去除供者骨髓中的T淋巴细胞可有效防止移植物抗宿主病(GVHD)的发生.用含大肠杆菌胞嘧啶脱氨酶(EC-CD)基因的高滴度逆转录病毒(1.5×105 CFU/ml)感染小鼠T淋巴细胞,G418筛选,获得稳定表达CD基因的T/ pCD2细胞.经PCR和RT-PCR方法检测证明CD基因已成功地导入T淋巴细胞中并有效表达.分别用不同浓度的5-FCyt作用于T/ pCD2及T淋巴细胞,光镜下观察不同时间细胞数目变化及MTT法检测细胞活性.结果表明,5-FCyt浓度大于1 μmol/L时,即对T/ pCD2细胞有显著的杀伤作用,而对正常T淋巴细胞基本无毒性,且T/ pCD2细胞在加入药物后生存时间(3~5 d)明显短于未转染的T淋巴细胞(大于14 d).
T-lymphocyte depletion of the marrow graft can prevent graft versus-host disease(GVHD) effectively. T-lymphocytes of mice were transfected with high-titer (1.5×105 CFU/ml) retrovirus supernatant, and by selection with G418 positive cell clones (T/pCD2) were obtained. PCR and RT-PCR showed that CD gene was transferred into the T-lymphocyte and expressed successfully. T-lymphocyte and T/pCD2 cells were exposed to different doses of 5-FCyt for various hours,and were then observed under light microscope and the viability of these cells was determined by MTT colorimetric cell proliferation assay. The results showed that retrovirus-mediated CD gene transferred to T/ pCD2 cells could confer them high sensitivity to 5-FCyt, and T/ pCD2 cells exposed to 5-FCyt (>1 μmol/L) can be killed. But 5-FCyt was not harmful to normal T-lymphocyte cells. The survival time of T/ pCD2 cells in the presence of 5-FCyt(3~5 d) was significantly shorter than that of T-lymphocyte(>14 d).
宋艳斌,伍志坚,尹芳,马文丽,杨光彩,伍柏松,徐钤. CD自杀基因系统对T淋巴细胞作用的实验研究[J].生物化学与生物物理进展,2000,27(5):524-528
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