bcr-abl融合基因真核表达载体诱导小鼠特异性免疫应答

目的在小鼠体内外研究bcr-abl融合基因真核表达载体诱导的特异性免疫应答,探索肿瘤免疫治疗的新途径。方法构建表达bcr-abl融合基因cDNA片段的真核细胞质粒pVbcr-abl,将 pVbcr-abl质粒用纳米颗粒聚乙烯亚胺包裹后给BALB／c小鼠肌内注射,检测小鼠血清中bcr-abl特异性抗体水平。小鼠免疫后20 d皮下接种具有相同遗传背景的SP2／0／ber-abl细胞(H-2~d),观察小鼠生存情况、移植肿瘤的生长情况和肿瘤细胞浸润情况。利用免疫组织化学方法观察肿瘤组织中T淋巴细胞浸润情况;流式细胞术分析免疫小鼠脾脏T细胞亚群的变化;LDH释放法检测脾脏细胞毒性T淋巴细胞(CTL)活性。结果成功构建真核表达载体pVbcr-abl,bcr-abl融合基因cDNA片段在真核细胞中可得到高效表达。pVbcr-abl免疫BALB／c小鼠能激活机体免疫系统,诱导产生特异性抗体和特异性CTL活性,并形成特异性免疫保护力。免疫小鼠的移植肿瘤形成时间、表面出现破溃时间、生长速度明显降低,荷瘤生存时间明显延长。免疫小鼠移植肿瘤组织中有大量CD3~+T细胞浸润。免疫小鼠的脾细胞杀伤SP2／0／bcr-abl细胞的细胞毒活性明显升高。小鼠脾脏T细胞亚群发生改变,CIM~+／ CD8~+细胞比值升高到1．54±0．29。结论pVbcr-abl真核表达载体除能在小鼠体内诱导产生特异性抗体以外,还能诱导高水平特异性CTL活性,直接杀伤肿瘤细胞,抑制肿瘤细胞的生长速度。

Recombinant eukaryotic expression plasmid of bcr-abl gene fragment induces specific immune response in mice

OBJECTIVE: To study the specific immune response induced by a recombinant eukearyotic expression plasmid encoding bcr-abl fusion gene fragment so as to explore new immunotherapy in mouse. METHODS: A recombinant eukaryotic vector pVbcr-abl expression cDNA fragment of bcr-abl fusion gene was constructed and used to immunize BALB/c mice. Serum level of bcr-abl specific antibody was detected by enzyme-linked immunosorbent assay (ELISA). Twenty days later the immunized mice were subcutaneously inoculated SP2/0/bcr-abl cells. The survival time, tumor growth time and lymphocytic infiltration were observed. T cells infiltration into tumor tissue was analyzed by immunohistochemistry. Changes of T cell subset in the spleen of mice was analyzed by fluorescent-activated cell sorting (FACS) and the cytotoxicity T lymphocyte (CTL) activity in spleen by lactate dehydrogenase (LDH)-release assay. RESULTS: The eukaryotic expression vector pVbcr-abl was constructed successfully, and highly expressed the cDNA fragment of bcr-abl fusion gene. The BALB/c mice immunized with the vector could generate the specific antibody and CTL, resulting in a specific immunoprotection. There were dramatic differences in the tumor-forming time, tumor ulcer appearring time and tumor-growing speed between the immunized and the control groups. The mice had longer survival time in the immunized group than in the control group. There were a large amount of CD3(+) T cells infiltration in tumor tissue of the immunized mice. The spleen cells from the immunized mice had higher CTL activity with a alteration of T cell subset, the CD4(+)/CD8(+) ratio being 1.54 +/- 0.29, higher than that of control group (1.18 +/- 0.30). CONCLUSION: The recombinant eukaryotic expression plasmid pVbcr-abl can induce in vivo not only the generation of specific antibody, but also high level of specific CTL activity, resulting in killing the SP2/0/bcr-abl tumor cells directly and inhibiting the tumor growth.