OAZI-1蛋白复合物在小鼠体内诱导特异性抗肿瘤效应的研究

摘要

目的:在实验动物的水平上分析来源于肿瘤细胞的鸟氨酸脱羧酶抗酶抑制因子-1(Ornithine decarboxylase antizyme inhibitor-1,OAZI-1)蛋白复合物能否在小鼠体内诱导特异性抗肿瘤效应.方法:用包被有OAZI-1抗体的免疫磁珠从B16-F1小鼠黑色素瘤细胞中分离OAZI-1蛋白复合物,用此复合物免疫小鼠后,再在小鼠皮下接种B16-F1活细胞,然后观察接种瘤在小鼠体内的成瘤及生长状况.ELISA法用于检测免疫小鼠血清中IFN-γ含量.乳酸脱氢酶释放实验(LDH)检测免疫小鼠脾脏淋巴细胞对B16-F1细胞的杀伤效应.上述动物实验用原核表达纯化的OAZI-1蛋白和PBS免疫的小鼠作为对照.结果:与对照小鼠相比,接种OAZI-1蛋白复合物的小鼠脾淋巴细胞(效应细胞)对B16-F1黑素瘤细胞(靶细胞)具有更强的杀伤能力.在三种不同的效:靶比下(10:1、50:1、100:1),该组小鼠脾淋巴细胞对靶细胞的杀伤活性分别为46.2%、59.5%和92.5%,显著性高于接种纯化OAZI-1蛋白组(36.1%、26.8%和45.9%)和接种PBS组(24.6%、24.0%和27.2%)小鼠脾淋巴细胞.此外,接种OAZI-1蛋白复合物的小鼠血清中抗肿瘤细胞因子IFN-γ含量(538.3 pg/ml)也显著性高于接种纯化OAZI-1蛋白组(256.2 pg/ml)和接种PBS组(131.0 pg/ml)小鼠.上述方法免疫的小鼠在皮下再接种B16-F1活细胞后,免疫OAZI-1蛋白复合物组小鼠成瘤率为40%,而PBS组和纯化OAZI-1蛋白组小鼠成瘤率为100%,且接种瘤在OAZI-1蛋白复合物免疫小鼠体内生长更为缓慢.结论:从B16-F1肿瘤细胞中分离的OAZI-1蛋白复合物中可能含有肿瘤抗原,用此复合物接种小鼠能在实验动物体内诱导抗肿瘤免疫杀伤活性.%Objective:To analyze whether the OAZI-1 (ornithine decarboxylase antizyme inhibitor-1) protein complex isolated from tumor cells could induce specific antitumor effects in the experiment mice .Methods:OAZI-1 protein complexes were isolated from B16-F1 melanoma cells by immune magnetic beads coated with OAZI-1 antibody and used as the vaccine to immune the C 57BL/6 mice.After immunization,the mice were inoculated subcutaneously with live B 16-F1 cells and then tumor formation and growth were ob-served.ELISA was used to determine the level of cytokine IFN-γin the serum of immunized mice.Lactate dehydrogenase assay (LDH) was performed to evaluate killing effect of spleen lymphocytes on B 16-F1 cells.The mice immunized by purified OAZI-1 from prokaryotic expression and PBS were used as controls in the animal experiment .Results: Compared with the control mice ,the spleen lymphocytes ( effector cells ) from the mice inoculated with OAZI-1 protein complexes had stronger killing ability on B 16-F1 cells (target cells).At three different effector:target ratio (10:1,50:1,100:1),the killing ability of these spleen lymphocytes were 46.2%, 59.5%and 92.5% respectively,which was significantly higher than the spleen lymphocytes from the mice inoculated with purified AZIN-1 protein (36.1%,26.8% and 45.9%) or inoculated with PBS (24.6%,24.0% and 27.2%).In addition,the content of serum anti-tumor cytokine IFN-γwas also significantly higher in the mice inoculated with OAZI-1 protein complexes (538.3 pg/ml) than the mice inoculated with purified AZIN-1 ( 256.2 pg/ml ) or with PBS ( 131.0 pg/ml ) .When B16-F1 live cells were subcutaneously inoculated into the immunized mice described above ,the tumor formation rate was only 40%in the mice immunized with OAZI-1 protein complex ,but 100%in the mice immunized with PBS or purified OAZI-1.The growth of inoculated tumors in the mice immunized with OAZI-1 protein complex was also much slower than the control mice .Conclusion:The results in this study suggest that the OAZI-1 protein complex isolated from B 16-F1 tumor cells could contain some tumor antigens .When used as tumor vaccine to inoculate mice ,this complex can induce anti-tumor immune killing activity in experimental animals .