中文摘要：

目的对广西眼镜蛇毒中磷脂酶A2（PLA2）进行分离纯化,测定其对肝星状细胞HSC-T6的增殖抑制作用。方法采用Sephadex G-50凝胶层析柱、CM-Sepharose CL-6B离子交换柱、Macro-prep High S预装柱结合的方法分离广西眼镜蛇粗毒,经平板法测定各峰的PLA2活性;经SDS-PAGE电泳鉴定终产物纯度并测定分子量,NanoLC-ESI-MS/MS鉴定其组分;CCK-8法测定PLA2对肝星状细胞（HSC-T6）的增殖抑制作用,确定其凋亡的最小毒性浓度。结果 Sephadex G-50凝胶层析柱、CM-Sepharose CL-6B离子交换柱、Macro-prep High S预装柱层析法,得到第Ⅲ峰具PLA2活性,且达到电泳纯,经NanoLCESI-MS/MS鉴定其为PLA2,分子量约为14.06kD;PLA2在0~1μg/ml的浓度下对HSC-T6细胞具有一定的促增殖作用,2μg/ml时细胞数达到最大值,4~16μg/ml时对细胞生长有抑制作用,且随浓度增大细胞数降低。结论采用Sephadex G-50、CM-Sepharose CL-6B、Macro-prep High S预装柱结合的方法对广西眼镜蛇毒进行分离纯化,得到电泳纯且具PLA2活性的磷脂酶A2;广西眼镜蛇毒PLA2对肝星状细胞HSC-T6增殖有抑制作用,PLA2对HSC-T6细胞的最小毒性浓度为2μg/ml。

英文摘要：

Objective Purification of phospholipase A2 from Guangxi cobra venom and determination its proliferation inhibition of hepatic stellate cells（HSC-T6）. Methods Using Sephadex G-50 gel chromatography column,CM-Sepharose CL-6Bion exchange column,and Macro-prep High S pre-loaded column with the method of separation of guangxi cobra toxin,the plate method for determining the PLA2 activity of each peak;via the SDS-PAGE electrophoresis appraisal end product purity and the determination of molecular weight,the NanoLC-ESI-MS/MS to identify its components;the inhibition effect of PLA2 on proliferation of hepatic stellate cells（HSC-T6）was determined by CCK-8,and finding out the cause of its apoptosis minimal toxicity concentration. Results Sephadex G-50 gel chromatography column,CM-Sepharose CL-6Bion exchange column,and Macro-prep High S pre-loaded column,get the peak III were PLA2 activity and achieving electrophoresis purity,and identification by the NanoLCESI-MS/MS,the molecular weight is about 14.06kD;the PLA2 can facilitate the proliferation of HSC-T6 cell at the concentration of 0~1μg/ml.It can achieve the most cell at 2μg/ml,and there is inhibition effects on the cell growth when the concentration is 4~16μg/ml.In this scope,the cell amount can be reduced along with the increase of concentration. Conclusion Using Sephadex G-50 gel chromatography column,CM-Sepharose CL-6B ion exchange column,and Macro-prep High S pre-loaded column with the method of separation of guangxi cobra toxin,getting electrophoresis pure and PLA2 activity of phospholipase A2;the PLA2 of Guangxi cobra venom has inhibitory effect on the proliferation of hepatic stellate cells（HSC-T6）,and the minimum toxicity concentration of PLA2 to HSC-T6 cells was 2μg/ml.