抗VEGF发夹状核酶基因抑制VEGF表达及卵巢癌细胞增殖的实验研究

Experimental Study of Anti-VEGF Hairpin Ribozyme Gene Inhibiting Expression of VEGF and Proliferation of Ovarian Cancer Cells

背景与目的:研究表明血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)在与上皮性卵巢癌有关的血管形成过程中可能发挥着重要作用。目前,核酶策略已成为一种基因治疗的策略,用于阻断肿瘤形成或肿瘤的生长与转移。VEGF在卵巢肿瘤形成中的作用尚不十分清楚。我们试图采用抗VEGF发夹状核酶基因阻断卵巢癌中VEGF的自分泌和/或旁分泌通路,以检测其对卵巢癌细胞增殖的影响。方法:采用脂质体介导的方法将自行设计和构建的抗VEGF发夹状核酶基因真核表达载体转染人卵巢癌SKOV3细胞,采用G418筛选获得阳性克隆;RNA斑点杂交检测核酶基因在卵巢癌细胞中的表达;免疫组化、间接免疫荧光染色及流式细胞仪结合免疫荧光检测转染前后卵巢癌细胞中VEGF表达;通过MTT、细胞贴壁克隆形成实验、软琼脂克隆形成实验及细胞周期分析观察其对卵巢癌细胞增殖的影响。结果:与SKOV3细胞相比,转染后的SKOV3-RZ细胞VEGF表达明显降低(P<0.01);细胞生长减慢,细胞贴壁克隆形成及软琼脂克隆形成率明显降低犤(12.7±1.4)%和(9.4±2.0)%比(30.1±1.9)%和(24.8±1.5)%,P<0.001犦;G1期细胞显著增多(77.6%比57.9%,P<0.01),S期细胞显著减少(17.0%比29.9%,P<0.01)。结论:抗VEGF发夹状核酶基因可显著抑制卵巢癌细胞SKOV3增殖;

Background & Objective: Growth of solid tumor and tumor metastases are critically dependent on angiogenesis. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, has been identified as one of the most potent inducers of tumor associated angiogenesis. studies have shown that VEGF plays an important role in angiogenesis which is associated with epithelial ovarian cancer. Until now, many strategies for gene therapy have been developed. Among them is Ribozyme-based therapeutics for cancer which might be devised to inhibit tumor growth or prevent metastases. Angiogenesis is required for sustained tumor growth, making the VEGF pathway another promising target for either small molecule or nucleic acid-based therapeutics. Little is known about the role of VEGF in ovarian tumorigenecity. We propose to block the autocrine and /or paracrine pathway of VEGF in ovarian cancer using anti-VEGF hairpin ribozyme gene to see whether the growth of tumor cells could be inhibited and to further exploit its mechanisms. Methods: Anti VEGF hairpin ribozyme gene eukaryotic expression vector was introduced into ovarian cancer SKOV3 cells by lipofectin mediation and positive clones were screened by G418;Ribozyme expression was confirmed by RNA dot blot; The VEGF expression of SKOV3 cells before or after transfection were detected by immunohistochemical and immunofluorescence and flow cytometer immunofluorescene methods, MTT, colony forming , soft agar colony forming, and FCM were used to observe the effect of proliferation to ovarian cancer cells. Results: VEGF expression decreased distinctly in SKOV3 RZ cells. The growth of uransfected SKOV3 RZ cells were slower, The average colony forming efficiency and soft agar colony forming efficiency of SKOV3 RZ cells(12.7±1.4 and 9.4±2.0,respectively) decreased distinctly (P< 0.001). The SKOV3 RZ cells of G1 stage increased(P< 0.01), the SKOV3 RZ cells of S stage were reduced(P< 0.01). Conclusions: Anti VEGF hairpin ribozyme gene can inhibit the proliferation of ovarian cancer SKOV3 cells. This provides a experimental basis for cure human ovarian cancer with antiangiogenesis method.