CAJ | 학술논문

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100μl(对照组)、2×108 pfu/100μl Ad-PUMA(PUMA组)、TMZ 10 mg/kg(TMZ组)和2×108 pfu/100μl Ad-PUMA+TMZ 10 mg/kg(联合组),每组8只.治疗20d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MGMTmRNA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.
목적 통과체내실험탐토외원성p53상조조망조공인자(PUMA)대인류뇌효질류세포생장적영향급기증강효질류세포대체막서알(TMZ)민감성적궤제.방법 건립인류뇌효질류세포주U87MG라서피하이식류모형,장효질류라서용수궤구조법수궤분위사조,우건모2주후,분별자복강주사0.9%NaCl용액100μl(대조조)、2×108 pfu/100μl Ad-PUMA(PUMA조)、TMZ 10 mg/kg(TMZ조)화2×108 pfu/100μl Ad-PUMA+TMZ 10 mg/kg(연합조),매조8지.치료20d후처사라서,부복측량원위종류체적、억류솔.TUNEL검측종류세포조망정황급계산조망지수(AI).반정량RT-PCR화Western blot법검측DNA손상수복단백6-양-갑기조표령DNA갑기전이매(MGMT)mRNA화MGMT단백표체정황.결과 치료20 d시대조조、PUMA조、TMZ조、연합조유발종류류체적분별위(3.68±0.09)、(2.63±0.13)、(2.13±0.07)、(0.97±0.02)cm3,사조체적진행량량비교차이유통계학의의(P균＜0.05).치료조억류솔분별위28.5%、42.1%、73.6%,량량비교차이유통계학의의(P균＜0.05).TUNEL염색병계산AI:대조조(2.0±1.2)%、Ad-PUMA조(11.4±2.6)%、TMZ조(7.6±3.2)%、연합조(20.6±8.6)%,진행량량비교차이유통계학의의(P균＜0.05).반정량RT-PCR화Western blot법검측현시MGMTmRNA화MGMT단백재TMZ조중표체최고,여기타삼조비교차이유통계학의의(P균＜0.05).결론 Ad-PUMA연합TMZ구유협동억제효질류생장작용,기궤제가능여Ad-PUMA촉진조망화억제MGMT표체유관.
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.