摘要
目的:制备慢病毒载体为基础的野生型及突变型单纯疱疹病毒胸苷激酶(HSV-TK/HSV-sr39TK)基因工程T细胞(TK+T及sr39TK+T细胞),观察给予前体药物丙氧鸟苷/无环鸟苷后小鼠T淋巴细胞对其敏感性及存活情况。方法:构建含HSV-TK/HSV-sr39TK及内部核糖体进入位点序列(IRES)和绿色荧光蛋白基因(GFP)的双顺反子慢病毒载体;采用磷酸钙沉淀法将慢病毒载体三质粒转染293T细胞,收集病毒上清感染刀豆蛋白刺激的小鼠T淋巴细胞,给予不同浓度的前体药物GCV/ACV,用Cell Counting Kit(CCK-8)等方法检测T细胞的存活率。结果:转染293T细胞后,病毒滴度达106/U/ml以上;含不同启动子的病毒载体转染效率CMV>PUB>PGK,对小鼠T淋巴细胞的感染效率以CMV启动子最高;IC50值的测定显示sr39TK+T细胞对GCV和ACV的敏感性较TK+T细胞分别提高约2.5和17.4倍;ACV及GCV浓度在1～10μmol/Lsr39TK+T细胞活率下降最为明显,ACV组细胞存活率由(98.4±2.7)%下降为(49.9±5.9)%,GCV组由(97.9%±2.7)%下降为(33.7±5.3)%,P<0.05,随ACV及GCV浓度的增加,细胞活率下降趋势有所减弱;TK+T细胞对GCV敏感,细胞活率由(97.9±2.7)%下降为(38.4±5.5)%,(P<0.05),对ACV不敏感,细胞活率由(98.4±2.7)%下降为(73.8±7.4)%,P>0.05。结论:成功构建了HSV-TK/HSV-sr39TK基因工程T细胞,不同启动子对慢病毒载体在293T细胞的转染效率、转导自杀基因在T淋巴细胞内的表达均有影响;HSV-sr39TK+T细胞对GCV和ACV的敏感性高于TK+T细胞。
Objective：The genetic engineering T lymphocytes containing herpes simplex virus thymidine kinase gene （TK^＋T cells） or mutant herpes simplex virus thymidine kinase（HSV-sr39TK） gene（ sr39 TK^＋ T cells） were generated and the survival rate of T cells after giving the predrug Ganciclovir/Acyclovir（GCV/ACV） was observed. Methods：The bicistronic lentiviral vectors containing HSV-TK/HSV-sr39TK and internal ribosomal entry site-green fluorescent protein gene （IRES-GFP） were constructed. Human embryonic kidney 293T cells were co-transfected by the three-plasmid system of lentiviral vector including the packaging plasmid ANRF, the vector plasmid and the envelope plasmid VSV-G by calcium phosphate DNA precipitation, the mouse T lymphecytes stimulated by concanavalin A were infected by the virus supernatant and diversity concentration predrug GCV/ACV was co-incubated with TK^＋ T cells or sr39TK^＋T cells, the killing effects was estimated by Cell Count kit-8 （CCK-8）. Results：The titres of lentiviral vector were above 10^6 IU/ml after packaging. The efficiency of different internal promoters to drive the expression of the target gene was CMV 〉 PUB 〉 PGK. The determination of IC50 showed that the sensitivity of sr39TK^＋ T cells to GCV and ACV were higher than TK ^＋ T cells about 2. 5 and 17.4 times respectively. The survival rates of sr39TK^＋ T cells declined significantly when the concentrations of ACV/GCV were between 1 - 10μmol/L, The T cell survival rates in ACV group declined from （98.4 ± 2.7） % to （49. 9 ± 5.9） % and GCV group from （97.9% ±2. 7）% to （33.7 ±5. 3）% ,P 〈0. 05. When the concentrations of ACV/GCV were increased, further decline ofT cell survival rates became unobvious. TK^＋ T ceils were sensitive to GCV, the survival rate of T cells declined from （97.9 ± 2.7 ） % to （38.4 ± 5.5 ） %, （ P 〈 0.05 ） but they were not sensitive to ACV, the survival rate of T cells declined from （ 98.4 ± 2.7 ） % to （73. 8 ± 7.4） %, （ P 〉 0. 05 ）. Conclusions： The genetic engineering T cells containing HSV-TK/HSV-sr39TK genes were generated. Different internal promoters had different effects on transfection efficiency and driving the expression of HSV-TK/HSV-sr39TK genes in T cells. HSV-sr39TK^＋T cells were more sensitive to GCV and ACV than TK^＋T cells.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2007年第11期999-1003,共5页
Chinese Journal of Immunology
基金
国家自然科学基金资助项目(编号:30170389)
江苏省"135"医学重点人才基金资助项目(编号RC2002080)
关键词
单纯疱疹病毒胸苷激酶
突变型
野生型
基因工程T细胞
慢病毒载体
Herpes simplex virus thymidine kinase gene
Wild-type
Mutant-type
Genetic engineering T lymphocytes
Lentiviral vector
