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为了构建稳定表达人OX4 0L基因的L92 9细胞株 ,初步研究OX4 0L信号通路对T细胞的共刺激效应。TRIzol一步法抽提人成熟DC总RNA ,RT PCR扩增出OX4 0L基因 ,双酶切装入逆转录病毒载体pEGZ Term ,与辅助病毒载体脂质体法共转染包装细胞 2 93T ,用其培养上清感染L92 9细胞 72h后 ,经Zeocin筛选出稳定表达OX4 0L分子的L92 9细胞株 ;采用3 H TdR掺入实验、细胞凋亡、细胞周期等方法研究OX4 0L信号对T细胞体外培养的共刺激作用。结果显示 ,成功地构建了含OX4 0L基因的重组逆转录病毒载体 ;经转染包装细胞 2 93T后 ,筛选获得能稳定高表达人OX4 0L蛋白的L92 9转基因细胞 ;OX4 0L转基因细胞对体外培养的T细胞具有促增殖、活化和抗凋亡的作用 ;同时 ,OX4 0 /OX4 0L信号与CD2 8/B7信号具有协同作用
In order to construct L92 9 cell line stably expressing human OX4 OL gene, the co-stimulatory effect of OX4 0L signaling pathway on T cells was preliminary studied. TRIzol one-step extraction of human mature DC total RNA, RT-PCR amplification of OX4 0L gene, double digestion into the retroviral vector pEGZ Term, co-transfected with helper virus vector liposome packaging cells 2 93T, with its The supernatant was infected with L92 9 cells for 72h, and the L929 cells stably expressing OX4 OL were screened by Zeocin. The T cells were cultured in vitro by 3 H TdR incorporation, apoptosis and cell cycle Costimulatory effect. The results showed that recombinant retroviral vector containing OX4 0L gene was successfully constructed. After transfection of packaging cells with 293T, L92 9 transgenic cells stably overexpressing human OX4 OL protein were screened out. OX4 0L transgenic cells were cultured in vitro Of T cells have the role of promoting proliferation, activation and anti-apoptosis; at the same time, OX4 0 / OX4 0L signal has a synergistic effect with CD2 8 / B7 signal