RNA干扰EGFR基因对骨肉瘤细胞影响的实验研究
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摘要
骨肉瘤在原发恶性骨肿瘤中最常见，主要见于15～25岁少年，男多于女约一倍。该瘤恶性程度甚高，预后极差，可于数月内出现肺部转移，截肢后3～5年存活率仅为5～20％。发生在股骨下端及胫骨上端的约占所有骨肉瘤的四分之三，其它处如肱骨、股骨上端、腓骨、脊椎、髂骨等亦可发生。多数为溶骨性，也有少数为成骨性。肿瘤多处于骨端，偶发生于骨干或骨骺。尽管目前肿瘤治疗手段的日益改善，但对于大多数晚期骨肉瘤患者真正有效的治疗措施十分有限，化疗仅对部分患者有效。因此，临床迫切需要高效低毒的新治疗手段。
目前，骨肉瘤的发病机制仍不清楚。但同其它肿瘤一样，癌基因，抑癌基因，凋亡相关基因，生长因子及受体的异常表达及细胞内信号传导的紊乱被认为与骨肉瘤的发生和转移密切相关。表皮生长因子受体(EGFR)，是目前最受注目的肿瘤治疗药物的靶位。大量研究表明，EGFR酪氨酸激酶区域是调节肿瘤细胞增殖、侵袭、血管生成、转移和凋亡信号通路的重要部分。表皮生长因子EGF与EGFR的结合导致EGFR的二聚体化和胞内区磷酸化，进而引发一系列的级联放大反应，从而调控和传递骨肉瘤的生物学行为的信号。研究发现EGFR在人骨肉瘤组织中过表达，且其表达水平和恶性预后相关，EGFR抑制剂有望被开发成为骨肉瘤的崭新治疗途径。
RNA干扰(RNAi)是由双链RNA(dsRNA)引发的转录后基因沉默机制(PTGS)，dsRNA经RNA沉默复合体(RISC)降解成21-23nt的小片段(siRNA)，并以其为模板，特定位点、特定间隔降解与之序列互补的mRNA，目前已成功用于基因功能和信号传递系统上下游分子相互关系的研究。本研究拟通过采用RNAi技术，探讨利用体外化学合成siRNA和构建质粒载体的方法是否能有效沉默骨肉瘤细胞EGFR基因表达，阻断EGFR所介导的恶性生物学行为，从而为骨肉瘤的基因治疗提供新策略。
目的
探讨采用RNAi技术是否能有效抑制骨肉瘤MG63细胞株EGFR表达，以及EGFR基因下调后骨肉瘤MG63细胞生物学特性的改变，以期为肿瘤治疗提供新的思路和实验依据。
材料与方法
首先通过免疫组化、RT-PCR和western blot测定MG63细胞株EGFR的表达情况。体外合成短发夹状shRNA—EGFR单链，并通过退火、回收、质粒酶切、连接、转化、挑克隆、提质粒、测序等步骤，构建和鉴定重组质粒载体psilencer-2.1-shRNA-EGFR，转染MG63细胞株。建立实时荧光定量RT-PCR定量检测EGFRmRNA的表达量的方法。用实时荧光定量RT-PCR检测shRNA对靶mRNA沉默效率；用western—blot法检测蛋白表达变化情况；用四甲基偶氮唑盐(MTT)比色分析检测细胞体外增殖率情况；通过DAPI染色观察干扰后的细胞调亡情况；透射电镜和原位末端标记TUNEL法观察细胞凋亡形态学改变；Annexin V/P I双标记法检测细胞的早期和晚期凋亡率改变。
结果
我们建立了EGFR基因干扰载体并建立荧光定量PCR检测技术平台。通过免疫组化、RT-PCR和western blot证实MG63细胞株存在EGFR高表达。Psilencer2.1-shRNA-EGFR-Lipofectamine 2000复合物转染细胞可引发EGFR序列特异性基因沉默效应，与RT-PCR、Western Blot分析得到了一致的结果。干扰组骨肉瘤细胞与对照组比较，shRNA-EGFR组EGFR基因水平在12h、24h、48h、60h和72h分别下调16.2％、27.3％、47.7％、52.3％及54.5％(P＜0.01)；shRNA-EGFR组EGFR蛋白表达水平也明显下调。MTT法检测EGFR对MG63细胞增殖抑制率在12h、24h、48h、60h和72h分别为12.6％、30.0％(P＜0.01)、39.1％(P＜0.01)、47.1％(P＜0.01)和62.1％(P＜0.01)。shRNA-EGFR诱发的调亡细胞细胞核DAPI染色明显增强，胞核缩小，核膜皱缩，染色质凝聚、边缘化，晚期染色质破碎。TUNEL检测RNA干扰48h后的细胞，可见明显的棕色细胞，表明凋亡细胞的存在。经EGFR干扰48h的人MG63细胞，电镜观察发现干扰后细胞细胞核体积缩小，胞浆内出现大小不等的空泡，细胞浆浓缩，整个细胞固缩，并可见脱落到细胞外的凋亡小体。Annexin V／P I检测EGFR干扰MG63细胞，干扰前凋亡检测值为1.9％，坏死为0.7％；干扰后24h凋亡检测值为12.6％，坏死为5.39％；48h凋亡凋亡检测值为31.6％，坏死为5.1％，72h凋亡凋亡检测值为36.2％，坏死为24.3％。
结论
1．MG63细胞株存在EGFR高表达。2．shRNA-EGFR可序列特异性下调MG63细胞EGFR基因水平，显著降低EGFR蛋白表达。3．shRNA-EGFR重组质粒通过抑制EGFR表达可抑制细胞增生和诱发调亡，从而为MG63基因治疗提供了新策略。
Osteosarcoma is the most predominant type of malignant bone tumor in childrenand adolescents between 15-25 years old. The prevalance of osteosarcoma in male istwo times than female. The prognosis of osteosarcoma is worse. 15-20％of patientshave clinically detectable metastases espesilly in lung at initial diagnosis. The 3-5year survival rate is only 5-20％even after amputation. Lesions deteted in the distalfemur and proximal tibia are account for 75％of total cases in osteosarcoma. Thepathology changes in osteosarcoma are mainly osteolytic. Despite treatment advances,chemotherapy is only marginally effective in most advanced cases. For those patientsrefractory to or intolerant of the current chemotherapy, treatment options are limited.Hence, more effective therapy with fewer side effects is needed.
At present, the machanisiam of osteosarcoma is still unclear. The abnormalexpression of oncogene, suppressor gene, and apoptosis related gene, growth factorreceptor and disorder of intracellar signal pathway were regarded as to have key rolewith metastasis occur in osteosarcoma. Epidermal growth factor receptor (EGFR) is aglycoprotein with a molecular weight of 170,000 to 180,000. It is an intrinsictyrosine-specific protein kinase, which is stimulated upon epidermal growth factor(EGF) binding. EGFR signaling involved in cell growth, angiogenesis, DNA repair,and autocrine growth regulation in MG63 as well as in a wide spectrum of human cancer cells. Thus, it has recently emerged as an innovative target for thedevelopment of new cancer therapy.
RNA interference (RNAi) is an evolutionarily conserved process in whichrecognition of double-stranded RNA (dsRNA) ultimately leads to posttranscriptionalsuppression of gene expression. This suppression is mediated by short hair RNA(shRNA), which induces specific degradation of mRNA through complementary basepairing. In several model systems, ie: mostly in lower order animals, this naturalresponse has been developed into a powerful tool for the investigation of genefunction. Recently it was discovered that introducing synthetic 21-23 nucleotidedsRNA duplexes into mammalian cells could efficiently silence gene expression. Inthis study, we investigated the possibility whether RNAi could silence EGFR gene incommonly used MG63 cancer cell lines. We also assessed the degree of EGFR genesilencing and its functional outcome in terms of effects on cell proliferation andgrowth inhibition in vitro.
Objective
To investigate whether RNA interference (RNAi) could induce gene silencing inosteosarcoma (MG63) cells; to evaluate the degree of epidermal growth factor (EGFR)receptor gene silencing and its effect on functional outcome of MG63 cells.
Meterials and methods
To determine whether EGFR is overexpressed and the effect of RNAi on EGFRgene in MG63 cells, EGFR gene RNAi lentivirus vector was constructed, MG63 celllines were transfected with target sequence-specific shRNA as well as variouscontrols. IHC and Western Blot were used to measure the expression of EGFR proteinin MG63 cells and reduction the EGFR protein in EGFR gene knocked down MG63cells. Quantitative reverse-transcriptase PCR was used to detect the expression andsilencing of the EGFR gene level. MTT assay, DAPI, TUNEL, transmission electronmicroscope (TEM) and Annexin V/P I double stain analysis were used to assess thefunction effects of EGFR gene science.
Results
We have demonstrated here EGFR gene was overexpressed in MG63 cells. InMG63 cell line, we displayed sequence specific silencing of the EGF receptor with16.2％、27.3％、47.7％、52.3％and 54.5％(P＜0.01)in 12h、24h、48h、60h and72h respectivly of down-regulation of EGF receptor gene mRNA. The Quantitativereverse-transcriptase PCR technique was built.The expression of EGFR gene proteinis also downregulated significantly. Compare with the control group, the cellproliferation suppresion rate in the EGFR gene silence group in MG63 is 12.6％、30.0％(P＜0.01)、39.1％(P＜0.01)、47.1％(P＜0.01) and 62.1％(P＜0.01) in 12h、24h、48h、60h and 72h respectivly with MTT assay. The reduction in EGFR causedMG63 cells apoptosis, apoptotic MG63 cells with condensed or fragmented nucleistained by DAPI were observed and Brown stained apoptosis cells were seen byTUNEL and apoptosis bodies was shown by TEM also. The ratio of apoptosis werechanged from 1.9％to 12.6％, 31.6％and％36.2％respectively. Differencebetween control group and other groups had statistical significance (P＜0.05).
Conclusion
These sequence specific shRNA-EGFR showed a blockbuster effect indownregulation of EGFR gene expression, inhibition of the cellular proliferation andmotility and arrestment of the cell cycle. The successful application of dsRNA-EGFRto reverse the neoplastic phenotype of EGFR overexpressing cells extends the list ofavailable therapeutic modalities in the treatment of human cancer. Our results suggestthat RNAi-mediated silencing of EGFR may provide an opportunity to develop a newtreatment strategy for osteosarcoma.
目前，骨肉瘤的发病机制仍不清楚。但同其它肿瘤一样，癌基因，抑癌基因，凋亡相关基因，生长因子及受体的异常表达及细胞内信号传导的紊乱被认为与骨肉瘤的发生和转移密切相关。表皮生长因子受体(EGFR)，是目前最受注目的肿瘤治疗药物的靶位。大量研究表明，EGFR酪氨酸激酶区域是调节肿瘤细胞增殖、侵袭、血管生成、转移和凋亡信号通路的重要部分。表皮生长因子EGF与EGFR的结合导致EGFR的二聚体化和胞内区磷酸化，进而引发一系列的级联放大反应，从而调控和传递骨肉瘤的生物学行为的信号。研究发现EGFR在人骨肉瘤组织中过表达，且其表达水平和恶性预后相关，EGFR抑制剂有望被开发成为骨肉瘤的崭新治疗途径。
RNA干扰(RNAi)是由双链RNA(dsRNA)引发的转录后基因沉默机制(PTGS)，dsRNA经RNA沉默复合体(RISC)降解成21-23nt的小片段(siRNA)，并以其为模板，特定位点、特定间隔降解与之序列互补的mRNA，目前已成功用于基因功能和信号传递系统上下游分子相互关系的研究。本研究拟通过采用RNAi技术，探讨利用体外化学合成siRNA和构建质粒载体的方法是否能有效沉默骨肉瘤细胞EGFR基因表达，阻断EGFR所介导的恶性生物学行为，从而为骨肉瘤的基因治疗提供新策略。
目的
探讨采用RNAi技术是否能有效抑制骨肉瘤MG63细胞株EGFR表达，以及EGFR基因下调后骨肉瘤MG63细胞生物学特性的改变，以期为肿瘤治疗提供新的思路和实验依据。
材料与方法
首先通过免疫组化、RT-PCR和western blot测定MG63细胞株EGFR的表达情况。体外合成短发夹状shRNA—EGFR单链，并通过退火、回收、质粒酶切、连接、转化、挑克隆、提质粒、测序等步骤，构建和鉴定重组质粒载体psilencer-2.1-shRNA-EGFR，转染MG63细胞株。建立实时荧光定量RT-PCR定量检测EGFRmRNA的表达量的方法。用实时荧光定量RT-PCR检测shRNA对靶mRNA沉默效率；用western—blot法检测蛋白表达变化情况；用四甲基偶氮唑盐(MTT)比色分析检测细胞体外增殖率情况；通过DAPI染色观察干扰后的细胞调亡情况；透射电镜和原位末端标记TUNEL法观察细胞凋亡形态学改变；Annexin V/P I双标记法检测细胞的早期和晚期凋亡率改变。
结果
我们建立了EGFR基因干扰载体并建立荧光定量PCR检测技术平台。通过免疫组化、RT-PCR和western blot证实MG63细胞株存在EGFR高表达。Psilencer2.1-shRNA-EGFR-Lipofectamine 2000复合物转染细胞可引发EGFR序列特异性基因沉默效应，与RT-PCR、Western Blot分析得到了一致的结果。干扰组骨肉瘤细胞与对照组比较，shRNA-EGFR组EGFR基因水平在12h、24h、48h、60h和72h分别下调16.2％、27.3％、47.7％、52.3％及54.5％(P＜0.01)；shRNA-EGFR组EGFR蛋白表达水平也明显下调。MTT法检测EGFR对MG63细胞增殖抑制率在12h、24h、48h、60h和72h分别为12.6％、30.0％(P＜0.01)、39.1％(P＜0.01)、47.1％(P＜0.01)和62.1％(P＜0.01)。shRNA-EGFR诱发的调亡细胞细胞核DAPI染色明显增强，胞核缩小，核膜皱缩，染色质凝聚、边缘化，晚期染色质破碎。TUNEL检测RNA干扰48h后的细胞，可见明显的棕色细胞，表明凋亡细胞的存在。经EGFR干扰48h的人MG63细胞，电镜观察发现干扰后细胞细胞核体积缩小，胞浆内出现大小不等的空泡，细胞浆浓缩，整个细胞固缩，并可见脱落到细胞外的凋亡小体。Annexin V／P I检测EGFR干扰MG63细胞，干扰前凋亡检测值为1.9％，坏死为0.7％；干扰后24h凋亡检测值为12.6％，坏死为5.39％；48h凋亡凋亡检测值为31.6％，坏死为5.1％，72h凋亡凋亡检测值为36.2％，坏死为24.3％。
结论
1．MG63细胞株存在EGFR高表达。2．shRNA-EGFR可序列特异性下调MG63细胞EGFR基因水平，显著降低EGFR蛋白表达。3．shRNA-EGFR重组质粒通过抑制EGFR表达可抑制细胞增生和诱发调亡，从而为MG63基因治疗提供了新策略。
Osteosarcoma is the most predominant type of malignant bone tumor in childrenand adolescents between 15-25 years old. The prevalance of osteosarcoma in male istwo times than female. The prognosis of osteosarcoma is worse. 15-20％of patientshave clinically detectable metastases espesilly in lung at initial diagnosis. The 3-5year survival rate is only 5-20％even after amputation. Lesions deteted in the distalfemur and proximal tibia are account for 75％of total cases in osteosarcoma. Thepathology changes in osteosarcoma are mainly osteolytic. Despite treatment advances,chemotherapy is only marginally effective in most advanced cases. For those patientsrefractory to or intolerant of the current chemotherapy, treatment options are limited.Hence, more effective therapy with fewer side effects is needed.
At present, the machanisiam of osteosarcoma is still unclear. The abnormalexpression of oncogene, suppressor gene, and apoptosis related gene, growth factorreceptor and disorder of intracellar signal pathway were regarded as to have key rolewith metastasis occur in osteosarcoma. Epidermal growth factor receptor (EGFR) is aglycoprotein with a molecular weight of 170,000 to 180,000. It is an intrinsictyrosine-specific protein kinase, which is stimulated upon epidermal growth factor(EGF) binding. EGFR signaling involved in cell growth, angiogenesis, DNA repair,and autocrine growth regulation in MG63 as well as in a wide spectrum of human cancer cells. Thus, it has recently emerged as an innovative target for thedevelopment of new cancer therapy.
RNA interference (RNAi) is an evolutionarily conserved process in whichrecognition of double-stranded RNA (dsRNA) ultimately leads to posttranscriptionalsuppression of gene expression. This suppression is mediated by short hair RNA(shRNA), which induces specific degradation of mRNA through complementary basepairing. In several model systems, ie: mostly in lower order animals, this naturalresponse has been developed into a powerful tool for the investigation of genefunction. Recently it was discovered that introducing synthetic 21-23 nucleotidedsRNA duplexes into mammalian cells could efficiently silence gene expression. Inthis study, we investigated the possibility whether RNAi could silence EGFR gene incommonly used MG63 cancer cell lines. We also assessed the degree of EGFR genesilencing and its functional outcome in terms of effects on cell proliferation andgrowth inhibition in vitro.
Objective
To investigate whether RNA interference (RNAi) could induce gene silencing inosteosarcoma (MG63) cells; to evaluate the degree of epidermal growth factor (EGFR)receptor gene silencing and its effect on functional outcome of MG63 cells.
Meterials and methods
To determine whether EGFR is overexpressed and the effect of RNAi on EGFRgene in MG63 cells, EGFR gene RNAi lentivirus vector was constructed, MG63 celllines were transfected with target sequence-specific shRNA as well as variouscontrols. IHC and Western Blot were used to measure the expression of EGFR proteinin MG63 cells and reduction the EGFR protein in EGFR gene knocked down MG63cells. Quantitative reverse-transcriptase PCR was used to detect the expression andsilencing of the EGFR gene level. MTT assay, DAPI, TUNEL, transmission electronmicroscope (TEM) and Annexin V/P I double stain analysis were used to assess thefunction effects of EGFR gene science.
Results
We have demonstrated here EGFR gene was overexpressed in MG63 cells. InMG63 cell line, we displayed sequence specific silencing of the EGF receptor with16.2％、27.3％、47.7％、52.3％and 54.5％(P＜0.01)in 12h、24h、48h、60h and72h respectivly of down-regulation of EGF receptor gene mRNA. The Quantitativereverse-transcriptase PCR technique was built.The expression of EGFR gene proteinis also downregulated significantly. Compare with the control group, the cellproliferation suppresion rate in the EGFR gene silence group in MG63 is 12.6％、30.0％(P＜0.01)、39.1％(P＜0.01)、47.1％(P＜0.01) and 62.1％(P＜0.01) in 12h、24h、48h、60h and 72h respectivly with MTT assay. The reduction in EGFR causedMG63 cells apoptosis, apoptotic MG63 cells with condensed or fragmented nucleistained by DAPI were observed and Brown stained apoptosis cells were seen byTUNEL and apoptosis bodies was shown by TEM also. The ratio of apoptosis werechanged from 1.9％to 12.6％, 31.6％and％36.2％respectively. Differencebetween control group and other groups had statistical significance (P＜0.05).
Conclusion
These sequence specific shRNA-EGFR showed a blockbuster effect indownregulation of EGFR gene expression, inhibition of the cellular proliferation andmotility and arrestment of the cell cycle. The successful application of dsRNA-EGFRto reverse the neoplastic phenotype of EGFR overexpressing cells extends the list ofavailable therapeutic modalities in the treatment of human cancer. Our results suggestthat RNAi-mediated silencing of EGFR may provide an opportunity to develop a newtreatment strategy for osteosarcoma.
引文
1. Tunn PU, Reichardt P. Chemotherapy for Osteosarcoma without High-Dose Methotrexate: A 12-Year Follow-Up on 53 Patients.Onkologie. 2007; 30(5): 228-32.
2. Lamoureux F, Trichet V, Chipoy C, et al. Recent advances in the management of osteosarcoma and forthcoming therapeutic strategies. Expert Rev Anticancer Ther. 2007; 7(2): 169-81.
3. Hughes DP, Thomas DG, Giordano TJ, et al. Cell surface expression of epidermal growth factor receptor and Her-2 with nuclear expression of Her-4 in primary osteosarcoma. Cancer Res. 2004; 64(6):2047-53.
4. Hughes DP, Thomas DG, Giordano TJ, et al. Essential erbB family phosphorylation in osteosarcoma as a target for CI-1033 inhibition. Pediatr Blood Cancer. 2006; 46(5):614-23.
5. Freeman BB 3rd, Daw NC, Geyer JR, et al. Evaluation of gefitinib for treatment of refractory solid rumors and central nervous system malignancies in pediatric patients. Cancer Invest. 2006;24(3):310-7.
6. Ferguson WS, Goorin AM. Current treatment of osteosarcoma. Cancer Investig,, 2001; 19: 292-315.
7. Meyer WH, Malawer MM. Osteosarcoma. Clinical features and evolving surgical and chemotherapeutic strategies. Pediatr Clin N Am, 1991;38: 317-48.
8. Provisor AJ, Ettinger LJ, Nachman JB, et al Treatment of nonmetastatic osteosarcoma of the extremity with preoperative and postoperative chemotherapy: a report from the Children's Cancer Group. J Clin Oncol, 1997;15: 76-84.
9 Bacci G, Ferrari S, Bertoni F, et al Long-term outcome for patients with nonmetastatic osteosarcoma of the extremity treated at the Istituto Ortopedico Rizzoli according to the Istituto Ortopedico Rizzoli/Osteosarcoma-2 Protocol: an updated report. J Clin Oncol, 2000; 18: 4016-27.
10. Kager L, Zoubek A, Potschger U, et al Primary metastatic osteosarcoma: presentation and outcome of patients treated on neoadjuvant cooperative osteosarcoma study group protocols. J Clin Oncol, 2003;21: 2011-8.
11. Bacci G, Ferrari S, Longhi A, et al Relationship between dose-intensity of treatment and outcome for patients with osteosarcoma of the extremity treated with neoadjuvant chemotherapy. Oncol Rep, 2001; 8: 883-8.
12. Goorin A, Andersen J. Experience with multiagent chemotherapy for osteosarcoma. Improved outcome. Clin Orthop, 1991: 22-8,.
13. Bacci G, Briccoli A, Ferrari S, et al Neoadjuvant chemotherapy for osteosarcoma of the extremities with synchronous lung metastases: treatment with cisplatin, Adriamycin and high dose of methotrexate and ifosfamide. Oncol Rep, 2000; 7: 339-46.
14. Arteaga CL, Baselga J. Tyrosine kinase inhibitors: Why does the current process of clinical development not apply to them? Cancer Cell, 2004; 5:525-531.
15. Holbro T, Civenni G, Hynes NE. The ErbB receptors and their role in cancer progression. Exp Cell Res, 2003; 284:99-110.
16. Langer, C. J.. Mind Your Elders: Therapeutic Implications of Epidermal Growth Factor Receptor Inhibition in Older Patients With Advanced Non-Small-Cell Lung Cancer. JCO, 2007; 25: 751-753.
17. Rowinsky EK. The erbB family: Targets for therapeutic development against cancer and therapeutic strategies using monoclonal antibodies and tyrosine kinase inhibitors. Annu Rev Med 55, 2004; 433-457.
18. Kang JH, Park KK, Lee IS, et al. Proteome analysis of responses to ascochlorin in a human osteosarcoma cell line by 2-D gel electrophoresis and MALDI-TOF MS. J Proteome Res. 2006; 5(10):2620-31.
19. Drattstrom D, Wester K, Bergqvist M, et al. HER-2, EGFR, COX-2 expression status correlated to microvessel density and survival in resected non-small cell lung cancer. Acta Oncol, 2004; 43: 80-86.
20. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med, 2004; 350:2129-2139.
21. Dubey, S., Powell, C. A. Update in Lung Cancer 2006. Am. J. Respir. Crit. Care Med. 2007; 175: 868-874.
22. Carette JE, Graat HC, Schagen FH, et al. A conditionally replicating adenovirus with strict selectivity in killing cells expressing epidermal growth factor receptor. Virology. 2007; 361(1):56-67.
23. Dai FH, Chen Y, Ren CC, et al. Construction of an EGF receptor-mediated histone H1(0)-based gene delivery system.J Cancer Res Clin Oncol. 2003; 29(8):456-62..
24. Witlox MA, Van Beusechem VW, Grill J, et al. Epidermal growth factor receptor targeting enhances adenoviral vector based suicide gene therapy of osteosarcoma. J Gene Med. 2002;4(5):510-6.
25. Kenny, P. A., Bissell, M. J. Targeting TACE-dependent EGFR ligand shedding in breast cancer. J. Clin. Invest. 2007;117: 337-345.
26. Toschi, L., Cappuzzo, F. Understanding the New Genetics of Responsiveness to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors. The Oncologist, 2007; 12:211-220.
27. Shamovsky I, Ivannikov M, Kandel ES, Gershon D, Nudler E. RNA-mediated response to heat shock in mammalian cells. Nature. 2006; 440(7083):556-60.
28. Wischnewski F, Pantel K, Schwarzenbach H. Promoter demethylation and histone acetylation mediate gene expression of MAGE-A1, -A2, -A3, and -AI2 in human cancer cells. Mol Cancer Res. 2006; 4(5): 339-49.
29. Patzel V, Rutz S, Dietrich I, Koberle C, Scheffold A, Kaufmann SH. Design of siRNAs producing unstructured gnide-RNAs results in improved RNA interference efficiency. Nat Biotechnol. 2005; 23(11): 1440-4.
30．刘彬，丁卫，谷静丽，等．EGFR及IGF-1对UMR106细胞增殖及p微管蛋白的影响．生物化学杂志，1995，11(14)：406-11．
31. Fang MA, Kujubu DA, Hahn TJ. The effects of prostaglandin E2, parathyroid hormone, and epidermal growth factor on mitogenesis, signaling, and primary response genes in UMR 106-01 osteoblast-like cells. Endocrinology 1992, 131(5): 2113-9.
32. Heldin CH, Ostman A, Eriksson A, et al. Platelet-derived growth factor: isoform-specific signalling via heterodimeric or homodimeric receptor complexes. Kidney Int. 1992, 41(3): 571-4.
33. Abdiu A, Abdiugren S, Larsson SE, et al. growth factor-AB on sarcoma growth in vitro Effects of human platelet-derived and in vivo. Cancer Lett. 1999, 141(1—2): 39-45.
34．宝建中，郑建明，周霖，等．癌基因蛋白P21，P185，EGFR及LN，Ⅳ型胶原在骨肉瘤中的表达肿瘤防治研究，1999，26(4)：265-6．
35. Stirling D. Qualitative and quantitative PCR: a technical overview. Methods Mol Biol, 2003; 226:181—184.
36. Pusterla N, Madigan JE, Leutenegger CM. Real-time polymerase chain reaction: a novel molecular diagnostic tool for equine infectious diseases. J Vet Intern Med, 2006; 20(1):3-12.
37. Khalil SH. Molecular hematology. Qualitative to quantitative techniques. Saudi Med J. 2005, 26(10): 1516-1522.
38. Chen Z, Yung WK. A reliability test of standard-based quantitative PCR: vs endogenous standards. Mol. Cell Probes, 2000, 14(2): 127-135.
39. Vickers TA, Koo S, Bennett CF, Crooke ST, Dean NM, Baker BF. Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J Biol Chem. 2003;278:7108-7118.
40. Zhang X, Shan P, Jiang D, Noble PW, Abraham NG, Kappas A, Lee PJ. Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis. J Biol Chem. 2004;279:10677-10684.
41. Ge Q, McManus MT, Nguyen T, Shen CH, Sharp PA, Eisen HN, Chen J. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc Natl Acad Sci USA. 2003; 100:2718-2723
42. Fire A, Xu SQ, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. [J]. Nature,1998; 391:806-811.
43. Nakanishi Y, Kodama J, Yoshinouchi M, et al. The expression of vascular endothelial growth factor and transforming growth factor-beta associates with angiogenesis in epithelial ovarian cancer [J]. Int J Gyneeol Pathol, 1997;16(3):256-262.
44. Chen CHA, Cheng WF, Lee CHN, et al. Serum vascular endothelial growth factor in epithelial ovarian neoplasms: correlation with patient survival[J]. Gyneeol Oneol, 1999, 74: 235-240.
45. Montgomery MK, Xu S, Fire A. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans[J]. Proc Natl Acad Sei USA, 1998, 95(26): 15502-47.
46. Elbashir SM, Harborth J, Lendeekel W, et al. Duplexes of 21-nueleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature, 2001; 411(6836): 494-498.
47. Wittig JC, Biekels J, Priebat D, et aL. Osteosareoma: a multidisciplinary approach to diagnosis and treatment[J]. Am Faro Physician, 2002, 65(6): 1123.
48 郭爱林，张盈华 成骨肉瘤患者TH1／TH2亚性漂移研究 中华实验外科杂志．1999，16(2)．-140-141
49．郭爱林，范清宇 儿童恶性骨肿瘤患者Th1／Th2亚群漂移的研究 中华儿科杂志．2000，38(5)．-316-317
50. van der Veen AH, de Witt JH, Eggermont AM, et al. TNF—a1Pha augments intratumoural concentrations of doxorubicin in TNF—alpha—based isolated limb Perfusionin rat sarcoma models and enhances anti—tumour effects[J]. Br J Cancer, 2000, 82(4):973.
51. Luksch R, Perotti D, Cefalo G, et al. Immunomodulation in a treatment program including pre- and post-operative interleukin-2 and chemotherapy for childhood osteosarcoma. [J] Tumori. 2003 May-Jun;89(3):263-8.
52. Southam CM, Marcove RC, Levin AG, et al. Proceedings: Clinical trial of autogenous tumor vaccine for treatment of osteogenic sarcoma. [J] Proc Natl Cancer Conf. 1972;7:91-100.
53．王臻 彭磊 等 骨肉瘤细胞与活化B淋巴细胞融合疫苗的制备及其诱导的抗瘤活性 中华骨科杂志．2001，21(10)：622-625
54. Worth LL, Jia SF, Zhou Z, et al. Intranasal therapy with an adenoviral vector containing the murine interleukin12 gene eradicates osteosarcoma lung metastases[J]. Clin Cancer Res, 2000, 6(9):3713-3718.
55. de Wilt JH, Bout A, Eggermout AM, et al. Adenovirus mediated interleukin3 beta gene transfer by isolated limb perfusion inhibits growth of limb sarcoma in rats[J]. Hum Gene Ther, 2001, 12(5): 489-502.
56. Swarthout JT, Culver KW. Bystander effect mediated regression of osteosarcoma via retrovial transfer of the herpes simplex virus thymidine kinase and human interleukin2 genes[J]. Cancer Gene Ther, 2000, 7(2): 187-196.
57. Hodge JW, Mclaughlin JP, Kantor JA, et al. Diversified prime and boost protocol susing recombint vaccinia virus and recombinant non replication avian pox virus to enhance T cell immunity and anti tumor responses [J] Vaccine, 1997, 15(4):759
58. Rosenberg SA, Lotze MT, Yang JC, et al. Experience with the use of high dose interleuken2 in the treatment of 652 cancer patients[J]Ann Surg, 1999, 210(4):474
59. Plouet J, Schilling J, Gospodarowicz D, Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cell [J]. EMBO j, 1989, 8(12):3801.
60. Dvorak HF, Brown LF, Detmar M, et al. Vascular permeability factor Nascular endothelial growth factor, microvascular hyperpermeability and angilgenesis[J]. Am J Pathol, 1995, 146(5):1029.
61. Yancopoulos GD, Davis S, Gale NW, et al. Vascular-specific growth factors and blood vessel formation [J]. Nature, 2000, 407(6801):242
62. Folkman J, Shing Y, Angiogensis [J]. J Biol chem.1992, 267(16): 10931
63. Park JE, Keller GA, Ferrara N, Tbe vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity or extracellular matrix-bound VEGF [J]. Mol Biol Cell, 1993, 4(12): 1317
64. Petruzzelli GJ, Benefild j, et al, Heparin-binding growth factor (s) derived from head and neck squamous cell carcinomas induces endothelial cell proliferations. Head Neck, 1997 Oct; 19(7):576-82
65. Nakashema T, Hudson JM et al. Antisense inhibition of vascular endothelial groth factor in human head and neck squamous cell carcinoma Head Neck, 20004;22(5):483-8
66. Jussila L, Alitalo K, Vascular growth factor and lymphangiogenesis. Phyiol Rev, 2002 Jul 82(3):673-700
67. Darzynkiewicz Z, Bruno S, Del Bino G, et al. Features of apoptotie cells measured by folw cytometry. [J] Cytometry, 1992, 13(8):795-808.
68. Arends MJ, Morris RG, Wyllie AH. Apoptosis-The role of the endonuclease.[J] Am J Pathol, 1990, 136(3):593-608.
69. Afanasyev VN, Korlo BA, Matylevich NP, et al, The use of folw cytomtry for the investigation of cell death.[J] Cytometry, 1993, 14(6);603-609.
70. Tyagi S, Kramer FRet al. Molecular beacons: probes than fluoresce upon hybridization. [J] Nat. Biotechnol. 1996, 14: 303-308.
71. Silhavy D, Monar A, Lucioloi A, et al, A viral protein suppresses RNA silencing and binds silencing generated, 21 to 25 nucleotide double stranded RNAs. [J] EMBO Journal, 2002, 21(12): 3070-3080.
72. Yang D, Buchholz F, Huang Z, Goga A, Chen CY, Brodsky FM, and Bishop JM, Short RNA duplexes produced by hydrolysis with Eschefichia coli RNase Ⅲ mediate effective RNA interference in mammalian cells, [J] Proc Nail Acad Sci U S A: 2002, 99:9942-7
73. Hu W, Myers C, Kilzer J, Pfaff S, and Bushman F, Inhibition ofretroviral pathogenesis by RNA interference. [J] Curr, Biol, 2002; 12:1301-1306.
74. Caplen N J, Parrish S, Imani F, Fire A, et al. Specific inhibition of gene expression by small double stranded RNAs in invertebrate and vertebrate systems. [J] Proceeedings of the National Academy of Sciences of the United Stated of America, 2001, 98(17): 9742-9747.
75. Haasnoot PCJ, Cupac D, Berkhout B, Inhibition of virus replication by RNA interference, [J] Biomed, Sci 2003; 10: 607-616.
76. Vickers TA, Koo S, Frank Bennett C, et al, Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense atents. [J] Bilo Chem, 2003, 278(9): 7108-7118.
77. Shuey D J, McCallus DE, Giordano T, RNAi: gene-silencing in therapeutic intervention. [J] Drugdiscovery Today, 2002, 7(20): 1040-1046.
78. Hernann MT, Fridman JS, Zifou JT, et al, An epi-allelic sseries of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. [J] Nat Genetics, 2003, 33(3): 396-400.
79. Jiang M, Milner J, Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNAi interference, [J] Oncogene, 2002, 21 (39): 6041-6048.
80. Lu PY, Xie FY, Woodle MC, et al. siRNA-mediated anti-tumorigenesis for drug target validation and therapeutics, [J] Curt Opin Mol Ther, 2003, 5(3): 225-234.
2. Lamoureux F, Trichet V, Chipoy C, et al. Recent advances in the management of osteosarcoma and forthcoming therapeutic strategies. Expert Rev Anticancer Ther. 2007; 7(2): 169-81.
3. Hughes DP, Thomas DG, Giordano TJ, et al. Cell surface expression of epidermal growth factor receptor and Her-2 with nuclear expression of Her-4 in primary osteosarcoma. Cancer Res. 2004; 64(6):2047-53.
4. Hughes DP, Thomas DG, Giordano TJ, et al. Essential erbB family phosphorylation in osteosarcoma as a target for CI-1033 inhibition. Pediatr Blood Cancer. 2006; 46(5):614-23.
5. Freeman BB 3rd, Daw NC, Geyer JR, et al. Evaluation of gefitinib for treatment of refractory solid rumors and central nervous system malignancies in pediatric patients. Cancer Invest. 2006;24(3):310-7.
6. Ferguson WS, Goorin AM. Current treatment of osteosarcoma. Cancer Investig,, 2001; 19: 292-315.
7. Meyer WH, Malawer MM. Osteosarcoma. Clinical features and evolving surgical and chemotherapeutic strategies. Pediatr Clin N Am, 1991;38: 317-48.
8. Provisor AJ, Ettinger LJ, Nachman JB, et al Treatment of nonmetastatic osteosarcoma of the extremity with preoperative and postoperative chemotherapy: a report from the Children's Cancer Group. J Clin Oncol, 1997;15: 76-84.
9 Bacci G, Ferrari S, Bertoni F, et al Long-term outcome for patients with nonmetastatic osteosarcoma of the extremity treated at the Istituto Ortopedico Rizzoli according to the Istituto Ortopedico Rizzoli/Osteosarcoma-2 Protocol: an updated report. J Clin Oncol, 2000; 18: 4016-27.
10. Kager L, Zoubek A, Potschger U, et al Primary metastatic osteosarcoma: presentation and outcome of patients treated on neoadjuvant cooperative osteosarcoma study group protocols. J Clin Oncol, 2003;21: 2011-8.
11. Bacci G, Ferrari S, Longhi A, et al Relationship between dose-intensity of treatment and outcome for patients with osteosarcoma of the extremity treated with neoadjuvant chemotherapy. Oncol Rep, 2001; 8: 883-8.
12. Goorin A, Andersen J. Experience with multiagent chemotherapy for osteosarcoma. Improved outcome. Clin Orthop, 1991: 22-8,.
13. Bacci G, Briccoli A, Ferrari S, et al Neoadjuvant chemotherapy for osteosarcoma of the extremities with synchronous lung metastases: treatment with cisplatin, Adriamycin and high dose of methotrexate and ifosfamide. Oncol Rep, 2000; 7: 339-46.
14. Arteaga CL, Baselga J. Tyrosine kinase inhibitors: Why does the current process of clinical development not apply to them? Cancer Cell, 2004; 5:525-531.
15. Holbro T, Civenni G, Hynes NE. The ErbB receptors and their role in cancer progression. Exp Cell Res, 2003; 284:99-110.
16. Langer, C. J.. Mind Your Elders: Therapeutic Implications of Epidermal Growth Factor Receptor Inhibition in Older Patients With Advanced Non-Small-Cell Lung Cancer. JCO, 2007; 25: 751-753.
17. Rowinsky EK. The erbB family: Targets for therapeutic development against cancer and therapeutic strategies using monoclonal antibodies and tyrosine kinase inhibitors. Annu Rev Med 55, 2004; 433-457.
18. Kang JH, Park KK, Lee IS, et al. Proteome analysis of responses to ascochlorin in a human osteosarcoma cell line by 2-D gel electrophoresis and MALDI-TOF MS. J Proteome Res. 2006; 5(10):2620-31.
19. Drattstrom D, Wester K, Bergqvist M, et al. HER-2, EGFR, COX-2 expression status correlated to microvessel density and survival in resected non-small cell lung cancer. Acta Oncol, 2004; 43: 80-86.
20. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med, 2004; 350:2129-2139.
21. Dubey, S., Powell, C. A. Update in Lung Cancer 2006. Am. J. Respir. Crit. Care Med. 2007; 175: 868-874.
22. Carette JE, Graat HC, Schagen FH, et al. A conditionally replicating adenovirus with strict selectivity in killing cells expressing epidermal growth factor receptor. Virology. 2007; 361(1):56-67.
23. Dai FH, Chen Y, Ren CC, et al. Construction of an EGF receptor-mediated histone H1(0)-based gene delivery system.J Cancer Res Clin Oncol. 2003; 29(8):456-62..
24. Witlox MA, Van Beusechem VW, Grill J, et al. Epidermal growth factor receptor targeting enhances adenoviral vector based suicide gene therapy of osteosarcoma. J Gene Med. 2002;4(5):510-6.
25. Kenny, P. A., Bissell, M. J. Targeting TACE-dependent EGFR ligand shedding in breast cancer. J. Clin. Invest. 2007;117: 337-345.
26. Toschi, L., Cappuzzo, F. Understanding the New Genetics of Responsiveness to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors. The Oncologist, 2007; 12:211-220.
27. Shamovsky I, Ivannikov M, Kandel ES, Gershon D, Nudler E. RNA-mediated response to heat shock in mammalian cells. Nature. 2006; 440(7083):556-60.
28. Wischnewski F, Pantel K, Schwarzenbach H. Promoter demethylation and histone acetylation mediate gene expression of MAGE-A1, -A2, -A3, and -AI2 in human cancer cells. Mol Cancer Res. 2006; 4(5): 339-49.
29. Patzel V, Rutz S, Dietrich I, Koberle C, Scheffold A, Kaufmann SH. Design of siRNAs producing unstructured gnide-RNAs results in improved RNA interference efficiency. Nat Biotechnol. 2005; 23(11): 1440-4.
30．刘彬，丁卫，谷静丽，等．EGFR及IGF-1对UMR106细胞增殖及p微管蛋白的影响．生物化学杂志，1995，11(14)：406-11．
31. Fang MA, Kujubu DA, Hahn TJ. The effects of prostaglandin E2, parathyroid hormone, and epidermal growth factor on mitogenesis, signaling, and primary response genes in UMR 106-01 osteoblast-like cells. Endocrinology 1992, 131(5): 2113-9.
32. Heldin CH, Ostman A, Eriksson A, et al. Platelet-derived growth factor: isoform-specific signalling via heterodimeric or homodimeric receptor complexes. Kidney Int. 1992, 41(3): 571-4.
33. Abdiu A, Abdiugren S, Larsson SE, et al. growth factor-AB on sarcoma growth in vitro Effects of human platelet-derived and in vivo. Cancer Lett. 1999, 141(1—2): 39-45.
34．宝建中，郑建明，周霖，等．癌基因蛋白P21，P185，EGFR及LN，Ⅳ型胶原在骨肉瘤中的表达肿瘤防治研究，1999，26(4)：265-6．
35. Stirling D. Qualitative and quantitative PCR: a technical overview. Methods Mol Biol, 2003; 226:181—184.
36. Pusterla N, Madigan JE, Leutenegger CM. Real-time polymerase chain reaction: a novel molecular diagnostic tool for equine infectious diseases. J Vet Intern Med, 2006; 20(1):3-12.
37. Khalil SH. Molecular hematology. Qualitative to quantitative techniques. Saudi Med J. 2005, 26(10): 1516-1522.
38. Chen Z, Yung WK. A reliability test of standard-based quantitative PCR: vs endogenous standards. Mol. Cell Probes, 2000, 14(2): 127-135.
39. Vickers TA, Koo S, Bennett CF, Crooke ST, Dean NM, Baker BF. Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis. J Biol Chem. 2003;278:7108-7118.
40. Zhang X, Shan P, Jiang D, Noble PW, Abraham NG, Kappas A, Lee PJ. Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis. J Biol Chem. 2004;279:10677-10684.
41. Ge Q, McManus MT, Nguyen T, Shen CH, Sharp PA, Eisen HN, Chen J. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc Natl Acad Sci USA. 2003; 100:2718-2723
42. Fire A, Xu SQ, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. [J]. Nature,1998; 391:806-811.
43. Nakanishi Y, Kodama J, Yoshinouchi M, et al. The expression of vascular endothelial growth factor and transforming growth factor-beta associates with angiogenesis in epithelial ovarian cancer [J]. Int J Gyneeol Pathol, 1997;16(3):256-262.
44. Chen CHA, Cheng WF, Lee CHN, et al. Serum vascular endothelial growth factor in epithelial ovarian neoplasms: correlation with patient survival[J]. Gyneeol Oneol, 1999, 74: 235-240.
45. Montgomery MK, Xu S, Fire A. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans[J]. Proc Natl Acad Sei USA, 1998, 95(26): 15502-47.
46. Elbashir SM, Harborth J, Lendeekel W, et al. Duplexes of 21-nueleotide RNAs mediate RNA interference in cultured mammalian cells[J]. Nature, 2001; 411(6836): 494-498.
47. Wittig JC, Biekels J, Priebat D, et aL. Osteosareoma: a multidisciplinary approach to diagnosis and treatment[J]. Am Faro Physician, 2002, 65(6): 1123.
48 郭爱林，张盈华 成骨肉瘤患者TH1／TH2亚性漂移研究 中华实验外科杂志．1999，16(2)．-140-141
49．郭爱林，范清宇 儿童恶性骨肿瘤患者Th1／Th2亚群漂移的研究 中华儿科杂志．2000，38(5)．-316-317
50. van der Veen AH, de Witt JH, Eggermont AM, et al. TNF—a1Pha augments intratumoural concentrations of doxorubicin in TNF—alpha—based isolated limb Perfusionin rat sarcoma models and enhances anti—tumour effects[J]. Br J Cancer, 2000, 82(4):973.
51. Luksch R, Perotti D, Cefalo G, et al. Immunomodulation in a treatment program including pre- and post-operative interleukin-2 and chemotherapy for childhood osteosarcoma. [J] Tumori. 2003 May-Jun;89(3):263-8.
52. Southam CM, Marcove RC, Levin AG, et al. Proceedings: Clinical trial of autogenous tumor vaccine for treatment of osteogenic sarcoma. [J] Proc Natl Cancer Conf. 1972;7:91-100.
53．王臻 彭磊 等 骨肉瘤细胞与活化B淋巴细胞融合疫苗的制备及其诱导的抗瘤活性 中华骨科杂志．2001，21(10)：622-625
54. Worth LL, Jia SF, Zhou Z, et al. Intranasal therapy with an adenoviral vector containing the murine interleukin12 gene eradicates osteosarcoma lung metastases[J]. Clin Cancer Res, 2000, 6(9):3713-3718.
55. de Wilt JH, Bout A, Eggermout AM, et al. Adenovirus mediated interleukin3 beta gene transfer by isolated limb perfusion inhibits growth of limb sarcoma in rats[J]. Hum Gene Ther, 2001, 12(5): 489-502.
56. Swarthout JT, Culver KW. Bystander effect mediated regression of osteosarcoma via retrovial transfer of the herpes simplex virus thymidine kinase and human interleukin2 genes[J]. Cancer Gene Ther, 2000, 7(2): 187-196.
57. Hodge JW, Mclaughlin JP, Kantor JA, et al. Diversified prime and boost protocol susing recombint vaccinia virus and recombinant non replication avian pox virus to enhance T cell immunity and anti tumor responses [J] Vaccine, 1997, 15(4):759
58. Rosenberg SA, Lotze MT, Yang JC, et al. Experience with the use of high dose interleuken2 in the treatment of 652 cancer patients[J]Ann Surg, 1999, 210(4):474
59. Plouet J, Schilling J, Gospodarowicz D, Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cell [J]. EMBO j, 1989, 8(12):3801.
60. Dvorak HF, Brown LF, Detmar M, et al. Vascular permeability factor Nascular endothelial growth factor, microvascular hyperpermeability and angilgenesis[J]. Am J Pathol, 1995, 146(5):1029.
61. Yancopoulos GD, Davis S, Gale NW, et al. Vascular-specific growth factors and blood vessel formation [J]. Nature, 2000, 407(6801):242
62. Folkman J, Shing Y, Angiogensis [J]. J Biol chem.1992, 267(16): 10931
63. Park JE, Keller GA, Ferrara N, Tbe vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity or extracellular matrix-bound VEGF [J]. Mol Biol Cell, 1993, 4(12): 1317
64. Petruzzelli GJ, Benefild j, et al, Heparin-binding growth factor (s) derived from head and neck squamous cell carcinomas induces endothelial cell proliferations. Head Neck, 1997 Oct; 19(7):576-82
65. Nakashema T, Hudson JM et al. Antisense inhibition of vascular endothelial groth factor in human head and neck squamous cell carcinoma Head Neck, 20004;22(5):483-8
66. Jussila L, Alitalo K, Vascular growth factor and lymphangiogenesis. Phyiol Rev, 2002 Jul 82(3):673-700
67. Darzynkiewicz Z, Bruno S, Del Bino G, et al. Features of apoptotie cells measured by folw cytometry. [J] Cytometry, 1992, 13(8):795-808.
68. Arends MJ, Morris RG, Wyllie AH. Apoptosis-The role of the endonuclease.[J] Am J Pathol, 1990, 136(3):593-608.
69. Afanasyev VN, Korlo BA, Matylevich NP, et al, The use of folw cytomtry for the investigation of cell death.[J] Cytometry, 1993, 14(6);603-609.
70. Tyagi S, Kramer FRet al. Molecular beacons: probes than fluoresce upon hybridization. [J] Nat. Biotechnol. 1996, 14: 303-308.
71. Silhavy D, Monar A, Lucioloi A, et al, A viral protein suppresses RNA silencing and binds silencing generated, 21 to 25 nucleotide double stranded RNAs. [J] EMBO Journal, 2002, 21(12): 3070-3080.
72. Yang D, Buchholz F, Huang Z, Goga A, Chen CY, Brodsky FM, and Bishop JM, Short RNA duplexes produced by hydrolysis with Eschefichia coli RNase Ⅲ mediate effective RNA interference in mammalian cells, [J] Proc Nail Acad Sci U S A: 2002, 99:9942-7
73. Hu W, Myers C, Kilzer J, Pfaff S, and Bushman F, Inhibition ofretroviral pathogenesis by RNA interference. [J] Curr, Biol, 2002; 12:1301-1306.
74. Caplen N J, Parrish S, Imani F, Fire A, et al. Specific inhibition of gene expression by small double stranded RNAs in invertebrate and vertebrate systems. [J] Proceeedings of the National Academy of Sciences of the United Stated of America, 2001, 98(17): 9742-9747.
75. Haasnoot PCJ, Cupac D, Berkhout B, Inhibition of virus replication by RNA interference, [J] Biomed, Sci 2003; 10: 607-616.
76. Vickers TA, Koo S, Frank Bennett C, et al, Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense atents. [J] Bilo Chem, 2003, 278(9): 7108-7118.
77. Shuey D J, McCallus DE, Giordano T, RNAi: gene-silencing in therapeutic intervention. [J] Drugdiscovery Today, 2002, 7(20): 1040-1046.
78. Hernann MT, Fridman JS, Zifou JT, et al, An epi-allelic sseries of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo. [J] Nat Genetics, 2003, 33(3): 396-400.
79. Jiang M, Milner J, Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNAi interference, [J] Oncogene, 2002, 21 (39): 6041-6048.
80. Lu PY, Xie FY, Woodle MC, et al. siRNA-mediated anti-tumorigenesis for drug target validation and therapeutics, [J] Curt Opin Mol Ther, 2003, 5(3): 225-234.