抑制EphA7基因对肝癌细胞SMMC-7721裸鼠移植瘤血管生成的影响

Effects of silencing EphA7 gene on angiogenesis of SMMC-7721 cell xenograft in nude mice

目的 采用RNA干扰（RNAi）技术观察EphA7基因抑制对肝癌细胞SMMC-7721裸鼠移植瘤组织中血管生成的影响.方法 EphA7基因特异性小干扰RNA （siRNA）重组质粒采用脂质体介导法转染肝癌细胞SMMC-7721.裸鼠移植瘤模型采用左上肢皮下注射的方法建立,根据注射细胞的不同分为转染组、空载体转染组和未转染组.35 d后获取移植瘤组织,采用实时荧光定量聚合酶链反应（Real-time PCR）检测各组移植瘤组织中EphA7 mRNA的表达,免疫组织化学和蛋白印迹（Western blot）法检测各组裸鼠移植瘤组织中血管内皮生长因子（VEGF）和血管生成素-2（Ang-2）的蛋白表达并计算微血管密度（MVD）.结果 Real-time PCR结果显示,与空载体转染组（0.390 0±0.086 3）和未转染组（0.421 9±0.0595）比较,转染组裸鼠移植瘤中EphA7 mRNA （0.191 1±0.020 4）的表达明显受到抑制,差异有统计学意义（P＜0.05）.免疫组织化学和Western blot结果显示,转染组裸鼠移植瘤组织中VEGF和Ang-2阳性染色的细胞明显较少且染色较浅,其蛋白的表达（0.360 9±0.075 2、0.334 7±0.088 2）明显低于空载体转染组（0.490 8±0.084 4、0.510 8±0.1048）和未转染组（0.555 9±0.075 4、0.5640±0.178 7）,差异有统计学意义（P＜0.05）.微血管密度计算显示,转染组MVD平均值为42.88±12.80,明显低于空载体转染组（60.63±12.13）和未转染组（64.38±10.51）,差异有统计学意义（P＜0.05）.结论 抑制EphA7基因能下调肝癌细胞SMMC-7721裸鼠移植瘤中VEGF和Ang-2的表达,EphA7基因可能通过调节VEGF和Ang-2的表达实现对肝癌细胞血管生成的调控.

Objective To investigate the effects of RNA interference targeting EphA7 gene on angiogenesis of SMMC-7721 cell xenograft in nude mice.Methods Recombinant plasmid of EphA7 gene-targeting small interfering RNA （siRNA） was transfected into SMMC-7721 cells by LipofectamineTM 2000.The nude mice tumor model was established by subcutaneous injection of hepatic cancer cells in the left upper limb of the mice.The nude nice were then divided into transfected group,empty vector transfected group and untransfected group according to the different cells injected.The xenograft tumor tissues were obtained 35 days later.Real-time PCR assay was used to detect the expression of EphA7 mRNA in tumor tissues in different groups.Immunohistochemistry and Western blot assay were used to detect the expression of vascular endothelial growth factor （VEGF） and Angiopoietin-2 （Ang-2） protein in tumor tissues of different groups and the microvessel density （MVD） was calculated also.Results Real-time PCR results showed that compared with empty vector transfected group （0.390 0 ± 0.086 3） and untransfected group （0.421 9 ± 0.059 5）,the expression of EphA7 mRNA in transfected group （0.191 1 ±0.020 4） was remarkably suppressed.Immunohistochemistry showed that VEGF and Ang-2 protein positive staining cells in tumor tissues of transfected group were less and lighter staining.Western blot assay showed that compared with empty vector transfected group （0.490 8 ± 0.084 4,0.510 8 ± 0.104 8） and untransfected group （0.555 9 ±0.075 4,0.564 0 ±0.178 7）,the expression VEGF and Ang-2 protein in transfected group （0.360 9 ± 0.075 2,0.334 7 ± 0.088 2） were remarkably suppressed （P ＜ 0.05）.Microvessel density calculation shows that the average value of MVD of the transfected group （42.88 ± 12.80） was significantly lower than that of empty vector transfected group （60.63 ± 12.13） and untransfected group （64.38 ±10.51） （P＜0.05）.Conclusion The inhibition of EphA7 gene can down regulate the expression of VEGF and Ang-2 in SMMC-7721 hepatoma cell line.EphA7 may regulate angiogenesis of hepatocellular carcinoma through a regulation of the expression of VEGF and Ang-2.