CAJ | 학술논문

目的构建携带SEA基因的选择性增殖腺病毒,体外实验观察SEA基因的表达及其刺激淋巴细胞对肿瘤的杀伤功能.方法构建一种由端粒酶(hTERT)和缺氧反应元件(HIF)双重启动的携带SEA的选择性增殖腺病毒,通过逆转录-聚合酶链反应(RT-PCR)检测SEA在肿瘤细胞内mRNA表达,免疫荧光定位SEA表达于肿瘤细胞,Western blot测定蛋白表达,显微镜下动态观测淋巴细胞与肿瘤细胞共培养.酶联免疫吸附试验(ELISA)检测白细胞介素(IL)-4分泌.结果琼脂糖电泳252 bp处可见清晰条带;免疫荧光及Western blot用考马斯亮蓝染色显示在27 kDa附近可见蛋白清晰表达;淋巴细胞与肿瘤细胞共培养12、24、48 h实验组肿瘤细胞明显少于对照组,ELISA检测IL-4分泌量实验组均高于对照组(t=2.585 P＜0.05).结论携带SEA基因的选择性腺病毒成功构建并表达目的基因,证明对肿瘤细胞的杀伤作用.
목적 구건휴대SEA기인적선택성증식선병독,체외실험관찰SEA기인적표체급기자격림파세포대종류적살상공능.방법 구건일충유단립매(hTERT)화결양반응원건(HIF)쌍중계동적휴대SEA적선택성증식선병독,통과역전록-취합매련반응(RT-PCR)검측SEA재종류세포내mRNA표체,면역형광정위SEA표체우종류세포,Western blot측정단백표체,현미경하동태관측림파세포여종류세포공배양.매련면역흡부시험(ELISA)검측백세포개소(IL)-4분비.결과 경지당전영252 bp처가견청석조대;면역형광급Western blot용고마사량람염색현시재27 kDa부근가견단백청석표체;림파세포여종류세포공배양12、24、48 h실험조종류세포명현소우대조조,ELISA검측IL-4분비량실험조균고우대조조(t=2.585 P＜0.05).결론 휴대SEA기인적선택성선병독성공구건병표체목적기인,증명대종류세포적살상작용.
Objective SEA gene construct carrying the selective adenovirus, in vitro experimental observation SEA gene expression and to stimulate anti-tumor function of lymphocytes. Methods Building a dual boot by telomerase, and HIF-carrying SEA selective adenovirus by real-time quantitative polymerase chain reaction (PCR) detection of SEA in the nucleic acid expression in tumor cells, immune fluorescence positioned SEA expressed in tumor cells, Western blotting determination of protein expression, observed under a microscope, dynamic co-culture of lymphocytes and tumor cells, enzyme linked immu-nosorbent assay (ELISA) detection of interleukin (IL)-4 secretion. Results SEA levels of success in the nucleic acid and protein expression, cell ELISA and flow cytometry proved to be CD3+ T lymphocytes significantly stimulated proliferation, lymphocytes and tumor cells co-cultured tumor cells 12, 24, 48 hours the experimental group significantly lower than the control group, ELISA detection of IL-4 secretion experi-mentsgroup were higher than that (t = 3.585 P < 0.05 ). Conclusion The choice of carrying SEA gene adenovirus gene was successfully constructed and expressed a preliminary verification of the tumor cells in vitro.