摘要
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
To construct the tranfected cell line expressing the human OX40L gene and to study the costimulation of the gene signals to T cells,the total RNA was isolated from mature dendritic cells (DC) with TRIzol,and the OX40L gene was amplified by RT-PCR,digested with restriction endonuclease Pst I and BamH I, and inserted into retrovirus vector pEGZ-Term. The recombinant vector together with its two helper virus vectors was cotransfected into the package cell 293T with LipfectAMINE 2000. Then the supernatant of the 293T cell culture was used to infect L929 cells,and after 72 hours cultivation,the L929 cell clone stably expressing the OX40L molecule were screened in the presence of Zeocin (500 μg/ml). The costimulation of OX40L signal to T cells in vitro was studied by means of 3 H-TdR incorporation assay,cell apoptosis and cell cycle analysis. It was found that the full-length of OX40L gene was successfully cloned,and the recombinant retrovirus vector carrying the OX40L gene was constructed. The screened L929 cell clone could stably express the human OX40L gene on the cell membrane. The transfected cells could promote the costimulation of T cells in vitro. Meanwhile,there existed a synergic effect between OX40/OX40L and CD28/B7 signals.
出处
《现代免疫学》
CAS
CSCD
北大核心
2004年第2期99-103,共5页
Current Immunology
基金
国家"973"基金资助项目 ( 2 0 0 1CB5 10 0 3 )
江苏省医学重点实验室资助项目
