蛇毒神经生长因子对大鼠肝星状细胞增殖、凋亡及肝纤维化相关蛋白质表达的影响

目的探讨眼镜蛇毒神经生长因子(NGF)对大鼠肝星状细胞(HSC-T6)增殖、凋亡以及蛋白质表达差异的影响,进一步为蛇毒NGF抗肝纤维化提供依据。方法实验分为对照组(单纯HSC-T6培养)和实验组(NGF进行干预)。应用MTT检测眼镜蛇毒NGF对HSC-T6细胞增殖抑制率;流式细胞技术检测眼镜蛇毒NGF对HSC-T6细胞凋亡的影响;双向电泳技术观察HSC-T6细胞蛋白质表达变化;质谱鉴定差异表达的蛋白。结果 MTT结果显示NGF对HSC-T6细胞增殖具有明显抑制作用(5 mg.L-1NGF时抑制率为48.2%±1.9%,P<0.01);流式细胞仪也有同样的发现,NGF(5 mg.L-1)干预组的凋亡率21.15%±3.31%明显高于对照组的2.7%±1.55%(P<0.05);比较分析空白对照组和NGF作用组的2-DE图谱,找到差异蛋白质点47个,其中与对照组相比在NGF作用组表达上调22个,下调25个;质谱鉴定出9个蛋白质,其中4个表达上调,5个表达下调。结论蛇毒NGF能抑制大鼠肝星状细胞(HSC-T6)的增殖,诱导其凋亡,并且影响HSC-T6细胞蛋白质的表达。

Effects of cobra venom NGF on cell proliferation, apoptosis and liver fibrosis protein expression in rat hepatic stellate cells

Aim To investigate the effects of cobra venom nerve growth factor(NGF)on cell proliferation,apoptosis and the differences of protein expression in rat hepatic stellate cell(HSC-T6),and to further provide the basis for the treatment of anti-liver fibrosis.Methods The experimental cells were divided into the control group(simple HSC-T6 culture) and the experimental group(NGF intervention);MTT was used to test the effect of cobra venom NGF on HSC-T6 cells viability;flow cytometry was adopted to detect HSC-T6 cell apoptosis after adding the cobra venom NGF;the protein expression changes of HSC-T6 cell were observed with two-dimensional electrophoresis;the differentially expressed proteins were identified by mass spectrometry.Results MTT assay showed that NGF significantly inhibited the proliferation of HSC-T6 cells(5 mg·L-1NGF inhibition ratio is 58.2%±1.9%,P<0.01).It was also found in flow cytometry that apoptotic ratio of HSC-T6 was 21.15%±3.31%(5 mg·L-1NGF),which was obviously higher than in control(2.7%±1.55%,P<0.05).After a comparative analysis of control group and NGF group,47 differential protein spots were found,among which 22 protein spots were up-regulated and 25 protein spots were down-regulated.9 proteins were identified by mass spectrometry,four of which were up-regulated and 5 were down-regulated.Conclusion Cobra venom NGF can inhibit cells proliferation,induce cell apoptosis,and affect the protein expression in rat hepatic stellate cell(HSC-T6).