突变型胸苷激酶对鼠C6胶质瘤细胞的杀伤效应

Killing effect of mutant herpes simplex virus type 1 thymidine kinase on rat C6 glioma cells

目的观察比较突变型单纯疱疹胸苷激酶（HSV1-sr39tk）／更昔洛韦（GCV）和野生型HSV1-tk／GCV对鼠C6胶质瘤细胞的杀伤效应。方法利用真核表达载体转染目的基因到C6细胞，RT—PCR鉴定。通过MTT实验和活体植瘤，比较各组对GCV敏感性。结果成功获得分别转染有HSV1-tk（C6／tk）和HSV-sr9tk（C6／sr9tk）的C6细胞。GCV浓度在0～400μmol／L时，C6／sr39tk细胞存活率由（99．96±3．54）％下降到（4．75±1．79）％；C6／tk细胞存活率从（100．03±2．95）％降至（59．16±3．48）％，未转染组细胞存活率则从（100．29±1．20）％到（83．62±7．56）％。同-GCV浓度时，各组间细胞存活率差异有统计学意义（P〈0．05）。各组细胞在活体均能致瘤，GCV治疗10d后，C6、C6／tk和C6／sr39tk组肿瘤大小分别为（1287．24±364．84）mm^3、（928．47±165．61）mm^3和（574．08±107．72）mm^3，较治疗前均有一定程度的生长，但后两组生长较前组明显减缓，以C6／sr39tk组生长减缓最明显。组间比较差异有统计学意义（P〈0．05）。结论体外和体内实验证实HSV1-sr39tk比HSV1-tk对GCV更敏感，可提高其与GCV所组成的自杀基因系统对C6胶质瘤细胞的杀伤效应。

Objective To explore the killing effect of mutant herpes simplex virus type 1 thymidine kinase （HSV1-sr39tk） and its wild type （HSV1-tk） on rat C6 glioma cells after Ganciclovir （GCV） treatment. Methods Eukaryotic expressing vector pcDNA3.1 containing HSV1-tk or HSVl-sr39tk was transfected into C6 cells and identified with RT-PCR. GCV killing efficiencies after gene transfer of HSV1-tk or HSVl-sr39tk were observed and compared in vitro and in vivo. Results 308bp DNA fragment was amplified through RT-PCR in both cells transfected with HSVI-tk （C6/tk） and those with HSVl-sr39tk （C6/sr39tk）. As the prodrug GCV concentrations increasing from 0μmol/L to 400μmol/L, the cell survival rates in C6/sr39tk group declined from （ 99. 96 ± 3.54 ） % to （4. 75 ± 1.79 ） %, while in C6/tk group from （ 100. 03 ± 2. 95 ） % to （59. 16± 3.48） % and in C6 group from（ 100. 29 ±1.20） % to （ 83.62 ±7.56）%. There was a significant difference in killing effect among C6,C6/tk and C6/sr39tk group after GCV treatment（ P 〈 0.05）. In vivo experiment, the rate of tumor formation was 100%. After 10 day treatment of GCV following tumor formation, tumors in animals implantated with C6/sr39tk,C6/tk or C6 got an average volume equaling （574. 08 ± 107.72） mm^3 , （ 928.47 ± 165.61 ） mm^3 and （ 1287.24± 364. 84 ） mm^3 respectively. The most powerful tumor growth inhibition was observed in C6/sr39tk ＋ GCV group, with an inhibition rate of tumor growth about 55.40%. Conclusion C6 cells transfeeted with HSV1-sr39tk were more sensitive to GCV than those with HSV1-tk both in vitro and in vivo, and thus HSV1 - sr39tk can be considered as a mean to improve the overall efficacy of the HSV1 - tk/GCV suicide gene system.