siRNA沉默VEGF基因对大肠癌细胞生物学特性的影响

目的研究VEGF和受体FLT-1、FLK-1在大肠癌组织中的表达及与临床病理因素之间的关系;观察SiRNA干扰VEGF基因对人大肠癌细胞系Caco-2生物学特性的影响。方法应用免疫组织化学S?P法,检测82例大肠癌及14例大肠正常黏膜组织中VEGF和FLT-1、FLK-1的表达;以脂质体lip-2000为载体,用针对VEGF特异靶点的siRNA转染入人大肠癌细胞系Caco-2后,免疫细胞化学S-P法、Western Blot检测VEGF蛋白表达变化,MTT法检测对细胞增殖的影响;流式细胞技术检测细胞凋亡。结果 (1)大肠癌组织VEGF和受体FLT-1、FLK-1阳性表达率显著高于正常大肠组织(P0.05)。VEGF的表达在有无淋巴结转移组和不同的Duke’s分期中亦有显著差异(P0.05)。(2)VEGF-siRNA转染Caco-2细胞后,免疫细胞化学结果显示转染VEGF-siRNA组阳性细胞呈弱阳性表达,明显减弱;Westernblot结果显示VEGF-siRNA组细胞的蛋白条带亮度明显降低;MTT检测VEGF-siRNA组细胞生长出现明显的抑制(P0.05),不同时段(24h、48h、72h)肿瘤细胞增殖抑制率之间有显著差异(P0.05);流式细胞术检测VEGF-siRNA组出现明显亚二倍体峰,凋亡率显著高于正常对照组(P0.01)。结论 VEGF在大肠癌的进展过程中起重要作用。siRNA干扰VEGF基因能有效抑制人大肠癌细胞系VEGF蛋白表达,细胞增殖能力减弱,促进细胞凋亡。以VEGF基因为靶点,利用siRNA技术进行基因治疗有可能成为一种新的有效手段。

INFLUENCE OF siRNA-MEDIATED SILENCING VEGF GENE ON BIOLOGICAL CHARACTERISTICS OF CACO-2 CELLS

Objective To investigate the expression and clinical significance of vascular endothelial growth factor（VEGF） and its receptor FLT-1 and FLK-1 in colorectal carcinoma, and the effect of blocking VEGF expression with a specific small interfering RNA （siRNA） on the characteristics of human colorectal carcinoma cell line Caco-2. Methods The expression of VEGF and FLT-1,FLK-1 was detected by immunohistochemical S-P method in 82 cases of colorectal carcinoma and 14 cases of normal colorectal tissue. To knockdown VEGF expression, a small interfering RNA targeting VEGF was synthesized and transfected into Caco-2 cell with lipofectamine 2000. The expression of VEGF protein was detected by immunocytochemical staining （S-P method） and Western blot analysis. The effect of suppressing proliferation of cells was detected by MTT assay.The effect of inducing cell apoptosis was detected by flow cytometry（FCM）. Results （1） The expression of VEGF and FLT-1,FLK-1 was significantly higher in tumor tissue than in normal tissue（P0.05）.The expression of VEGF was positively correlated with lymph node metastasis and clinical stages（P0.05）.（2） Immunocytochemistry showed in the VEGF-siRNA group that cytoplasm exhibited weakly positive expression.Western blot indicated that VEGF-siRNA cell protein band was significantly lower. MTT test showed that VEGF-siRNA cell growth inhibition was significant（P0.05）.Tumor cell proliferation rates were significantly different at the different times （24h, 48h, 72h） （P0.05）.Flow cytometry showed that the VEGF-siRNA group had an apparent hypodiploid peak （P0.01）.Conclusion VEGF plays an important role in the progress of colorectal carcinoma. Blocking VEGF expression with RNAi technology can significantly reduce VEGF protein level and suppress proliferation of human colorectal cancer Caco-2 cells and induce apoptosis to a certain degree.The application of RNA interference technology targeting VEGF may become a new effective gene therapy for colorectal cancer.