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目的:研究眼镜蛇毒因子(CVF)耗竭体内补体对Thy-1肾炎大鼠肾小球系膜细胞(GMCs)凋亡病变及其生长阻滞和DNA损伤诱生蛋白45α(Gadd45α)基因表达的影响。方法:利用抗胸腺细胞抗血清(ATS)复制大鼠Thy-1肾炎(Thy-1 N)模型。大鼠随机分为正常对照组、Thy-1 N组和CVF+Thy-1 N组。取各组大鼠血清检测补体CH50活性。另取各组大鼠肾皮质,行电镜和TUNEL染色检查判断其肾组织细胞凋亡的病理变化;用RT-PCR和real-time PCR检测大鼠肾组织凋亡相关基因,即Gadd45α mRNA丰度;用Western blotting检测Gadd45α蛋白表达水平。结果:CVF+Thy-1 N组补体CH50活性明显下降,与Thy-1 N组和正常对照组相比差异明显(P<0.01)。电镜和TUNEL染色观察到Thy-1 N组大鼠GMCs有凋亡病变,而CVF+Thy-1 N组凋亡病变则显著减轻。Thy-1 N组大鼠肾皮质Gadd45α mRNA 40min开始上调,3h达到峰值,而用CVF处理的Thy-1 N大鼠,Gadd45α mRNA丰度则明显低于Thy-1N组(P<0.01);Thy-1 N组Gadd45α蛋白水平3h时显著增多,而CVF+Thy-1 N Gadd45α蛋白水平明显低于Thy-1 N组(P<0.01)。结论:CVF耗竭补体可显著减轻大鼠Thy-1肾炎凋亡病变并抑制Gadd45α基因的表达。
OBJECTIVE: To investigate the effects of complement depletion of cobra venom factor (CVF) on apoptosis, growth arrest and expression of Gadd45α gene in glomerular mesangial cells (GMCs) of rats with Thy-1 nephritis . Methods: Rat Thy-1 nephritis (Thy-1 N) model was replicated by antithymocyte antiserum (ATS). Rats were randomly divided into normal control group, Thy-1 N group and CVF + Thy-1 N group. Take each group of rat serum detection of complement CH50 activity. In addition, renal cortex of each group was examined by electron microscopy and TUNEL staining to determine the pathological changes of renal cell apoptosis. RT-PCR and real-time PCR were used to detect the expression of Gadd45α mRNA The protein expression of Gadd45α was detected by Western blotting. Results: The CH50 activity of complement in CVF + Thy-1 N group was significantly lower than that in Thy-1 N group and normal control group (P <0.01). The apoptosis of GMCs in Thy-1 N group was observed by electron microscopy and TUNEL staining, while the apoptosis in CVF + Thy-1 N group was significantly reduced. The expression of Gadd45α mRNA in Thy-1 N group increased at 40 min and peaked at 3 h, while the mRNA expression of Gadd45α in Thy-1 N group was significantly lower than that in Thy-1 N group (P <0.01) The protein level of Gadd45α in -1N group was significantly increased at 3h, while the protein level of CVF + Thy-1N Gadd45α was significantly lower than that in Thy-1N group (P <0.01). Conclusion: CVF depletion of complement can significantly reduce the apoptosis of Thy-1 nephritis rats and inhibit the expression of Gadd45α gene.