RNA干扰技术下调CEACAMl基因表达对胶质瘤血管生成的影响

目的：利用RNA干扰技术下调胶质瘤细胞中癌胚抗原相关细胞黏附分子1（ CEACAM1）基因的表达情况，探讨胶质瘤细胞CEACAM1表达变化后其培养上清液诱导人脐静脉内皮细胞（ HUVEC）增殖、血管生成的情况及其可能机制。方法设计并化学合成针对CEACAM1的3对siRNA，脂质体法瞬时转染SHG44细胞，收集细胞培养上清液作为条件培养基。分别采用RT－PCR检测RNA干扰后细胞中CEACAM1及血管内皮生长因子（ VEGF） mRNA表达的变化情况，ELISA法检测上清液中VEGF蛋白的含量。 MTT比色法及Transwell侵袭实验检测共培养刺激后HUVEC增殖及迁移能力的变化，体外血管生成实验检测体外血管生成能力。结果与正常对照组和阴性对照组相比，转染CEACAM1－siRNA后胶质瘤细胞中CEACAM1 mRNA的表达水平下调（P＜0．01），以CEACAM1－siRNA3组最为显著。 RNA干扰后SHG44细胞VEGF mRNA及上清液中的VEGF蛋白含量均减少，HUVEC的增殖受到抑制，迁移及体外血管生成能力下降。结论 CEACAM1－siRNA能够下调SHG44细胞中CEACAM1 mRNA的表达，并通过减少VEGF分泌，从而影响HUVEC的增殖、迁移和体外血管生成能力。

Effect of CEACAM1 gene RNAi on angiogenesis of glioma in vitro

Objective To investigate the effect of supernatant from carcinoembryonic antigen -related cell adhesion molecule 1（CEACAM1）gene RNAi glioma SHG44 cells on human umbilical vein endothelial cell proliferation and angiogenesis in vitro .Methods Three pairs of specific siRNA targeting CEACAM 1 were de-signed and synthesized , and then transiently transfected into SHG 44 cells via cathodolyte liposome transfection method.The supernatant from cultured glioma SHG 44 cells was collected as the conditional medium 48 h after transfection.RT-PCR was used to detect CEACAM1 and VEGF expression at mRNA level after CEACAM1 gene RNAi.The expression of VEGF in supernatant was measured by ELISA .The proliferation of HUVEC was assessed by MTT method after co-cultured with supernatant .The migration of HUVEC was examined by using a Transwell assay.The ability of angiogenesis was measured by capillary -like network formation assay .Results Forty -eight hours after the 3 pairs of specific CEACAM1 siRNA were transfected into SHG44 cells,RT-PCR results showed that the expression of CEACAM 1 mRNA was significantly inhibited compared with those in the normal control and the negative control groups （P〈0.01）.The most significant interference effect was CEACAM 1-siR-NA3.Expression of VEGF mRNA was remarkably reduced ,and expression of VEGF protein in the supernatant was also reduced after transfection of CEACAM 1-siRNA for 48 h.The proliferation of HUVEC was inhibited , HUVEC migration and tube capillary -like formation were decreased .Conclusion CEACAM1-siRNA can ef-fectively suppress the expression of CEACAM 1 mRNA in human glioma SHG44 cells,may inhibit HUVEC prolif-eration,migration and tube capillary -like formation via down -regulated the secretion of VEGF in vitro