Norrin基因对人视网膜微血管内皮细胞增殖和周期的影响

目的：体外培养并鉴定人视网膜微血管内皮细胞（HRCECs），并探讨Norrin基因高表达对HRCECs增殖和周期的影响。方法： 体外分离、培养并鉴定人视网膜微血管内皮细胞，抗第Ⅷ因子相关抗原抗体鉴定细胞。利用脂质体lipofectamine 2000将AP-3myc-hNorrin/pRK5质粒转染HRCECs，RT-PCR、免疫组化和Western blotting方法检测Norrin/myc的表达确定转染效率。采用MTT法测定转染后细胞的增殖能力，流式细胞术分析其细胞周期变化。结果： 所培养的细胞第Ⅷ因子相关抗原免疫组化为强阳性。AP-3myc-hNorrin/pRK5质粒转染后 24 h，RT-PCR显示实验组Norrin表达水平明显高于阴性对照组(P<0.01)。转染后48h免疫组化及Western blotting均显示实验组细胞Myc表达强于阴性对照组。MTT法显示细胞增殖高于对照组，细胞周期检测示实验组G2期细胞高于阴性对照组，P<0.01。结论：脂质体能成功转染AP-3myc-hNorrin/pRK5进入HRCECs。Norrin高表达能促进HRCECs的增殖，并促进细胞DNA合成，提示Norrin在视网膜血管生成过程中可能具有重要作用。

Effects of Norrin gene on the proliferation and cell cycle of human retinal capillary endothelial cells

AIM: To isolate,cultivate and identify human retinal capillary endothelial cells(HRCECs),and to assess the effects of high expression of Norrin gene on the proliferation and cell cycle of HRCECs.METHODS: The cultured cells were identified with anti-factor VIII related antigen.AP-3myc-hNorrin/pRK5 were transfected into cultured HRCECs in vitro by lipofectamine 2000.Their transfection efficiency were measured by RT-PCR,immunohistochemistry and Western blotting,respectively.Its effects on cell proliferation and cell cycle were detected.RESULTS: The cultured cells were identified with immunochemically positive brown staining.In comparison with those of the controls,the Norrin expression in experimental group was significantly increased on mRNA and protein levels(showed by the myc tag) after 48 h.The cell number of experimental group was larger than that in the control group with statistically significant differences.Flow cytometry showed the cells in G2 phase were mainly increased(P<0.01).CONCLUSION: The plasmid AP-3myc-hNorrin/pRK5 is successfully transfected into HRCECs by lipofectamine 2000.Norrin gene improves the proliferation ability of HRCECs by promoting the synthesis of DNA.Norrin may have an important role in the retinal angiogenesis,which may provide a new gene target in the treatment of retinal vascular disorders.