摘要
目的观察1,25二羟基维生素D3[1,25(OH)2D3]对高糖诱导的人肾小球系膜细胞株增殖能力、周期分布影响,并探讨其机制。方法取对数生长期人肾小球系膜细胞并分为6组,干预组加入30 mmol/L的D-葡萄糖和10^-8 mol/L的1,25(OH)2D3,1,25(OH)2D3组加入10^-8 mol/L的1,25(OH)2D3,高糖组加入30 mmol/L的D-葡萄糖,高渗组加入25 mmol/L的甘露醇,LY294002组加入2μg/mL的PI3K特异性抑制剂(LY294002),正常对照组未加任何刺激物,各组培育48 h。采用CCK-8法检测细胞450 nm波长处吸光度值(A450),并计算细胞增殖抑制率;采用流式细胞术检测细胞周期分布,并计算系膜细胞增殖指数(Mc增殖指数);采用Western blotting法检测细胞Akt、p-Akt、mTOR、p-mTOR蛋白,并计算p-Akt/Akt、p-mTOR/mTOR蛋白相对表达量比值。结果与正常对照组比较,1,25(OH)2D3组、LY294002组细胞A450值降低,高糖组细胞A450值升高(P均<0.05);与高糖组比较,干预组细胞A450值降低(P均<0.05)。与正常对照组比较,1,25(OH)2D3组和LY294002组S期、G2/M期细胞数及高糖组G1期细胞数增多,1,25(OH)2D3组和LY294002组G1期细胞数及高糖组S期、G2/M期细胞数减少(P均<0.05);与高糖组比较,干预组G1期细胞数减少,而S期、G2/M期细胞数增多(P均<0.05)。与正常对照组比较,1,25(OH)2D3组和LY294002组p-Akt/Akt、p-mTOR/mTOR蛋白相对表达量比值降低,高糖组p-Akt/Akt、p-mTOR/mTOR蛋白相对表达量比值升高(P均<0.05);与高糖组比较,干预组p-Akt/Akt、p-mTOR/mTOR蛋白相对表达量比值降低(P均<0.05)。结论1,25(OH)2D3可抑制高糖诱导的人肾小球系膜细胞的增殖能力,并将细胞阻滞于G1/S期,作用机制可能与其下调PI3K/Akt/mTOR通路有关。
Objective To investigate the effects of 1,25(OH)2D3 on the proliferation and cell cycle distribution of human mesangial cells in the high glucose environment and its mechanism.Methods Human mesangial cells at logarithmic growth stage were divided into six groups,including the blank control group,high glucose group(treated with 30 mmol/LD-glucose),hypertonic group(treated with 25 mmol/L mannitol),1,25(OH)2D3 group[treated with 10^-8 mol/L 1,25(OH)2D3],1,25(OH)2D3 intervention group[treated with 30 mmol/L D-glucose+10^-8 mol/L 1,25(OH)2D3],and LY294002 group(treated with 2μg/mLLY294002),and then they were cultured at 37℃saturated humidity and 5%carbon dioxide for 48 hours.The A450 values were measured by CCK-8 assay as well as the proliferation inhibitory rates;the cell cycle distribution was measured by the flow cytometry,and the mesangial cell proliferation index(Mc proliferation index)was calculated;Western blotting was used to detect cellular Akt,p-Akt,mTOR,and p-mTOR proteins,and the relative expression ratios of p-Akt/Akt and p-mTOR/mTOR protein.Results Compared with the normal control group,the A450 value of cells in the 1,25(OH)2D3 group and LY294002 group decreased,and the A450 value of cells in the high glucose group increased(P<0.05).Compared with the high glucose group,the A450 value of cells in the intervention group decreased(P<0.05).Compared with the normal control group,the number of cells in the S phase and G2/M phase increased in the 1,25(OH)2D3 group and LY294002 group.The number of cells in the G1 phase increased in the high glucose group.The number of cells in the G1 phase decreased in 1,25(OH)2D3 group and LY294002 group.The number of cells in the S phase and G2/M phase decreased in the high glucose group(P<0.05).Compared with the high glucose group,the number of cells in the G1 phase decreased,while the number of cells in the S phase and G2/M phase increased in the intervention group(P<0.05).Compared with the normal control group,the relative expression ratio of p-Akt/Akt,p-mTOR/mTOR prote
作者
王丽媛
唐小铁
戚婷
于洋
崔学彬
徐洁
WANG Liyuan;TANG Xiaotie;QI Ting;YU Yang;CUI Xuebing;XU Jie(School of Medicine,Wuhan University of Science and Technology,Wuhan 430000,China)
出处
《山东医药》
CAS
2020年第24期22-26,共5页
Shandong Medical Journal
基金
湖北省卫生和计划生育委员会联合基金立项面上项目(WJ2018H0107)。
