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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。
OBJECTIVE: To isolate and purify Nerve Growth Factor (NGF) from cobra venom and observe the effects of NGF on the proliferation and apoptosis of hepatic stellate cells HSC-T6, and to provide basis for anti-hepatic fibrosis treatment of snake venom NGF . METHODS: NAC from Naja naja is purified by two-step column chromatography with shephadex G-75 and CM Sepharose CL-6B. The activity of each peak was determined by PC12 cells. The purity of NGF- Relative molecular mass. Twenty-four hours after HSC-T6 treatment, HSC-T6 cells were treated with NGF and untreated HSC-T6 cells respectively. MTT assay was used to detect the effect of NGF on the viability of HSC-T6 cells. HE staining, UV- TUNEL, flow cytometry of cobra venom NGF on the apoptosis of HSC-T6 cells. Results: Cobra venom was identified by PC-12 cells in the first Ⅵ peak with NGF activity; SDS-PAGE detected electrophoresis pure, relative molecular mass of 22.3KD; NGF on HSC-T6 cell proliferation was significantly inhibited (2μg / ml NGF inhibition (P <0.05). The inhibition rate of 6.25μg / ml NGF was 71.33% ± 1.53%, P <0.05). The apoptosis rate of NGF intervention group was 28.71% ± 1.59% (2ug / ml NGF) and 34.4% ± 2.49% (5μg / ml NGF) were significantly higher than that of the control group (15.85% ± 1.58%, P <0.05). Flow cytometry also found that the apoptosis rate of NGF intervention group was 16.12% ± 3.02% (2 μg / ml NGF) and 21.15% ± 3.31% (5 μg / ml NGF) were significantly higher than those in the control group (2.7% ± 1.55%, P <0.05). Conclusion: Cobra venom NGF can inhibit hepatic stellate HSC-T6 proliferation and induce apoptosis.