水通道蛋白-8对结肠癌细胞化疗药敏性的影响及意义  

Effects of AQP-8 overexpression on chemotherapeutic sensitivity of human colon cancer HT-29 cells in vitro

作　　者：杨明利 石晓明[2] 赵伟[2] 王建升[3] 

机构地区：[1]河北省武安市第一人民医院普外二科,056300 [2]河北省人民医院普外二科 [3]河北省人民医院检验科

出　　处：《河北医药》2015年第5期674-678,共5页Hebei Medical Journal

摘　　要：目的通过过表达水通道蛋白-8(AQP-8)真核表达载体,探讨AQP-8水通道蛋白对HT-29细胞凋亡、增殖及化疗药敏性的影响。方法取对数生长期的人结肠癌HT-29细胞株用于实验。构建AQP-8真核表达载体并转染HT-29细胞,通过Western blot检测GFP-AQP-8转染效率;采用SRB法检测3组细胞增殖抑制率;流式细胞术检测细胞周期分布和凋亡率;分光光度法检测细胞caspase-3活性;Real Time-PCR和Western blot检测PCNA和P53表达。转染前后,分别用5-氟尿嘧啶(5-Fu)和顺铂(DDP)刺激细胞,SRB检测3组细胞的增殖抑制率。结果 GFPAQP-8转染HT-29细胞后,Western blot结果显示,细胞AQP-8基因表达显著上调(P<0.05);SRB分析结果显示,细胞增殖抑制率显著增加(P<0.05);流式细胞术检测发现,处于G0/G1期的细胞数明显增加(P<0.05),S期的细胞数则显著降低(P<0.05);细胞凋亡率升高(P<0.05);细胞caspase-3活性增强(P<0.05);Real Time-PCR和Western blot结果显示,细胞增殖相关基因PCNA的mRNA和蛋白表达水平明显降低(P<0.05),而凋亡基因P53的mRNA和蛋白表达水平明显增加(P<0.05)。GFP-AQP-8转染联合化疗药物5-Fu处理、GFP-AQP-8转染联合化疗药物DDP处理HT-29细胞较转染对照空质粒组细胞增殖抑制率显著增加,联合用药组对细胞增殖的抑制率显著高于单独转染GFP-AQP-8组和单独给予5-Fu或DDP组(P<0.05)。结论过表达AQP-8可抑制HT-29细胞生长、促进其凋亡、提高细胞对5-Fu和DDP的化疗敏感性。Objective To investigate the effects of AQP-8 overexpression on the proliferation, apoptosis, and chemotherapeutic sensitivity of human colon cancer HT-29 cells in vitro. Methods Human colon cancer HT-29 cells in logarithm growth period were used in the study. AQP-8 eukaryotic expression vector was constructed and transfected into HT-29 ceils. Then transfection efficiency was detected by Western Blot;cell growth inhibition rate was determined by SRB assay;cell cycle and apoptosis rate were measured by flow cytometry （FCM）;the activities of caspase-3 were assessed by speetrophotometry. The expression levels of PCNA and P53 were determined by fluorescence real-time quantitative RT-PCR and Western Blot. The transfected and untransfected cells were cultured in the medium with 5-Fu or DDP, then the cell growth inhibition rate was detected by SRB assay. Results Western Blot showed that the expression levels of AQP-8 were up-regulated in colon cancer HT-29 cells after GFP-AQP-8 transfection （ P 〈 0. 05 ）. SRB showed that as compared with that in GFP-N1 control group, the proliferation inhibition rate was significantly increased in HT-29 ceils after GFP-AQP-8 transfection （ P 〈 0.05 ） ;FCM analysis showed that GFP-AQP-8 transfection increased the cell number in G0/G1 phase （ P 〈 0.05 ）,and decreased the cell number in S phase （ P 〈 0.05 ）, moreover, which induced HT-29 cell apoptosis （ P 〈 0.05 ）. The activities of caspase-3 were also increased after GFP-AQP-8 transfeetion. The results of fluorescence real-time quantitative RT-PCR and Western Blot showed that as compared with those in GFP-N1 negative control group, the expression levels of PCNA were significantly decreased,however,the expression levels of P53 were obviously increased （ P 〈 0.05 ） after GFP-AQP-8 transfection. The combination of GFP-AQP-8 transfection with 5-Fu or DDP increased the proliferation inhibition rate of HT-29 cells,moreover, the increase degree of proliferation inhibition rate was more obvious than that b

关 键 词：水通道蛋白8 结肠肿瘤 化疗敏感性 

分 类 号：R735.35[医药卫生—肿瘤]