眼镜蛇毒神经生长因子抑制肝星状细胞增殖并诱导其凋亡

目的:从眼镜蛇毒中分离纯化神经生长因子(Nerve Growth Factor,NGF),观察眼镜NGF对肝星状细胞HSC-T6增殖、凋亡活性的影响,进一步为蛇毒NGF在抗肝纤维化治疗提供依据。方法:采用shephadex G-75和CM Sepharose CL-6B二步柱色谱对眼镜蛇毒NGF进行纯化分离;PC12细胞测定各洗脱峰的活性,再用SDS-PAGE鉴定具有NGF活性洗脱峰的纯度和相对分子质量。实验设立空白对照和NGF处理组,分别作用于HSC-T6,培育相应时间,MTT检测眼镜蛇毒NGF对HSC-T6细胞活力影响;HE染色、紫外激光显微镜与透射电镜观察HSC-T6细胞的形态学变化;TUNEL、流式细胞技术检测眼镜蛇毒NGF对HSC-T6细胞凋亡的影响。结果:眼镜蛇毒经PC-12细胞鉴定第Ⅵ峰具有NGF活性;SDS-PAGE检测为电泳纯,相对分子质量为22.3KD;NGF对HSC-T6细胞增殖具有明显抑制作用(2μg/ml NGF的抑制率为49.66%±6.50%,P<0.05;6.25μg/ml NGF的抑制率为71.33%±1.53%,P<0.05);TUNEL法检测发现NGF干预组的凋亡率28.71%±1.59%(2ug/ml NGF)和34.4%±2.49%(5μg/mlNGF)明显高于对照组的15.85%±1.58%(P<0.05);流式细胞仪也有同样的发现,NGF干预组的凋亡率16.12%±3.02%(2 ug/mlNGF)和21.15%±3.31%(5μg/ml NGF)明显高于对照组的2.7%±1.55%(P<0.05)。结论:眼镜蛇毒NGF能抑制肝星状细胞HSC-T6增殖并诱导其凋亡。

Cobra Venom NGF Inhibits Proliferation and Induces Apoptosis of Hepatic Stellate Cell

Objective: Cobra venom NGF canbe isolate and purfity with a rapid method,and observe the effect of the proliferation and apoptosis after NGF add to the HSC-T6 cells.This provide the basis for the treatment of anti-liver fibrosis in further.Methods: NGF is separated from guangxi cobra venom through shephadex G-75 and CM Sepharose CL-6B column chromatography;PC12 cell assay the activity of the NGF elution peak,then identified NGF purity and relative molecular mass with SDS-PAGE;we establishment groups of control and NGF respectively,acting on the HSC-T6,nurturing the corresponding time points,MTT test HSC-T6 cells viability effected on cobra venom NGF;hematoxylin and eosin stain、PALM MicroBeam and transmission electron microscope Morphology observed the changes in HSC-T6 cells;Tunel、and flow cytometry detected HSC-T6 cell apoptosis after adding the cobra venom NGF;Results: The Cobra venom elution peak Ⅵ identified by PC-12 cells having NGF active;SDS-PAGE electrophoresis show the elution peak Ⅵ pure and the relatived molecular mass is 22.3KD;HSC-T6 cell proliferation is inhibited evidently(2 μg/mlNGF inhibition ratio is 49.66% ±6.50%;6.25 μg/mlNGF inhibition ratio is 71.33% ±1.53%,P<0.05);TUNEL shows apoptosis ratio of HSC-T6 is 28.71%±1.59%(2 μg/ml NGF) and 34.4% ±2.49 %(5 μg/mlNGF) that is obviously higer than control 15.85% ±1.58%,P<0.05;It is also founded in flow cytometry,apoptosis ratio of HSC-T6 is 16.12% ±3.02%(2 μg/ml NGF) and 21.15%±3.31%(5?g/mlNGF) that is obviously higer than control 2.7% ±1.55%,P<0.05.Conclusion: The cobra venom NGF inhibits HSC-T6 proliferation and induces apoptosis.