应用要点：通过构建人反义血管内皮生长因子基因真核表达载体，采用基因转染的方法将反义血管内皮生长因子载体转染入肾癌786-0细胞中，用G418进行筛选含反义血管内皮生长因子基因真核表达载体的细胞克隆并扩大培养，建立了稳定表达反义血管内皮生长因子的肾癌786-0细胞系。

同行评价：本文的创新之处在于将siRNA技术用于肾癌的研究，同时密切结合了临床需要，具有一定的实用性。

相关链接：反义基因治疗方式有反义寡核苷酸，反义表达载体包括质粒真核表达载体和腺病毒表达载体等。质粒真核表达载体则不存在上述问题，且更容易制备和大量的纯化，重复应用不产生DNA抗体，相比来说更富有优越性。

摘要
目的： 构建反义血管内皮生长因子基因表达载体，观察其对肾癌细胞血管内皮生长因子表达的影响。
方法：实验于2006-07/2007-03在郑州大学第三附属医院实验中心完成。①实验材料：质粒 pcDNA3.1(－)，宿主菌DH5α，肾癌细胞株786-0。②实验过程：克隆人血管内皮生长因子基因，将反义血管内皮生长因子基因定向克隆于质粒pcDNA3.1(－)表达载体，酶切鉴定；转染人肾癌细胞，并分别命名为反义血管内皮生长因子组和空载体组，未转染细胞命名为对照组；G418筛选阳性克隆。③实验评估：反转录-聚合酶链反应方法检测血管内皮生长因子mRNA的表达，免疫组织化学法检测血管内皮生长因子基因的蛋白表达，流式细胞术检测细胞周期，四甲基偶氮唑盐法检测细胞生长情况。
结果：①成功构建反义血管内皮生长因子基因表达载体。②反义血管内皮生长因子组血管内皮生长因子mRNA的表达受到抑制，明显低于空载体组和对照组血管内皮生长因子mRNA的表达(P < 0.01),空载体组血管内皮生长因子mRNA的表达没有受到影响。③反义血管内皮生长因子组的血管内皮生长因子蛋白表达明显降低，明显低于空载体组和对照组(P < 0.01)，空载体组和对照组血管内皮生长因子蛋白的表达差异无显著性。④反义血管内皮生长因子组细胞生长减慢，G1期细胞比例增加，S期细胞比例减少,反义血管内皮生长因子组细胞生长明显减慢。
结论：成功构建血管内皮生长因子的pcDNA3.1(－)反义基因表达载体，人反义血管内皮生长因子基因可明显降低肾癌细胞血管内皮生长因子基因在转录和翻译水平的表达，抑制肾癌细胞生长，为肾癌基因治疗提供一定的实验依据。
关键词：血管内皮生长因子类；肾肿瘤；基因表达

潘周辉，杨太森.人反义血管内皮生长因子基因载体构建及对肾细胞癌血管内皮生长因子表达的影响[J].中国组织工程研究与临床康复，2008，12(2):262-265 [www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-262(ps).pdf]

Construction of antisense vascular endothelial growth factor gene vector and its effect on the expression of vascular endothelial growth factor gene in human renal cell carcinoma

Abstract

AIM：Studies have shown that vascular endothelial growth factor (VEGF) is highly expressed in renal carcinoma. In this study, eukaryotic expression vector carrying human antisense VEGF gene was constructed to observe its effect on VEGF expression and growth of renal cell carcinoma.
METHODS: The experiment was conducted in the Experimental Center of Third Affiliated Hospital of Zhengzhou University from July 2006 to March 2007. ①VEGF gene was cloned. Antisense VEGF gene was inserted into eukaryotic expression vector pcDNA3.1(-), then identified using restrict enzyme. The vector was transfected into renal cell carcinoma and name by antisense VEGF group and empty vector group; non-transfected cells served as controls. The positive clone was selected by using G418. ②VEGF mRNA and protein expressions were detected using RT-PCR and immunohistochemical method. The growth of cell was measured by MTT and cell cycle by flow cytometry.
RESULTS: ①Antisense VEGF gene eukaryotic expression vector was successfully constructed. ②Compared with empty vector group and control group, the amount of VEGF mRNA expression was significantly decreased (P < 0.01), and the expression in empty vector group did not change a lot. ③The VEGF protein expression was remarkably decreased compared with empty vector group and control group (P < 0.01), but there was no significant difference between empty vector group and control group. ④The percent of G1 phase was increased and the percent of S phase decreased in antisense VEGF group, and the cell growth slowed down.
CONCLUSION: pcDNA3.1(-) antisense gene expression vector is successfully constructed. Antisense VEGF gene can significantly decrease the VEGF mRNA and protein expression in renal cell carcinoma and inhibit the growth of renal carcinoma. It provides experimental evidence for the gene therapy of renal carcinoma.

Pan ZH, Yang TS.Construction of antisense vascular endothelial growth factor gene vector and its effect on the expression of vascular endothelial growth factor gene in human renal cell carcinoma.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(2):262-265(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-2/2k-262(ps).pdf]