转4-1BBL基因小鼠肝癌细胞瘤苗体外诱导抗肿瘤免疫应答的研究

目的　研究转 4 1BBL基因小鼠肝癌细胞疫苗体外诱导淋巴细胞特异性杀伤活性及刺激同系小鼠脾细胞产生细胞因子 (IL 2、TNF α和GM CSF)的能力。方法　采用脂质体介导法将真核表达质粒 pCDNA3 1( ) m4 1BBL导入小鼠肝癌细胞Hepa1 6 ,经G4 18筛选后获得稳定高表达克隆 ,以丝裂霉素C(MMC)处理后 ,制成肿瘤细胞疫苗 (TCV) ,经体外与同系小鼠脾淋巴细胞共同培养后 ,测定淋巴细胞特异性杀伤活性及对脾细胞产生细胞因子 (IL 2、TNF α和GM CSF)的影响。结果转染 4 1BBL的Hepa1 6细胞能够高表达 4 1BBL蛋白 ,并且经MMC处理后制成的瘤苗在培养 4 8h仍能表达m4 1BBL。与野生型的Hepa1 6细胞相比 ,上述瘤苗能诱导淋巴细胞产生针对亲本的小鼠肝癌细胞Hepa1 6的特异性杀伤作用 (P <0 0 5 ) ,但是对于小鼠肝癌细胞H2 2及成纤维细胞NIH3T3无效 ;此瘤苗在体外能显著增强脾细胞分泌细胞因子IL 2、TNF α和GM CSF的能力。结论　转 4 1BBL基因小鼠肝癌细胞疫苗能诱导有效的抗肝癌免疫反应。

Antitumor immune responses induced by gene transfer of 4-1BBL into hepatocellular carcinoma Hepa1-6 in vitro

OBJECTIVE: To study the cytotoxic activity against tumor cells and cytokines production of spleen cells induced in vitro by murine 4-1BBL gene transfected Hepa1-6. METHODS: The eukaryotic expression vector pCDNA3.1(+)-m4-1BBL was transfected into murine hepatocellular carcinoma cell line Hepa1-6 by Liposomes. Then the transfected cells were selected in medium containing G418 (400 - 800 micro g/ml) and termed as Hepa1-6-m4-1BBL. The TCV-m4-1BBL was obtained by treating them with mitomycin (MMC). Cocultivation TCV with syngeneic murine spleen cells, then the lymphocytes were tested for cytotoxic activity against Hepa1-6-wt cells and the supernatants were harvested for detecting the cytokines (IL-2, TNF-alpha and GM-CSF). RESULTS: Hepa1-6-m4-1BBL cells expressed 4-1BBL protein with highest cell surface level. The 4-1BBL mRNA could still be detected in the cells when cultured 48 h after treated with MMC (80 mg/L). Comparing with TCV-Hepa1-6, the tumor cell vaccine derived from Hepa1-6-m4-1BBL (TCV-m4-1BBL) could induce a more efficient cytotoxic activity of syngeneic murine lymphocyte against its parental tumor cell Hepa1-6 (P < 0.05), but not against non-parental tumor cell H22 and NIH3T3. Higher levels of IL-2, TNF-alpha and GM-CSF were released by the splencytes after stimulated by TCV-m4-1BBL. CONCLUSIONS: These results suggest the expression of m4-1BBL by tumor cells is effective in inducing antitumor immune responses.