小鼠端粒酶蛋白亚单位转染树突状细胞体外诱导特异性抗肝癌细胞免疫应答

Induction of specific CTL elicited by dendritic cells transfected with recombinant adenovirus vector expressing the mouse telomerase reverse transcriptase in vitro

目的观察携带小鼠端粒酶蛋白亚单位（mTERT）基因重组腺病毒载体（AdmTERT）转染树突状细胞（DC）后诱发免疫效应细胞产生特异性抗肝癌细胞免疫应答的研究。方法用流式细胞仪分析及电镜观察培养6dDC的细胞表型及形态，Ad—mTERT重组腺病毒转染体外培养的小鼠DC，Western blot检测mTERT融合蛋白表达；用负载mTERT的DC刺激同型淋巴细胞，免疫磁珠分选CD8^＋T细胞做为效应细胞，小鼠肝癌细胞株（H22）及小鼠结肠癌细胞（CT26）作为靶细胞，用酶联免疫吸附试验（ELISA）及酶联免疫斑点法（ELISPOT）检测干扰素（IFN）-γ分泌量和释放抗原特异性IFN-γ的T细胞数，^51Cr释放法检测细胞毒性T淋巴细胞（CTL）对肝癌细胞的杀伤活性。结果细胞表型及形态观察证实小鼠骨髓来源的DC为成熟的树突状细胞；AdmTERT转染DC后能正确表达mTERT融合蛋白，用AdmTERT转染DC致敏的淋巴细胞IFN-γ分泌量（208．6μg／L）和分泌IFN—γ的特异性T细胞的数量（341／10^6脾细胞）都高于Ad—GFP转染的DC组（14．2μg/L，33／10^6脾细胞）和单纯DC组（12．1μg／L，19／10^6脾细胞，P〈0．05）。AdmTERT修饰DC刺激产生的效应T细胞在效靶比为90：1时，对H22细胞的杀伤率（54．2％）明显高于AdGFP致敏组（8．2％）和未致敏DC组（4．5％，P〈0．05），而对CT26细胞无明显杀伤作用。结论AdmTERT修饰的DC体外能够诱导出针对mTERT抗原特异性的CTL效应．可特异性杀伤mTERT阳性的肝癌细胞。

Objective To investigate the mouse telomerase reverse transcriptase （mTERT） specific CTL elicited by dendritic cells （DC） transfected with adenovirus vector expressing the mouse telomerase reverse transcriptase （AdmTERT） and its anti-tumor effect in vitro. Methods Flow cytometry and electron microscopy were used to observe DC phenotype and morphology. DC were transfeeted with AdmTERT. T cells were isolated from mouse spleen. After third stimulation, eytotoxicity of mTERT specific CTL was determined by using 51 Cr release assay. H22 and CT26 cells were used as stimulators, and CD8^ ＋ T cells as responders. IFN-γ/positive blots were developed and calculated following the kit＇ s instruction. IFN-γ ELISPOT assays for antigen specific IFN-γ production by T cells. Results Phenotype and morphous observe mouse bone marrow from DC was full-blown and express mTERT fusion protein. IFN-γ/secreted （208.6 μg/L） and IFN-γ secreting cells （341/106 spleen cell）elicited by the AdmTERT infected DCs were much stronger and higher than that by Ad-GFP group （14.2 μg/L,33/106 spleen cell）and DC group （12.1 μg/L, 19/106 spleen cell）. High cytotoxicity of mTERT specific CTL could be elicited by AdmTERT transfected DC groups （ 54.2% ） , in comparison, no specific lysis was observed for T cells coeultured with control groups. Conclusion DC transfeeted with adenovirus encoding mTERT can induce strong and functional mTERT antigen specific CTL and remarkable anti-tumor effect in vitro.