CAJ | 학술논문

目的构建转染人口腔鳞状细胞癌细胞株4-1-BBL基因,探讨其体外诱导抗肿瘤免疫的能力.方法通过脂质体法将重组载体pEGFP-4-1-BBL转染人口腔鳞状细胞癌细胞Tca8113,经G-418筛选及有限稀释后获得稳定高表达克隆,分别用反转录聚合酶链反应(RT-PCR)和蛋白质印迹法检测转染细胞中人4-1-BBL mRNA和蛋白的表达.制备肿瘤细胞疫苗.用淋巴细胞分离液分离纯化人外周血T淋巴细胞,用抗CD-3单抗预处理T细胞后,与转染及未转染人4-1-BBL的Tca8113疫苗混合培养.用CCK-8比色法检测细胞毒性T淋巴细胞(cytotoxic T lymphocytes,CTL)的杀伤活性;锥虫蓝计数法检测T细胞增殖,酶联免疫吸附实验检测培养上清液中自细胞介素(IL)-2、干扰素γ分泌水平.结果转基因Tca8113细胞能够稳定高表达人4-1-BBL.与野生型的Tca8113细胞相比较,转染人4-1-BBL的Tca8113细胞PCR扩增产物的大小约271 bp,能够显著增强T细胞增殖,促进IL-2、干扰素γ分泌,并能有效地诱导CTL产生对Tca8113细胞的特异性杀伤作用.结论转染4-1-BBL基因既能增强野生型Tca8113细胞的免疫原性,也能诱导T细胞产生有效的抗肿瘤免疫应答.
목적 구건전염인구강린상세포암세포주4-1-BBL기인,탐토기체외유도항종류면역적능력.방법 통과지질체법장중조재체pEGFP-4-1-BBL전염인구강린상세포암세포Tca8113,경G-418사선급유한희석후획득은정고표체극륭,분별용반전록취합매련반응(RT-PCR)화단백질인적법검측전염세포중인4-1-BBL mRNA화단백적표체.제비종류세포역묘.용림파세포분리액분리순화인외주혈T림파세포,용항CD-3단항예처리T세포후,여전염급미전염인4-1-BBL적Tca8113역묘혼합배양.용CCK-8비색법검측세포독성T림파세포(cytotoxic T lymphocytes,CTL)적살상활성;추충람계수법검측T세포증식,매련면역흡부실험검측배양상청액중자세포개소(IL)-2、간우소γ분비수평.결과 전기인Tca8113세포능구은정고표체인4-1-BBL.여야생형적Tca8113세포상비교,전염인4-1-BBL적Tca8113세포PCR확증산물적대소약271 bp,능구현저증강T세포증식,촉진IL-2、간우소γ분비,병능유효지유도CTL산생대Tca8113세포적특이성살상작용.결론 전염4-1-BBL기인기능증강야생형Tca8113세포적면역원성,야능유도T세포산생유효적항종류면역응답.
Objective To examine the activation and cytotoxieity of human peripheral blood T lymphocyte induced in vitro by human 4-1-BB ligand(4-1-BBL) gene transfected into tumor Tca8113 cells.Methods The eukaryotic expression vector pEGFP-h4-1-BBL was transfected into human oral carcinoma cell line Tca8113 by Lipofectamine<'TM>2000. The transfected cells were then selected in medium containing G-418, cloned by limited dilution and named as Tca8113-4-1-BBL Human 4-1-BBL mRNA and protein expression of transfected cells was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting respectively. The tumor cell vaccines (TCV) were obtained by treatment with mitomycin (MMC). Human peripheral blood mononuclenr cells (PBMC) were prepared from lymphoprep, and then stimulated with anti-CD-3 mAb and incubated with non-transfected or transfected TCV-TcaSI13 cells,respectively. The proliferation of T cells was evaluated by trypan blue exclusion; the CCK-8 was used to detect the cytotoxic effect of T lymphecytes. Meanwhile, the secretion of intefferom-γ (IFN-γ) and interleukin(IL)-2 in culture supernatant was detected by enzyme-linked immunosorbnent assay (ELISA).Results The Tca8113 cells trasfected by pEGFP-h4-1-BBL could express human 4-I-BBL efficiently. As compared with wild type Tca8113 cells, the transfected Tca8113 cells could markedly promote proliferation,IL-2 and IFN-γ production and cytotoxic activity of lymphocytos. Condusions The transfection of human 4-1-BBL gene in Tca8113 cells is effctive in enhancing its immunogenicity and inducing antitumor immune response in vitro.