SHH信号通路对肝癌细胞Hep3B中血管内皮生长因子表达的影响

The influence on VEGF expression by sonic hedgehog signaling pathway in hepatocellular carcinoma cell Hep3B

目的研究sonic hedgehog(SHH)信号通路对肝癌Hep3B细胞中血管内皮生长因子(VEGF)表达的调控。方法对Hep3B肝癌细胞设计VEGF基因的特异性引物,将扩增的基因片段克隆到T载体PMD19-T上,构建标准品质粒并建立标准曲线,建立实时荧光定量PCR(RT-PCR)的检测方法。后将Hep3B肝癌细胞分为对照组和研究组处理,对照组为空白对照,研究组采用特异性抑制剂cyclopamine阻断肝癌细胞中的SHH通路,均采用实时荧光定量PCR方法及免疫印迹试验两种方法测定VEGF的表达量。结果 RT-PCR显示,SHH通路特异性抑制剂cyclopamine可明显下调肝癌Hep3B细胞中VEGF mRNA表达(研究组与对照组分别为10.728±0.186、18.506±0.395,P<0.05),同时免疫印迹试验检测结果提示VEGF蛋白表达也明显降低(研究组与对照组分别为0.84±0.09、0.33±0.07,P<0.05)。结论 cyclosporine可抑制肝癌Hep3B中VEGF的表达,VEGF可能为SHH通路调控的下游基因之一。

Objective To investigate the effect and mechanism of sonic hedgehog（SHH）signaling pathway on the expression of vascular endothelial growth factor（VEGF）gene in hepatocellular carcinoma cell Hep3 B.Methods The special primers of targeted VEGF gene were designed.The aimed fragment of VEGF gene was amplified with PCR,then inserted into PMD19-T vector to obtain stander vector of VEGF gene.Using this vector to establish a stander curve and the method of fluorescent quantitative polymerase chain reaction（FQ-PCR）for detecting VEGF gene was established.Blocking the SHH signaling pathway with cyclopamine which was special blocking agent of this pathway in the study group.The expression of VEGF was detected by FQ-PCR and Western blot.Results RT-PCR results showed,cyclopamine could reduced the expression of VEGF mRNA in Hep3Bcell（the study group and control group were 10.728±0.186,18.506±0.395 respectively,P〈0.05）.Meanwhile Western blot RT-PCR results also showed that cyclopamine could reduced the expression of VEGF（the study group and control group were 0.84±0.09,0.33±0.07 respectively,P〈0.05.Conclusion The expression of VEGF could be inhibited by cyclopamine,It is indicated that VEGF gene is targeted gene of SHH signaling pathway.