HSV1-TK、HSV1-SR39TK及GCV、ACV对小鼠T淋巴细胞的杀伤作用研究  

The killing effects on mouse T lymphocytes mediated by HSV1-TK/HSV1-SR39TK-GCV/ACV system

作　　者：高飞[1] 徐开林[1] 潘秀英[1] 鹿群先[1] 杜冰[1] 

机构地区：[1]徐州医学院附属医院血液科

出　　处：《白血病．淋巴瘤》2007年第3期180-183,187,共5页Journal of Leukemia & Lymphoma

基　　金：国家自然科学基金(30170389);江苏省"135"医学重点人才项目(RC2002080);江苏省高校自然科学研究计划(02KJB320013)

摘　　要：目的观察慢病毒载体携带的单纯疱疹病毒1型胸苷激酶(HSV1-TK)和突变型单纯疱疹病毒1型胸苷激酶(HSV1-SR39TK)基因在小鼠T淋巴细胞的表达及前体药物丙氧鸟苷(GCV)、无环鸟苷(ACV)对小鼠T淋巴细胞的杀伤效应。方法用亚克隆技术将PCR扩增的HSV1-TK、HSV1-SR39TK基因分别连接至慢病毒载体,在TK、SR39TK基因后以内部核糖体进入位点(IRES)连接绿色荧光蛋白(GFP)基因,采用三质粒包装系统包装病毒,将病毒感染刀豆蛋白A激活的小鼠T淋巴细胞,然后通过CCK-8方法观察其对前体药物GCV、ACV的敏感性。结果慢病毒载体携带的HSV1-TK及HSV1-SR39TK基因均可在小鼠T淋巴细胞内高效表达,GCV和ACV对转导TK基因的淋巴细胞均有杀伤作用,含突变型TK基因的淋巴细胞对前体药物敏感性更高,后者对GCV的IC_(50)值比野生型小2.5倍,对ACV的IC_(50)值比野生型小17.4倍。结论表达HSV1-SR39TK基因的T细胞对GCV有更高的敏感性,对ACV也较敏感,应用HSV1-SR39TK基因进行防治GVHD的研究是更好的选择。Objective To investigate herpes simplex virus type 1 thymidine kinase (HSV1-TK) and mutant herpes simplex virus type 1 thymidine kinase (HSV1-SR39TK) genes delivered by lentiviral vectors expression in mouse T lymphocytes and the killing effects on mouse T lymphocytes mediated by HSV1-TK/ HSV1-SR39TK Ganciclovir/Acyclovir (GCV/ACV) system.Methods Subcloned the target genes amplified by PCR into lentiviral vectors then was followed by GFP reporter gene linked by EMCV IRES.Lentivirus were generated by transient three-plasmid transfection,The viral particles were used to infect the concanavalin A (Con A)stimulant T lymphocytes,the killing effects was estimated by cell counting Kit-8 (CCK-8),trypanblan methods.Results HSV1-TK and HSV1-SR39TK genes can both express effectively in mouse T lympho- cytes,TK+and SR39TK+T lymphocytes both can be killed by GCV and ACV,the SR39TK+T lymphocyte was more sensitive to GCV or ACV,The IC_(50) value of GCV to SR39TK^+T lymphocyte was 2.5 time smaller than that of TK^+T lymphocyte,and The IC_(50) value of ACV to SR39TK^+T lymphocyte was 17.4 time smaller than that of TK^+T lymphocyte.Conclusion As compare with the cell killing effects of HSV1-TK-GCV/ACV system,the HSV1-SR39TK enhance the sensitivity of GCV to infected T cells.In addition,SR39TK^+T lymphocytes were also sensitive to ACV.

关 键 词：移植物抗宿主病 慢病毒载体 HSV1-TK HSV1-SR39TK IRES GCV/ACV 

分 类 号：R96[医药卫生—药理学]