HSP70-肿瘤肽复合物的纯化及其对肝癌细胞株HepG-2增殖的作用

目的:应用快速蛋白液相色谱(FPLC)系统从肝癌组织中分离和纯4EHSP70-肽复合物,并研究其对肝癌细胞系HepG-2增殖的影响．方法:将组织进行匀浆、高速离心提取总蛋白后依次进行ConA-Sepharose亲和层析和 DEAE-Sephacel子交换层析分离纯化,所得蛋白经SDS-聚丙烯酰胺凝胶电泳和Western blot进行蛋白分子质量及性质鉴定,Bradford 法测定蛋白浓度;利用MTT方法检测HSP70- 肽复合物对HepG-2细胞增长的情况．结果:分离、纯化得到的蛋白经SDS-聚丙烯酰胺凝胶电泳、考马斯量蓝鉴定为单一带, 分子质量为70 kDa;Western blot结果证实为 HSP70,每10 g组织最终获得1．5 mg的HSP70; HSP70刺激组12,48,72 h与对照组的A值之间有显著差异(0．1 mg／L:t=-0．2500,P=0．00; t=-0．1777,P=0．001:t=-0．3094,P=0．001; 0．5 mg／L:t=-0．2878,P=0．00;t=-0．2044,P= 0．00;t=-0．3285,P=0．00;1 mg／L:t=-0．3118, P=0．00;t=-0．2592,P=0．00;t=-0．1994, P=0．025;5 mg／L:t=-0．4007,P=0．00;t= -0．1302,P=0．016;t=-0．2537,P=0．005),细胞存活率明显高于对照组(P<0．01)．结论:使用本分离纯化方法可获得高纯度 HSP70肽复合物;HSP70肽复合物可以促进 HepG-2细胞的生长．

Purification of heat shock protein 70 peptide complex and its effect on proliferation of HepG-2 cells

AIM: To separate and purify heat shock protein 70 (HSP70) peptide complex from hepatocellular carcinoma (HCC) tissues by fast protein liquid chromatography (FPLC), and investigate its effect on the proliferation of HepG-2 cells. METHODS: The mixture of proteins was derived from HCC tissues by means of splitting and centrifugation. Then the proteins were purified by affinity chromatography on concanavalin A-Sepharose and ion exchange chromatography with DEAE-Sephacel.The obtained protein was identified by SDS-PAGE and Western blot for its molecular weight and property. Bradford meth od was used to measure the concentration of the protein. The growth of HepG-2 cells, which were stimulated by HSP-70 peptide complex (0.1, 0.5, 1, and 5 mg/L), was observed by MTT assay. RESULTS: A protein band with a molecular weight of about 70 kDa was obtained and shown by SDS-PAGE, and it was confirmed to be the HSP70 by Western blot. Bradford method showed that a quantity of 1.5 mg HSP70 protein was obtained from every 10 g HCC tissues. After treatment with HSP-70 peptide complex, the value of optical density (OD) in HSP70 group was significantly higher at 24, 48, and 72 h than that in control group (0.1 mg/L: t = - 0.2500, P = 0.00; t = -0.1777, P = 0.001; t=-0.3094, P = 0.001; 0.5 mg/L: t=-0.2878, P=0.00;t=-0.2044,P= 0.00; t = -0.3285, P = 0.00; 1 mg/L: t = -0.3118, P = 0.00; t = -0.2592, P = 0.00; t = -0.1994, P = 0.025; 5 mg/L: t = -0.4007, P = 0.00; t = -0.1302, P = 0.016; t = -0.2537, P = 0.005), and the cell livability in HSP70 group was also significantly higher (P< 0.01). CONCLUSION: The pure HSP70-peptide complex is obtained, and it can promote the growth of HepG-2 cells.