慢病毒介导的突变型胸苷激酶对T淋巴细胞杀伤作用的实验研究  

In vitro killing effect of mutant thymidine kinase mediated by lentiviral vector on T lymphocytes

作　　者：徐开林[1] 潘秀英[1] 杨宇娟[1] 鹿群先[1] 李振宇[1] 何徐彭[1] 

机构地区：[1]徐州医学院附属医院血液科,221002

出　　处：《中华血液学杂志》2005年第11期678-681,共4页Chinese Journal of Hematology

基　　金：国家自然科学基金资助项目(30170389);江苏省"135"医学重点人才项目;江苏省高校自然科学研究计划资助项目(02KJB320013)

摘　　要：目的观察慢病毒介导的突变型单纯疱疹病毒胸苷激酶(HSV-sr39tk)及野生型HSV-tk对T细胞的体外杀伤作用,比较HSV-sr39tk/更昔洛韦(GCV)、HSV-sr39tk/阿昔洛韦(ACV)、HSV-tk/GCV、HSV-tk/ACV对T细胞存活率的影响。方法采用改良的磷酸钙沉淀法将包装质粒、包膜蛋白质粒和含目的基因的转移质粒共转染293T包装细胞,收集的病毒上清感染T细胞后,分别与不同浓度梯度的前体药物GCV和ACV作用4d后,MTT法测定细胞存活率。结果共转染293T细胞后获得较高滴度的慢病毒(2×106IU/ml)。前体药物GCV和ACV浓度在0～10.0μmol/L时转染HSV-sr39tk细胞(39tk+T细胞)存活率下降比较明显,GCV组细胞存活率由(96.04±3.23)%下降为(36.76±4.38)%,ACV组细胞存活率由(97.31±4.61)%下降为(43.75±8.99)%,而GCV和ACV浓度大于10.0μmol/L时,39tk+T细胞存活率下降趋势则减缓。统计显示39tk+T细胞对GCV、ACV均有敏感性(P值均<0.05);转染HSV-tk T细胞(tk+T细胞)对GCV有敏感性(P<0.05),而对ACV不具有敏感性(P>0.05);同一浓度时39tk+T细胞+GCV与tk+T细胞+GCV两组间T细胞存活率差异具有统计学意义(P<0.05)。结论慢病毒载体可高效、稳定地感染T细胞,同时不影响细胞的增殖,与野生型HSV-tk基因比较,表达突变型HSV-sr39tk的T细胞对GCV具有更高的敏感性,而且对ACV也具有敏感性。Objective To explore the killing effect of the mutant herpes simplex virus thymidine kinase （HSV-sr39tk） and its wild-type （HSV-tk） mediated by lentiviral vector on T lymphoeytes in vitro and compare T cell survival rate after GCV or ACV treatment. Methods The three-plasmid lentiviral vector system including packaging plasmid △NRF, envelope plasmid VSV-G and vector plasmid （pTK151 ＋ HSV- sr39tk or pTK151 ＋ HSV-tk） were eotransfeeted into human embryonic kidney 293T cells using modified calcium phosphate precipitation methods. The packaged virus was harvested 72 h later. The survival of T cells expressing HSV-sr39tk or HSV-tk was measured by MTT assay after 4 day-culture against a gradient of GCV or ACV concentrations. Results The three plasmids were effectively cotransfected and a high titre of lentivirus was obtained （2 × 10^6IU/ml）. 39tk^＋ T cell survival rates declined promptly when the prodrug GCV/ACV concentrations increased from 0 μmol/L to 10 μmoL/L. The T cell survival rates in GCV group declined from （96.04±3.23）% to （36.76±4.38）% while in ACV group from （97.31±4.61）% to （43.75±8.99）%. However, when GCV/ACV coneentrations were more than 10 μmol/L, further decline of 39tk ^＋ T cell survival rates beeame unobvious. The growth rate of 39tk^＋ T cell exposed to GCV or ACV was obviously lower than that in untransfected T cells （ P 〈 0.05 ）. Tk^＋ T cells were sensitive to GCV （ P 〈 0.05 ）, but not to ACV （P 〉0, 05）. There was a significant difference in killing effects between 39tk^＋ T cell ＋ GCV group and tk^＋ T cell ＋ GCV group （ P 〈 0.05 ）. Conclusion The lentiviral vectors containing HSV-sr39tk gene could infect T lymphocytes effectively and stably without affecting the proliferation of the transduced cell. In contrast to HSV-tk gene, T cells infected HSV-sr39tk were more sensitive not only to GCV but also to ACV.

关 键 词：慢病毒 突变型胸苷激酶 T淋巴细胞 杀伤作用 病毒感染 基因治疗 

分 类 号：R55[医药卫生—血液循环系统疾病]