重组腺病毒p53上调凋亡调控因子增强胶质瘤细胞对替莫唑胺敏感性的体内研究

 目的　通过体内实验探讨外源性p53上调凋亡调控因子（PUMA）对人类脑胶质瘤细胞生长的影响及其增强胶质瘤细胞对替莫唑胺（TMZ）敏感性的机制。方法　建立人类脑胶质瘤细胞株U87MG裸鼠皮下移植瘤模型，将胶质瘤裸鼠用随机区组法随机分为四组，于建模2周后，分别自腹腔注射0.9 % NaCl溶液100 μl（对照组）、2×108 pfu／100 μl Ad-PUMA（PUMA组）、TMZ 10 mg／kg（TMZ组）和2×108 pfu／100 μl Ad-PUMA + TMZ 10 mg／kg （联合组），每组8只。治疗20 d后处死裸鼠，剖腹测量原位肿瘤体积、抑瘤率。TUNEL检测肿瘤细胞凋亡情况及计算凋亡指数（AI）。半定量RT-PCR和Western blot法检测DNA损伤修复蛋白6-氧-甲基鸟嘌呤DNA甲基转移酶（MGMT）mRNA和MGMT蛋白表达情况。结果　治疗20 d时对照组、PUMA组、TMZ组、联合组诱发肿瘤瘤体积分别为（3.68±0.09）、（2.63±0.13）、（2.13±0.07）、（0.97±0.02）cm3，四组体积进行两两比较差异有统计学意义（P均<0.05）。治疗组抑瘤率分别为28.5 %、42.1 %、73.6 %，两两比较差异有统计学意义（P均＜0.05）。TUNEL染色并计算AI：对照组（2.0±1.2）%、Ad-PUMA组（11.4±2.6） %、TMZ组（7.6±3.2）%、联合组（20.6±8.6）%，进行两两比较差异有统计学意义（P均< 0.05）。半定量RT-PCR和Western blot法检测显示MGMT mRNA和MGMT蛋白在TMZ组中表达最高，与其他三组比较差异有统计学意义（P均＜0.05）。结论　Ad-PUMA联合TMZ具有协同抑制胶质瘤生长作用，其机制可能与Ad-PUMA促进凋亡和抑制MGMT表达有关。

Study on effect of recombinant adenovirus PUMA on sensitivity of human glioblastoma cells to temozolomide in vivo

Objective To investigate the inhibitive effects of Ad-PUMA combined with temozolomide on human glioblastoma cells growth in vivo experiments. Methods The nude mouse model with human glioblastoma cells subcutaneous transplantation was established. The mice were randomly divided into 4 groups to receive subcutaneous injection at the 14th day separately with: Normal saline 100 μl (control, n=8), Ad-PUMA 2×108 pfu/100 μl (PUMA group, n=8), 10 mg/kg TMZ (TMZ group, n=8) and 2×108 pfu/100 μl Ad-PUMA + 10 mg/kg TMZ (combined group, n=8). Mice were killed after 20 days treatment.Tumor volume, inhibition rates and apoptotic index (AI) were measured, meanwhile, apoptotic tumor cells were detected by TUNEL technology respectively. The expression of MGMT mRNA and MGMT protein were revealed by the methods of RT-PCR and Western blot. Results According to the order: control group, AdPUMA group, TMZ group, combined group, tumor volumes were (3.68±0.09), (2.63±0.13), (2.13±0.07),(0.97±0.02) cm3 respectively (P＜0.05); the inhibitive rates were 0, 28.5 %, 42.1%, 73.6 % respectively and AI were (2.0±1.2) %, (11.4±2.6) %, (7.6±3.2) %, (20.6±8.6) % (P＜0.05). The results of Western blot and RT-PCR showed that MGMT mRNA and MGMT protein levels in TMZ group were higher than other groups (all P＜0.01). Conclusion Ad-PUMA combined with TMZ greatly enhances the sensitivity of human glioblastoma cells to TMZ and could effectively inhibit the proliferation and promote the apeptosis of glioblastoma cells, its mechanism was probably related Ad-PUMA promote apoptosis and inhibit MGMT expression.