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-1 NGF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 rnviability; 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-1 NGF inhibition ratio is 58.2% ± 1.9% ,P <0.01 ).It was also found in flow cytometry that apop- totic ratio of HSC-T6 was 21.15% ± 3.31% ( 5 mg· L-1 NGF ), which was obviously higher than in control ( 2.7% ± 1.55% , P < 0.05 ).After a comparative a-nalysis 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 spectrome-try, four of which were up-regulated and 5 were down-rnregulated.Conclusion Cobra venom NGF can inhibit cells proliferation, induce cell apoptosis, and affect the protein expression in rat hepatic stellate cell( HSC- T6).
展开▼
