突变型胸苷激酶促进前体药物对小鼠T淋巴细胞的杀伤作用  

Promotion of T Lymphocyte Killing by Mutant Herpes Simplex Virus-1 Thymidine Kinase in Mouse

作　　者：李振宇[1] 董宁征[1] 赵益明[1] 阮长耿[1] 

机构地区：[1]苏州大学附属第一医院,江苏省血液研究所,卫生部血栓与止血重点实验室,江苏苏州215006

出　　处：《苏州大学学报（医学版）》2008年第5期720-723,882,共5页Suzhou University Journal of Medical Science

摘　　要：目的研制表达单纯疱疹病毒胸苷激酶基因(HSV1-TK)及其突变体HSV1-SR39TK的T淋巴细胞,探讨给予更昔洛韦(GCV)和阿昔洛韦(ACV)后T细胞的生存率。方法采用磷酸钙沉淀法将慢病毒载体三质粒系统共转染来源于人胚肾的293T细胞,表达HSV1-SR39TK和HSV1-TK的T细胞与不同浓度的GCV和ACV培养3d后,应用CCK-8法测定T细胞存活率。结果包装后慢病毒载体的滴度超过106IU/ml、ACV/GCV浓度在1～10μmol/L时,SR39TK+T细胞的存活率下降明显,ACV组T细胞存活率从(98.4±2.7)%下降至(49.9±5.9)%,GCV组从(97.9±2.7)%下降至(33.7±5.3)%,差异均有统计学意义(均P<0.05)。当ACV/GCV浓度进一步升高时,T细胞活率下降不明显。TK+T细胞对GCV敏感,T细胞活率从(97.9±2.7)%下降至(38.4±5.5)%,差异有统计学意义(P<0.05);但是对ACV不敏感,T细胞存活率从(98.4±2.7)%下降至(73.8±7.4)%,差异无统计学意义(P>0.05)。IC50值测定结果显示,SR39TK+T细胞对GCV和ACV的敏感性高于TK+T细胞。结论表达HSV1-SR39TK的T细胞对GCV和ACV的敏感性均较HSV1-TK高。突变型HSV1-TK在体外可促进前体药物介导的杀伤小鼠T细胞的作用。Objective To investigate generation of T lymphocytes that express herpes simplex virus-1 thymidine kinase （HSV1-TK, TK^＋T） and its mutant HSV1-SR39TK（SR39TK^＋T） and survival rate of T cells after giving ganeiclovir （GCV） or acyclovir （ACV）. Methods Human embryonic kidney 293T cells were co-transfected by the three-plasmid system of lentiviral vectors including packaging plasmid A NRF, vector plasmid and envelope plasmid VSV-G by means of calcium phosphate DNA precipitation. The survival of T cells expressing HSV-SR39TK or HSV-TK was estimated by cell count kit-8 （CCK-8） assay after 3 day culture against a gradient of GCV or ACV concentrations. Results The titres of lentiviral vector were above 10^6 IU/ml after packaging. The survival rate of SR39TK^＋T cells declined significantly when concentration of ACV/GCV was between 1-10 μmol/L, the T cell survival rate in ACV group declined from （98.4±2.7）% to （49.9±5.9）% and in the GCV group from （97.9%±2.7）% to （33.7±5.3）% （P〈0.05）. When concentration of ACV/GCV was increased, further decline of T cells survival rate became unobvious. TK^＋T cells were sensitive to GCV, 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, survival rate of T cells declined from （98.4±2.7）% to （73.8±7.4）% （P〉0.05）. Determination of IC50 showed that the sensitivity of SR39TK^＋T cells to GCV and ACV was higher than TK^＋T cells about 2.5 and 17.4 times respectively. Conclusion T lymphocytes expressing HSV1-SR39TK are more sensitive not only to GCV but also to ACV when compared with T cells expressing HSV1-TK. Mutant HSV1-TK will improve prodrug-mediated mouse T lymphocytes killing in vitro.

关 键 词：单纯疱疹胸苷激酶 突变型 野生型 T细胞 慢病毒 

分 类 号：R392[医药卫生—免疫学] Q755[医药卫生—基础医学]