热休克蛋白70的纯化及其抗小鼠肝癌作用的研究

Purification of hsp70 and its immunoprotective effect against mouse hepatoma

目的 建立热休克蛋白 70 (hsp70 )的分离方法 ,观察其在小鼠体内的特异抗瘤效应。方法 用经 43℃热处理 12h的H2 2细胞 ,制备裂解液 ,用DEAE5 2、ConA和ADP柱进行序贯分离 ,所得蛋白经电泳及Westernblot进行蛋白分子量及性质鉴定。用纯化的hsp70蛋白免疫A组C3H及C组C5 7BL/ 6小鼠 ,RPMI 16 40液免疫B组C3H及D组C5 7BL/ 6小鼠。第 15天A、B组小鼠用H2 2攻击 ,C、D组小鼠用EL 4攻击 ,观察各组小鼠肿瘤发生率及肿瘤大小。结果 纯化蛋白经鉴定确系相对分子质量约为 70 0 0 0的hsp70蛋白。A组小鼠肿瘤发生率为 0 / 6 ,而B组小鼠肿瘤发生率为 6 / 6。C组和D组小鼠的肿瘤发生率均为 6 / 6 ,两组小鼠第 35天的肿瘤大小差异无显著性。结论 用上述蛋白层析法可分离获得纯度高、并保留肿瘤抗原信息的hsp70蛋白。该蛋白免疫后的小鼠能抵抗同种肿瘤细胞的攻击 ,而对异种肿瘤细胞的抵抗能力有限。提示纯化蛋白中的抗原信息可能不是hsp70蛋白本身 ,而是其所携带的小分子多肽。

Objective To purify hsp70 protein and observe its anti tumor effect on mouse H22 hepatoma.Methods Cytosolic hsp70 protein of heat treated H22 cells was purified successively by chromatographic procedures, including DEAE 52 cellulose, ConA sepharose 4B and ADP argarose chromatography. The molecular weight and the identity of the purified hsp70 protein were confirmed by SDS PAGE and Western blot. Tumorigenicity of H22 hepatoma was examined in purified hsp70 treated C3H mice.Results A sharp stained protein band with a molecular weight of about 70 kD was obtained and shown to be hsp70 as confirmed by Western blot. In C3H mice challenged with H22 hepatoma, tumor developed in 6 of 6 mice while in hsp70 pretreated mice, none of the 6 H22 challenged mice developed tumor. Nevertheless, C57BL/6 mice pretreated with hsp70 or RPMI 1640 all developed tumor upon challenge with EL 4 lymphoma.Conclusion The results suggest that the immunoprotective effect of hsp70 protein is due not to hsp70 per se, but rather to the specific antigenic peptides it carries. Subject