腺病毒介导的白介素-24转移对脂多糖诱导的大鼠肾小球系膜细胞凋亡和周期调节蛋白p21、p27及CyclinE的影响

目的 探讨腺病毒介导的IL-24转移对脂多糖（LPS）诱导的大鼠肾小球系膜细胞（GMCs）和细胞周期调节蛋白p21、p27及CyclinE的影响。方法 采用10%FBS/DMEM培养293细胞，扩增腺病毒载体， GMCs培养4~6代细胞后用于分析。①观察IL-24对LPS诱导的GMCs凋亡影响：分为对照组、Ad.IL-24组、LPS组和LPS+ Ad.IL-24组，其中对照组和LPS组GMCs不转染携带IL-24腺病毒（Ad.IL-24），Ad.IL-24组和LPS+Ad.IL-24组以Ad.IL-24转染GMCs，LPS组和LPS+Ad.IL-24组加LPS培养，以流式细胞术检测培养后24和48 h的GMCs凋亡率；②考察IL-24对GMCs周期蛋白影响：对照组为5%FBS/DMEM培养GMCs，Ad-GFP组采用携带绿色荧光蛋白的对照载体（Ad.GFP）转染GMCs，Ad.IL-24+LPS组以Ad.IL-24转染GMCs,采用Western-blot检测p21、p27及CyclinE表达。结果 ①GMCs在培养后24和48 h细胞凋亡率：对照组分别为(0.86±0.15)%和(0.98±0.4)%；IL-24组分别为(1.02±0.22)%和(1.43±0.31)%；LPS组分别为(2.19±0.81)%和(2.49±0.12)%，LPS+Ad.IL-24组分别为(18.01±1.17)%、(26.82±5.01)%；2个观察时间点细胞凋亡率对照组与IL-24组差异无统计学意义， LPS组显著高于对照组（P＜0.05），LPS+ Ad.IL-24组细胞凋亡率最高（P＜0.05）。②Ad-GFP组p21、p27表达显著低于对照组（P<0.05），CyclinE表达显著高于对照组（P<0.05）；Ad.IL-24+LPS组p21、p27表达较Ad-GFP组显著上调（P<0.05），CyclinE表达较Ad-GFP组下调。结论 IL-24腺病毒载体对正常GMCs无影响，但可使LPS诱导增生的GMCs凋亡增加，其可能机制是上调关键细胞周期负调控蛋白p21、p27及下调CyclinE。

Effects of adenovirus mediated IL-24 gene transfer on apoptosis and cell cycle regulatory protein p21,p27 and CyclinE of rat gomerular mesangial cells induced by lipopolysaccharide

Objective To explore the effect of interleukin-24(IL-24) gene transfer on glomerular mesangial cells(GMCs) apoptosis and to find out the effect of IL-24 on cell cycle regulatory protein p21,p27 and CyclinE of GMCs induced by LPS.Methods 293 cells were cultured in 10%FBS/DMEM and Ad.IL-24 and Ad.GFP were amplifycated in 293 cells. GMCs were analysed after 4 to 6 generations. ①They were divided into four groups: control group, Ad.IL-24 group, LPS group and LPS+Ad.IL-24 group.And control group and LPS group weren't infected with Ad.IL-24, Ad.IL-24 group and LPS+Ad.IL-24 group GMCs were infected with Ad.IL-24,then LPS+Ad.IL-24 group GMCs were cultured in 5%FBS/DMEM with LPS(10 mg·L-1). The apoptosis of the GMCs was examined by AnnexinV/FITC flow cytometry; ②The effect of IL-24 on cell cycle regulatory protein p21, p27 and CyclinE of GMCs induced by LPS were determined. They were divided into three groups:control group, Ad-GFP group and IL-24 group. Control group GMCs were cultured in 5%FBS/DMEM. Ad-GFP group GMCs were infected with Ad.GFP and then cultured in 5%FBS/DMEM with LPS(10 mg·L-1). GMCs were infected with Ad.IL-24. The expressions of cell cycle regulatory protein p21, p27 and cyclinE were examined by Western-blotting.Results The GMCs were cultured for 24 hours and 48 hours. The apoptosis rate was (0.86±0.15)% and (0.98±0.4)% in the control group, (1.02±0.22)% and (1.43±0.31)% in the Ad. IL-24 group, (2.19±0.81)% and (2.49±0.12)% in the LPS group, (18.01±1.17)% and (26.82±5.01)% in LPS + Ad.IL-24 group. There was no difference between control group and Ad. IL-24 group, and the apoptosis rate of LPS group was higher than control group（P＜0.05). The apoptosis rate of LPS+Ad.IL-24 group was the highest while there was no change in Ad. IL-24 group（P＜0.05). ②The expressions of p21 and p27 were down-regulated while CyclinE expression was up-regulated in GMC by LPS（P<0.05）. Adenovirus mediated IL-24 gene transfer could abolish the role of LPS on regulation of p21, p27 and CyclinE expression（P<0.05）.Conclusion Adenovirus mediated exogenous IL-24 gene transfer could effectively inhibit LPS-induced proliferation of glomerular mesangial cells but had no effect on normal GMCs, and it could abolish the role of LPS on regulation of p21, p27 and CyclinE expression then increased the apoptosis of LPS-induced proliferation of GMCs.