shRNA抑制survivin基因表达对脐静脉血管内皮细胞增殖与凋亡的影响

目的：研究靶向存活素（survivin）的短发夹 RNA真核表达质粒（shRNA）对脐静脉血管内皮细胞增殖与凋亡的影响。方法合成靶向survivin的shRNA真核表达质粒及阴性对照质粒，用脂质体法将质粒转染至经 VEGF（50 ng／mL）处理的HUVEC细胞；转染48 h后，采用实时定量聚合酶链反应（PCR）和蛋白印迹法检测 HUVEC中survivin的mRNA表达及蛋白水平。四甲基偶氮唑蓝（MTT）法检测细胞增殖；TUNEL 法检测细胞凋亡。结果（1）survivin‐shRNA 质粒试验组与阴性对照shRNA质粒组及空白对照组比较，转染48 h后人脐静脉血管内皮细胞survivin的mRNA及蛋白表达水平明显下降（P＜0．05）。（2）与阴性对照shRNA组及空白对照组比较，转染后的人脐静脉血管内皮细胞增殖能力明显下降。转染后24、48、72 h生长抑制率分别为（13．53±3．91）％、（38．97±1．82）％、（65．75±1．83）％，于转染后72 h最为显著。（3）试验组凋亡率为（28．07±1．71）％，较阴性对照组（11．45±1．52）％和空白对照组（10．04±1．46）％显著增高（P＜0．05）。结论靶向survivin的shRNA质粒能通过下调survivin表达，进而抑制人脐静脉血管内皮细胞增殖并促进其凋亡。

Effects of shRNA-mediated survivin silencing on proliferation and apoptosis of human umbilical vein endothelial cells

Objective To investigate the effect of short hairpin RNA(shRNA) eukaryotic expression vector‐mediated silen‐cing of the survivin‐gene on proliferation and apoptosis of human umbilical vein endothelial cells(HUVEC) .Methods The shRNA vector targeting the survivin gene and negative control vector were transfected into human umbilical vein endothelial cells(HUVEC) incubated with 50 ng/mL of recombinant VEGF in vitro by lipofectamine 2000 .Transfection after 48 h ,the expression of survivin mRNA and protein was detected by quantitative real‐time PCR and Western blot ,respectively .HUVEC proliferation was assayed by four methylthiazolyl tetrazolium(MTT) and cell apoptosis was detected by TUNEL .Results (1)Transfection with survivin‐shR‐NA vector significantly down‐regulated the expression of survivin mRNA and protein as compared with the control group ,after transfection of 48 h(P<0 .05) .(2)After survivin‐shRNA vector transfected ,the proliferation of HUVEC decreased significantly . After transfection 24 ,48 ,72 h ,the growth inhibition rate were (13 .53 ± 3 .91)% ,(38 .97 ± 1 .82)% ,(65 .75 ± 1 .83)% respective‐ly ,at 72 hours after transfection was the most significant .(3)The apoptosis rate of experimental group was (28 .07 ± 1 .71)% , which was higher than the negative control group (11 .45 ± 1 .52)% and blank control group (10 .04 ± 1 .46)% (P<0 .05) .Conclu‐sion The shRNA‐mediated mediated silencing of the survivin‐gene could significantly inhibit proliferation and promote the apopto‐sis of rheumatoid arthritis synovial fibroblasts by regulating survivin expression .