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

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

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。

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＆lt;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＆lt;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）.