中文摘要：

为了得到高纯度眼镜蛇毒细胞毒素-4N，探索眼镜蛇毒中细胞毒素-4N（cytototxin-4N，CTX-4N）对大鼠肝星状细胞（hepatic stellate cells，HSC-T6）的增殖抑制作用。本研究采用DEAE-Sepharose CL-6B阴离子交换柱、SpehadexG-50凝胶层析柱、Macro-prepHighS阳离子交换柱结合的方法对眼镜蛇毒蛋白进行分离纯化。在每一步分离纯化过程中，采用CCK-8法检测各蛋白峰组分对HSC-T6细胞的增殖抑制作用活性，收集增殖抑制作用最强的CTX-4N峰。经SDS-PAGE电泳鉴定蛋白纯度，Nano-LC-ESI．MS／MS质谱方法鉴定其组分，Cell Counting Kit-8（CCK-8）试剂检测CTX-4N对肝星状细胞（HSC-T6）的增殖抑制作用，从而确定其药理作用。经DEAE-Sepharose CL-6B阴离子交换柱、SpehadexG-50凝胶层析柱、Macro-prepHighS阳离子交换柱分离纯化后得到一个电泳纯度的蛋白，蛋白质谱鉴定为CTX-4N，分子量约为9．605kD。不同浓度的CTX-4N作用HSC-T6细胞24h后，随着其浓度的增大对HSC-T6细胞增殖抑制作用越强，呈剂量-效应关系，IC殉为（12．836±0．045）μg／mL。因此，本研究建立一种眼镜蛇毒细胞毒素一4N的分离纯化方法，并确定了其对HSC-T6细胞的增殖抑制的药理作用随浓度的增加而增强，为进一步研究其药理作用提供一定的理论依据。

英文摘要：

In order to obtain the high purity of cobra venom cytotoxin-4N （CTX-4N） and to explore the anti-proliferative effect of cobra venom CTX-4N on rat hepatic stellate cells （HSC-T6）, the DEAE-Sepharose CL-6B anion exchange column, SpehadexG-50 gel chromatography column, Macro-prep High S cation exchange column were used for the separation and purification of protein in cobra venom. During each step of separation and purification, CCK-8 method was used to detect the activity of anti-proliferative effect of each protein peak on HSC-T6 cells, and the peak CTX-4N in strongest anti-proliferative effect was collected. SDS-PAGE was used to identify the protein purity, and the components were identified by Nano-LC-ESI-MS / MS mass spectrometry. And then Cell Counting Kit-8 （CCK-8） reagent was used to detect the anti-proliferative effect of CTX-4N on the hepatic stellate cells （HSC-T6） to determine their pharmacological effects. After separating and purifying byDEAE-Sepharose CL-6B anion exchange column, SpehadexG-50 gel filtration column, Macro-prep High S cation exchange column, we obtained the protein which was purified to electrophoretic purity. This protein was identified as CTX-4N via the Nano-LC-ESI-MS/MS Mass spectrometry method and its molecular weight was about 9.605 kD. HSC-T6 cells were treated with different concentrations of CTX-4N for 24 hours, and then we found that the anti-proliferative effect on HSC-T6 cells was stronger with the increase of concentration, showing a dose-effect relationship, and the IC50 was （12.836Э0.045） μg/mL. Therefore, this study established a method for the separation and purification of cobra venom Cytotoxin-4N, and confirmed that its pharmacological anti-proliferative effects on HSC-T6 cells was enhanced with the increase of its concentrations. It also provided some theory for further studying its pharmacological effect.