OMC685 cells climbed to the glass slice, and met the 30% and 70% convergence, the Endo-α-N-acetylgalactosaminidase were added into the culture medium by the ratio of 1:200. The AB-PAS stained slices were observed under optic microscope, to see whether the mucous layer had been degraded. The structure changes on the cellular surface were known by transmission electronic microscope.
Results: 1. Endo-α-N-acetylgalactosaminidase could specifically break down the mucus layer; 2. 1: 200 diluted Endo-α-N-acetylgalactosaminidase can be used in the test; 3. According to the results of ELISA test, the concentration of mucin 2 within the 48 hours culture medium was lowered significantly after the Endo-α-N-acetylgalactosaminidase had been added. This result demonstrated that Endo-α-N-acetylgalactosaminidase would be effective in the degradation of mucous layer; 4. The AB-PAS staining showed that the mucous layer was thicker on the slice where cancer cells climbed and grew to the 70% convergence, comparing to that on the slice where the cancer cells just reached 30% convergence. After treated with Endo-α-N-acetylgalactosaminidase, the mucous layer on the surface of 0MC685 cells decreased, and the cellular protuberances also drew back. Under scanning electronic mircrospcope, the reticular structures significant decreased, the cellular protuberances drew back. However, the wall-adherence status of cancer cells kept unchanged.
Conclusion: After cultured by Endo-α-N-acetylgalactosaminidase, the mucus layer on mucinous ovarian carcinoma cell was decomposed, and the mucin 2 level in the medium was depressed. So Endo-α-N-acetylgalactosaminidase can be used in further test.
PartⅢThe effect of mucus degradation on the anti-cancer function of a chemical agent: Taxol when it is used to treat the mucinous ovarian cancer Objective: To study the effect of mucous degradation on the anti-cancer function of Taxol in the treatment of mucinous ovarian cancer.
Methods: 1. In vitro experiments the cyto-toxicity on 0MC685 of Taxol was measured using MTT method and choosed the adaptive concentration of taxol; 2. Taxol was used to treat SKOV3 and CaoV3, and selected the control cell line; 3. The anti-cancer effects of Taxol were compared after 24, 48 and 72 hours' co-incubation together with Endo-α-N-acetylgalactosaminidase at 1:200 concentrations. Under the same experimental processes, the proliferation ability of OMC685 was measured with BrdU method; 4. The 48 hours culture medium of 0MC685 were added into the medium of SKOV3, and the proliferation ability of these cells were tested with MTT and BrdU method respectively, after 24、48、and 72 hours' treatment of Taxol; 5. In vivo experiments: when the subcutaneous cancer grew to 1 cm in diameter, which was about 4 weeks after 0MC685 cells had been inoculated into the nude mouse, the cancer tissue was taken out. It was cut into 3mm little cancer blocks, which were subsequently inoculated onto the surface of epiploon. Model mice were divided into four groups: Taxol group, Taxol plus 1:200 enzyme group, Taxol plus 1:40 enzyme group, and control group. The control group was used as the blank control. From the third day after inoculation, above agents (Taxol or enzyme) were given on every two days for 4 times. On the third day after the last time of anti-cancer treatment, mice were killed to examine the bulk of cancer tissues and their metastasis situations; 6. Selected 1:200 ratio as work concentration of Endo-α-N-acetylgalactosaminidase. Model mice were divided into three groups: Taxol group, Taxol plus 1:200 enzyme group, and control group, and were treated as above. On the eighth day after the last time of anti-cancer treatment, mice were killed to examine the bulk of cancer tissues and their metastasis situations.
Results: 1. Taxol can reach its effective therapeutic purpose at a concentration of 0.1 umol/L in vitro; 2. There were no significant distinctness among different concentration taxol treatment groups of CAOV3, and 0. 1、1μmol/L taxol can achieve effective therapeutic purpose. So SK0V3 was selected as control cell line, and concentration 0. 1μmol/L was used; 3. Comparing to the Taxol alone group, the proliferation-inhabiting rate of Taxol increased significantly after cancer cells had been co-incubated with Endo-α-N-acetylgalactosaminidase for 3 hours in Taxol plus enzyme group. The results kept unchanged whenever the proliferation ability were measured with MTT or BrdU method at several time points of 24、48、and 72 hours. Moreover, the statistical significance would increase with the extension of the observation interval; 4. On the contrary, after the 48 hours culture medium of OMC685 cells had been added into culture dishes of SKOV3 cells, the MTT and BrdU test could both demonstrate that the Taxol' s cyto-toxicity effect was weakened on SKOV3 cancer cells. Such a difference became more significant at the time point of 48 and 72 hours; 5. In 93.3% nude mice, the transplanted cancer cells could grow up to the visible cancer masses. In the control group, the volume of transplanted cancer mass was 46. 09±5.9 mm~3; in the Taxol alone group, the volume of transplanted cancer mass was 13. 57±1. 26 mm~3; in the Taxol plus 1:200 enzyme group, the volume of transplanted cancer mass was 9. 21±1. 2 mm~3; and in the Taxol plus 1:40 enzyme group, the volume of transplanted cancer mass was 7.7±0.66 mm~3. The differences between control group and Taxol group, as well as between Taxol group and Taxol plus enzyme group were significant. The difference between Taxol plus 1:200 enzyme group and Taxol plus 1:40 enzyme group was not significant. Therefore, 1:200 concentration was selected to use in vivo test; 6. Use 1:200 Endo-α-N-acetylgalactosaminidase as above, and kill the mice after had treated for 2 weeks. In the control group, the volume of transplanted cancer mass was 111.62±31.05 mm; in the Taxol group, the volume of transplanted cancer mass was 61.04±12.59 mm; and in the Taxol plus enzyme group, the volume of transplanted cancer mass was 34.76±5.46 mm. The differences between control group and Taxol group, as well as between Taxol group and Taxol plus enzyme group were significant(P<0.01). the cancer cell proliferation-inhibiting rate of Taxol group was 37.38%, compared with 53.46% of Taxol plus 1:200 enzyme group. The result showed that the anti-cancer effect is more obvious in Taxol plus enzyme group. No metastatic nodule of any other organ except the peritoneum cavity was found in all three experimental groups.
Conclusion: In vitro test and in vivo test both approve that Endo-α-N-acetylgalactosaminidase can effectively elevate the cancer cell proliferation-inhibiting rate of Taxol.
In summary, the mucous layer exists on the surface of human mucinous ovarian cancer, and Endo-α-N- acetylgalactosaminidase can effectively discompose it, which can be helpful to elevate the cancer cell proliferation- inhibiting rate of therapeutic remedies containing Taxol.
引文
1 Hornung R, Eppenberger U, Eppenberger S, et al. Analysis of potential prognostic factors in 111 patients with ovarian cancer[J]. Cancer Lett,2004, 206(1):97-106.
2 VivianeH, Roger AH, Nazar N, et al. Mucinous Epithelial Ovarian Cancer: A Separate Entity Requiring Specific Treatment[J]. J Clin Oncol, 2004,22(6): 1040-1044.
3 Bansil R, Stanley E, Lamont J. Mucin biophysics[J]. Annu Rev Physiol,1995, 57: 635-657.
4 Irimura T , Nakamori S , Mataushita Y, et al. Colorectal cancer metastasis determined by carbohydrate2mediated cell adhesion : Role of sialyl2Lex antigens[J]. Semin. Cancer Biol , 1993 , 4 : 319-324.
5 Tarek S, Robert JL, Anthony PC. Mucins and inflammatory bowel disease[J].Postgrad Med J, 2000, 76: 473-478.
6 Martensson S, Levery SB, Bendiak B. Neutral core oligosaccharides of bovine Submaxillary mucin: Use of lead tetraacetate in the cold for establishing Branch positions[J]. Eur J Biochem, 1998, 258: 603-622..
[1] McGuire V, Jesser CA, Whittemore AS. Survival among U.S.women with invasive epithelial ovarian cancer[J]. Gynecol Oncol 2002; 84:399 - 403.
[2] McGuire WP, Hoskins WJ, Brady MF, et al. Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer[J]. N Engl J Med 1996; 334: 1 -6.
[3] Piccart MJ, Bertelsen K, James K, et al. Randomized intergroup trial of cisplatin - paclitaxel versus cisplatin - cyclophosphamide in women with advanced epithelial ovarian cancer: three-year results[J]. J Natl Cancer Inst 2000; 92: 699-708.
[4] Muggia FM, Braly PS, Brady MF, et al. Phase III randomized study of cisplatin versus paclitaxel versus cisplatin and paclitaxel in patients with suboptimal stage III or IV ovarian cancer: a gynecologic oncology group study[J]. J Clin Oncol 2000; 18: 106-115.
[5] Neijt JP, Engelholm SA, Tuxen MK, et al. Exploratory phase III study of paclitaxel and cisplatin versus paclitaxel and carboplatin in advanced ovarian cancer[J]. J Clin Oncol 2000; 18: 3084-3092.
[6] International Collaborative Ovarian Neoplasm Group. Paclitaxel plus440 D. Pectasides et al[J]. Gynecol Oncol 97 2005; 97: 436-441.
[7] Hess V, Hern R, Nasiri N, et al. Mucinous epithelial ovarian cancer:a separate entity requiring specific treatment[J]. J Clin Oncol 2004;22: 1040-1044.
[8] Enomoto T, Kuragaki C, Yamasaki M, et al. Is clear cell carcinoma and mucinous carcinoma of the ovary sensitive to combination chemotherapy with paclitaxel and carboplatin[J]? Proc-Am Soc Clin Oncol 2003; 22:44 [abstr 1797].
[9] Shimizu Y, Nagata H, Kikuchi Y, et al. Cytotoxic agents active against mucinous adenocarcinoma of the ovary[J]. Oncol Rep 1998; 5: 99- 101.
[10] Shimizu Y, Umezawa S, Hasumi K. A phase II study of combined CPT-11 and mitomycin-C in platinum refractory clear cell and mucinous ovarian carcinoma[J]. Ann Acad Med Singapore 1998; 27: 650-656.
[11] Dimitrios P, George F, Gerasimos A, et al. Advanced stage mucinous epithelial ovarian cancer:The Hellenic Cooperative Oncology Group experience[J].Gynecol Oncol 2005;97:436-441.
[12]Andrews PA,Velury S,Mann SC,et al.Cis-Diamminedichloroplatinum (Ⅱ) accumulation in sensitive and resistant human ovarian carcinoma cells[J].Cancer Res 1988;48:68-73.
[13]Kasahara K,Fujiwara Y,Nishio K,et al.Metallothionein content correlates with the sensitivity of human small cell lung cancer cell lines to cisplatin[J].Cancer Res 1991;51:3237-3242.
[14]Kigawa J,Minagawa Y,Cheng X,et al.Gamma-glutamyl cysteine synthetase up-regulates glutathione and multidrug resistanceassociated protein in patients with chemoresistant epithelial ovarian cancer[J].Clin Cancer Res 1998;4:1737-1741.
[15]Pieretti M,Hopenhayn-Rich C,Khattar NH,et al.Heterogeneity of ovarian cancer:relationships among histological group,stage of disease,tumor markers,patient characteristics,and survival[J].Cancer Invest 2002;20:11-23.
[16]Hornung R,Eppenberger U,Eppenberger S,et al.Analysis of potential prognostic factors in 111 patients with ovarian cancer[J].Cancer Lett 2004;206(1):97-106.
[17]Viviane H,Roger AH,Nazar N,et al.Mucinous Epithelial Ovarian Cancer:A Separate Entity Requiring Specific Treatment[J].J Clin Oncol,2004,22(6):1040-1044.
[18]蒋家康,李绍刚。卵巢粘液性肿瘤组织发生的初步探讨[J]。临床与实验病理学杂志,2000年8月;16(4):280-282.
[19]Boman F,Buisine MP,W acrenier A,et al.Mucin gene transcripts in benign and borderline mucinous tumours of the ovary:an in situ hybridization study[J].J Pathol,2004,193(3):339-344.
[20]Ulbright TM,Roth LM,Stehman FB.Secondary ovarian neoplasia:A clinicopathologic study of 35 cases[J].Cancer,1984,53(5):1164-1174.
[21]Albarraein CT,Jafri J,Montag AG,et al.Differential expression of MUC2 and M UC5AC mucin genes in primary ovarian and metastatic colonic carcinoma[J].Hum Pathol 2004;31(6):672-677.
[22]Bansil R,Stanley E,Lamont J.Mucin biophysics.Annu Rev Physiol, 1995,57:635-657.
[23]Irimura T,Nakamori S,Mataushita Y,et al.Colorectal cancer metastasis determined by carbohydrate2mediated cell adhesion:Role of sialyl2Lex antigens[J].Semin.Cancer Biol 1993;4:319- 324.
[24]王自能,刘咏仪,楼湘莹,杨艳东.人卵巢粘液性肿瘤来源的再确认[J].电子显微学报 2005,24(6):581-583.
[25]Moniaux N,Escande F,Porchet N,et al.Structural organization and classification of the human mucin genes[J].Front Biosci 2001 Oct 1;6:D1192-1206.
[26]Tarek Shirazi,Robert JL,Anthony PC,et al.Mucins and inflammatory bowel disease[J].Postgrad Med J 2000;76:473-478.
[27]Aubert JP,Porchet N,Crepin M,et al.Evidence for different human tracheobronchial mucin peptides deduced from nucleotide cDNA sequences[J].Am J Respir Cell Mol Biol,1991;5(2):178.
[28]汪荣泉,房殿春,晋华源,等。胃癌及癌前病变组织中MUC1基因的表达及其意义[J]。中华医学杂志1999;79(6):443-444.
[29]Utsunomiya T,Yonezawa S,Sakamoto H,et al.Expression of MUC1 and MUC2 mucinsin gastric carcinomas:it relationship with the prognosis of the prognosis of the patients[J].Clin Cancer Res 1998;4(11):2605-2614.
[30]Baldus SE,Zirbes TK,Engel S,et al.Correlation of the immunohistochemical reactivity of mucin peptide cores MUC1 and MUC2 with the histopathological subtype and prognosis of gastric carcinomas[J].Int J Cancer 1998 Apr 17;79(2):133-138.
[31]Agrawal B,Krants MJ,Reddish M,et al.Cancer-associated MUC1 mucin inhibits human T-cell proliferation,which is reversible by IL-2[J].Nat Med 1998 4:43-49.
[32]Taylor KL,Mall AS,Barrard RA,et al.Immunohistochemical detection of gastric mucin in normal and disease states[J].Oncol Res 1998;10(9):465-473.
[33]乌兰娜,高积勇,李芬。粘蛋白MUC 2、MUC 5AC在卵巢上皮粘液性肿瘤中的表达及临床意义[J]。中国全科医学2003年8月8(16):1318-1320.
[34]Cornberg M,Enss ML,Wagner S.Differential mucin expression in the gastrointestinal tract[J].Cancer Lett 2001 Jul 10:71-80.
[35] Corfield AP, Carroll D, Myerscough N, et al. Mucin in the gastrointestinal tract in health and disease[J]. Front Biosci 2001 Oct 1;6: 1321-1357.
[36] Martensson S, Levery SB, Bendiak B. Neutral core oligosaccharides of bovine Submaxillary mucin: Use of lead tetraacetate in the cold for establishing Branch positions[J]. Eur J Biochem, 1998, 258:603-622.
[1]Huang C and Aminoff D.Enzymes that destroy blood group specificity.J Biol Chem:1972 247:6737-6742.
[2]Endo Y,Kobata,A.Partial purification and characterization of an endoα-N-acetylgalactosaminidase from the culture medium of Diplococcus pneumoniae.J Biochem 1976 80:1-8.
[3]Bhavanandan V,Umemoto J,and Davidson E.Characterization of an endoα-N-acetylgalactosaminidase from Diplococcus pneumoniae.Biochem.Biophys.Res.Commun.1976 70:738-745
[1]岳静,李静,刑辉,等。三种人卵巢癌动物模型的生物学特性比较[J]。现代妇产科进展,2002,11(5):334。
[2]吴秉铨,孙毓恺,郑洁,等。裸鼠体内建立的人类高转移癌系[J]。中华肿瘤杂志,1985,7(5):324。
[3]徐丛剑,金志军,丰有吉,等。人卵巢癌裸鼠腹腔及网膜移植的研究。现代妇产科进展,1997,6(2):111-113
1Menon U,Jacobs IJ.Ovarian cancer screening in the general population:current status.Int J Gynecol Cancer,2001,11([Suppl 1]:S3-6
2McGuire V,Jesser CA,Whittemore AS:Survival among U.S.women with invasive epithelial ovarian cancer.Gynecol Oncol,2002,84(3):399-403
3Jiang CX,Tan YB,Li EH,et al.Neuroendocrine differentiation in ovarian mutinous tumors.Chin Med J,2000,113(1):70-74
4Ingrid MR,Jaime P.Mucinous Tumors of the Ovary.Am J Surg Pathol,2002,26(2):139-152
5Maso LD,Canzonieri V,Talamini R,et al.Origin of ovarian cancer from benign cysts.Eur J Cancer Prey,2001,10(2):197-199
6Patricia MB,Esther O,Robert HY,et al.Ovarian Mucinous Carcinoids Including Some With a Carcinomatous Component.Am J Surg Pathol,2001,25(5):557-568
7Kenji O,Toshihiro T,Watsuhiko T,et al.Identification by cDNA Microarray of Genes Involved in Ovarian Carcinogenesis.Cancer Research,2000,15(1):5007-5011
8David MP,PenelopeMW,Victor S,et al.The Different Etiologies of Mucinous and Nonmucinous Epithelial Ovarian Cancers.gynecol Oncol,2003,88[Suppl 1]:S145-S148
9Hardiman P,Nieto JJ,MacLean AB.Infertility and ovarian.Gynecol Oncol,2000,76(1):1-2
10AllisonWK,Raymond RB,Valerie M,et al.Histologic types of epithelial ovarian cancer:have they different risk factors? Gynecol Oncol,2005,96(5):520-530
11Ohara N,Teramoto K.Successful treatment of pseudomyxoma peritonei with intraperitoneal 10 per cent dextrose,sizofiran and cisplatin.J Obstet Gynaecol,2002,22(2):223-230
12Sophie C,Philippe M,David A,et al.Lymph Node Disorders and Prognostic Value of Nodal Involvement in Patients Treated for a Borderline Ovarian Tumor: An Analysis of a Series of 42 Lymphadenectomies. Am College Surg, 2002,195(3):332-338
13 Hornung R, Eppenberger U, Eppenberger S, et al. Analysis of potential prognostic factors in 111 patients with ovarian cancer. Cancer Lett, 2004, 206(1):97-106
14 VivianeH, Roger AH, NazarN, et al. Mucinous Epithelial Ovarian Cancer: A Separate Entity Requiring Specific Treatment. J Clin Oncol, 2004, 22(6):1040-1044
15 Robert CY, Mark FB, Roberta KN, et al. Adjuvant Treatment for Early Ovarian Cancer:A Randomized Phase III Trial of Intraperitoneal 32P or Intravenous Cyclophosphamide and Cisplatin—A Gynecologic Oncology Group Study. J Cl in Oncol,2003, 21 (23):4350-4355
16 Favalli G, Odocino F, Torri V, et al. Early stage ovarian cancer: the Italian contribution to clinical research. An update. Int J Gynecol Cancer,2001,11 [Suppl 1] :S12- 19
17 Philippe M, Franklin J, Sophie C, et al. Lymph node involvement in epithelial ovarian cancer: analysis of 276 pelvic and paraaortic lymphadenectomies and surgical implications. J Am Coll Surg, 2003, 197(2):198-205
18 Morice P, Joulie F, Rey A, et al. Are nodal metastases in ovarian cancer chemoresistant lesions? Analysis of nodal involvement in 105 patients treated with preoperative chemotherapy. Eur J Gynaecol Oncol, 2004, 25(2):169-174
19 Masaki F, Noriyuki HT, NaokoU, et al. HER2 Is Frequently Over-expressed in Ovarian Clear Cell Adenocarcinoma: Possible Novel Treatment Modality Using Recombinant Monoclonal Antibody against HER2, Trastuzumab. Jpn J Cancer Res, 2002,93(6):1250-1257
20 Simon C, Pam M, Henry GB, et al. Estrogen Receptor Isoform Gene Expression in Ovarian Stromal and Epithelial Tumors. J Clin Endocrinol Metab, 2000, 85(3):1200-1205
2 VivianeH, Roger AH, Nazar N, et al. Mucinous Epithelial Ovarian Cancer: A Separate Entity Requiring Specific Treatment[J]. J Clin Oncol, 2004,22(6): 1040-1044.
3 Bansil R, Stanley E, Lamont J. Mucin biophysics[J]. Annu Rev Physiol,1995, 57: 635-657.
4 Irimura T , Nakamori S , Mataushita Y, et al. Colorectal cancer metastasis determined by carbohydrate2mediated cell adhesion : Role of sialyl2Lex antigens[J]. Semin. Cancer Biol , 1993 , 4 : 319-324.
5 Tarek S, Robert JL, Anthony PC. Mucins and inflammatory bowel disease[J].Postgrad Med J, 2000, 76: 473-478.
6 Martensson S, Levery SB, Bendiak B. Neutral core oligosaccharides of bovine Submaxillary mucin: Use of lead tetraacetate in the cold for establishing Branch positions[J]. Eur J Biochem, 1998, 258: 603-622..
[1] McGuire V, Jesser CA, Whittemore AS. Survival among U.S.women with invasive epithelial ovarian cancer[J]. Gynecol Oncol 2002; 84:399 - 403.
[2] McGuire WP, Hoskins WJ, Brady MF, et al. Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer[J]. N Engl J Med 1996; 334: 1 -6.
[3] Piccart MJ, Bertelsen K, James K, et al. Randomized intergroup trial of cisplatin - paclitaxel versus cisplatin - cyclophosphamide in women with advanced epithelial ovarian cancer: three-year results[J]. J Natl Cancer Inst 2000; 92: 699-708.
[4] Muggia FM, Braly PS, Brady MF, et al. Phase III randomized study of cisplatin versus paclitaxel versus cisplatin and paclitaxel in patients with suboptimal stage III or IV ovarian cancer: a gynecologic oncology group study[J]. J Clin Oncol 2000; 18: 106-115.
[5] Neijt JP, Engelholm SA, Tuxen MK, et al. Exploratory phase III study of paclitaxel and cisplatin versus paclitaxel and carboplatin in advanced ovarian cancer[J]. J Clin Oncol 2000; 18: 3084-3092.
[6] International Collaborative Ovarian Neoplasm Group. Paclitaxel plus440 D. Pectasides et al[J]. Gynecol Oncol 97 2005; 97: 436-441.
[7] Hess V, Hern R, Nasiri N, et al. Mucinous epithelial ovarian cancer:a separate entity requiring specific treatment[J]. J Clin Oncol 2004;22: 1040-1044.
[8] Enomoto T, Kuragaki C, Yamasaki M, et al. Is clear cell carcinoma and mucinous carcinoma of the ovary sensitive to combination chemotherapy with paclitaxel and carboplatin[J]? Proc-Am Soc Clin Oncol 2003; 22:44 [abstr 1797].
[9] Shimizu Y, Nagata H, Kikuchi Y, et al. Cytotoxic agents active against mucinous adenocarcinoma of the ovary[J]. Oncol Rep 1998; 5: 99- 101.
[10] Shimizu Y, Umezawa S, Hasumi K. A phase II study of combined CPT-11 and mitomycin-C in platinum refractory clear cell and mucinous ovarian carcinoma[J]. Ann Acad Med Singapore 1998; 27: 650-656.
[11] Dimitrios P, George F, Gerasimos A, et al. Advanced stage mucinous epithelial ovarian cancer:The Hellenic Cooperative Oncology Group experience[J].Gynecol Oncol 2005;97:436-441.
[12]Andrews PA,Velury S,Mann SC,et al.Cis-Diamminedichloroplatinum (Ⅱ) accumulation in sensitive and resistant human ovarian carcinoma cells[J].Cancer Res 1988;48:68-73.
[13]Kasahara K,Fujiwara Y,Nishio K,et al.Metallothionein content correlates with the sensitivity of human small cell lung cancer cell lines to cisplatin[J].Cancer Res 1991;51:3237-3242.
[14]Kigawa J,Minagawa Y,Cheng X,et al.Gamma-glutamyl cysteine synthetase up-regulates glutathione and multidrug resistanceassociated protein in patients with chemoresistant epithelial ovarian cancer[J].Clin Cancer Res 1998;4:1737-1741.
[15]Pieretti M,Hopenhayn-Rich C,Khattar NH,et al.Heterogeneity of ovarian cancer:relationships among histological group,stage of disease,tumor markers,patient characteristics,and survival[J].Cancer Invest 2002;20:11-23.
[16]Hornung R,Eppenberger U,Eppenberger S,et al.Analysis of potential prognostic factors in 111 patients with ovarian cancer[J].Cancer Lett 2004;206(1):97-106.
[17]Viviane H,Roger AH,Nazar N,et al.Mucinous Epithelial Ovarian Cancer:A Separate Entity Requiring Specific Treatment[J].J Clin Oncol,2004,22(6):1040-1044.
[18]蒋家康,李绍刚。卵巢粘液性肿瘤组织发生的初步探讨[J]。临床与实验病理学杂志,2000年8月;16(4):280-282.
[19]Boman F,Buisine MP,W acrenier A,et al.Mucin gene transcripts in benign and borderline mucinous tumours of the ovary:an in situ hybridization study[J].J Pathol,2004,193(3):339-344.
[20]Ulbright TM,Roth LM,Stehman FB.Secondary ovarian neoplasia:A clinicopathologic study of 35 cases[J].Cancer,1984,53(5):1164-1174.
[21]Albarraein CT,Jafri J,Montag AG,et al.Differential expression of MUC2 and M UC5AC mucin genes in primary ovarian and metastatic colonic carcinoma[J].Hum Pathol 2004;31(6):672-677.
[22]Bansil R,Stanley E,Lamont J.Mucin biophysics.Annu Rev Physiol, 1995,57:635-657.
[23]Irimura T,Nakamori S,Mataushita Y,et al.Colorectal cancer metastasis determined by carbohydrate2mediated cell adhesion:Role of sialyl2Lex antigens[J].Semin.Cancer Biol 1993;4:319- 324.
[24]王自能,刘咏仪,楼湘莹,杨艳东.人卵巢粘液性肿瘤来源的再确认[J].电子显微学报 2005,24(6):581-583.
[25]Moniaux N,Escande F,Porchet N,et al.Structural organization and classification of the human mucin genes[J].Front Biosci 2001 Oct 1;6:D1192-1206.
[26]Tarek Shirazi,Robert JL,Anthony PC,et al.Mucins and inflammatory bowel disease[J].Postgrad Med J 2000;76:473-478.
[27]Aubert JP,Porchet N,Crepin M,et al.Evidence for different human tracheobronchial mucin peptides deduced from nucleotide cDNA sequences[J].Am J Respir Cell Mol Biol,1991;5(2):178.
[28]汪荣泉,房殿春,晋华源,等。胃癌及癌前病变组织中MUC1基因的表达及其意义[J]。中华医学杂志1999;79(6):443-444.
[29]Utsunomiya T,Yonezawa S,Sakamoto H,et al.Expression of MUC1 and MUC2 mucinsin gastric carcinomas:it relationship with the prognosis of the prognosis of the patients[J].Clin Cancer Res 1998;4(11):2605-2614.
[30]Baldus SE,Zirbes TK,Engel S,et al.Correlation of the immunohistochemical reactivity of mucin peptide cores MUC1 and MUC2 with the histopathological subtype and prognosis of gastric carcinomas[J].Int J Cancer 1998 Apr 17;79(2):133-138.
[31]Agrawal B,Krants MJ,Reddish M,et al.Cancer-associated MUC1 mucin inhibits human T-cell proliferation,which is reversible by IL-2[J].Nat Med 1998 4:43-49.
[32]Taylor KL,Mall AS,Barrard RA,et al.Immunohistochemical detection of gastric mucin in normal and disease states[J].Oncol Res 1998;10(9):465-473.
[33]乌兰娜,高积勇,李芬。粘蛋白MUC 2、MUC 5AC在卵巢上皮粘液性肿瘤中的表达及临床意义[J]。中国全科医学2003年8月8(16):1318-1320.
[34]Cornberg M,Enss ML,Wagner S.Differential mucin expression in the gastrointestinal tract[J].Cancer Lett 2001 Jul 10:71-80.
[35] Corfield AP, Carroll D, Myerscough N, et al. Mucin in the gastrointestinal tract in health and disease[J]. Front Biosci 2001 Oct 1;6: 1321-1357.
[36] Martensson S, Levery SB, Bendiak B. Neutral core oligosaccharides of bovine Submaxillary mucin: Use of lead tetraacetate in the cold for establishing Branch positions[J]. Eur J Biochem, 1998, 258:603-622.
[1]Huang C and Aminoff D.Enzymes that destroy blood group specificity.J Biol Chem:1972 247:6737-6742.
[2]Endo Y,Kobata,A.Partial purification and characterization of an endoα-N-acetylgalactosaminidase from the culture medium of Diplococcus pneumoniae.J Biochem 1976 80:1-8.
[3]Bhavanandan V,Umemoto J,and Davidson E.Characterization of an endoα-N-acetylgalactosaminidase from Diplococcus pneumoniae.Biochem.Biophys.Res.Commun.1976 70:738-745
[1]岳静,李静,刑辉,等。三种人卵巢癌动物模型的生物学特性比较[J]。现代妇产科进展,2002,11(5):334。
[2]吴秉铨,孙毓恺,郑洁,等。裸鼠体内建立的人类高转移癌系[J]。中华肿瘤杂志,1985,7(5):324。
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