%0 Journal Article %T Drak2在高糖诱导胰岛β细胞凋亡中的作用 %A 王广宇 %A 吴国亭 %J 同济大学学报(医学版) %D 2016 %R 10.16118/j.1008-0392.2016.02.005 %X 目的 探讨Drak2在高糖诱导的胰岛β细胞凋亡中的作用。方法 不同浓度葡萄糖诱导刺激,模拟体外胰岛细胞糖毒性损伤后的细胞凋亡模型,通过CCK8检测葡萄糖作用后细胞的存活率；Western印迹法检测不同糖浓度时Drak2的表达情况,完成糖浓度的筛选。构建Drak2-pcDNA3.0质粒,应用基因转染技术构建Drak2过表达可诱导细胞系,Western印迹法分析糖诱导前后胰岛β细胞Drak2的表达。Real-Time PCR测定Drak2、Bax、Bcl-2的mRNA水平,流式细胞法测定诱导前后的凋亡情况。结果CCK8结果显示,随着培养液中糖浓度上升,Rinm 5mf凋亡逐渐增加,随着时间的增加(24、48、72h)凋亡也逐渐增加,呈一定的量效关系。Western印迹法显示在糖浓度22.2mmol/L培养72h时,Drak2的表达量最多。构建Drak2过表达,在转染6h后开始葡萄糖诱导,在高糖诱导72h时过表达组Drak2蛋白是正常组的6倍。Real-Time PCR显示,过表达组Drak 2 mRNA在葡萄糖诱导前开始增加,在高糖诱导72h时增高明显(P<0.05),凋亡相关基因BAX、Bcl-2表达均有增加(P<0.05)。流式细胞仪检测显示高糖诱导后,过表达组凋亡率明显高于正常组(P<0.05)。结论 Drak 2过表达促进高糖诱导的胰岛β细胞凋亡。</br>Objective To investigate the effect of Drak2 on apoptosis of pancreatic betacells induced by high glucose. Methods Murine Rin-m5f pancreatic β-cells were cultured in vitro and the cell apoptosis model was established by high glucose. Cell viability was detected by CCK8 method. The expression of Drak2 was analyzed by Western blotting. Drak2-pcDNA3.0 was transfected in Rin-m5f cells to establish stable Drak2-overexpressing cells. The expression of Drak2 induced by glucose in the both cell lines was confirmed by immunofluorescence staining. Drak2 mRNA, Bax mRNA, Bcl-2 mRNA were quantitated by Real-Time PCR. The apoptosis was deterrmined by flow cytometry. Results The viability of Rin-m5f cells was significantly reduced in a dose-dependent manner after exposure to glucose at the range of 0-55.5 mmol/L for 24,8 and 72 h; the expression of Drak2 was the highest when cells were cultured with glucose at 22.2 mmol/L for 72h. With glucose, the expression of Drak2 in over-expression cells was six times of the control cells as measured by Western blotting. Drak2 mRNA was increased before induction, which was highly significant after 72 h in over-expression group(P<0.05). From flow cytometric assay, the percent of apoptosis in over-expression group was highly significant, compared to the control group(P<0.05).Conclusion Drak2 overexpression results in increased β-cell apoptosis induced by glucose stimulation %K Drak2 葡萄糖 β细胞 凋亡< %K /br> %K Drak2 glucose β cell apoptosis %U http://tjyxxb.cnjournals.cn/ch/reader/view_abstract.aspx?file_no=20160205&flag=1