AQP9过表达增强顺铂诱导的肝癌SMMC-7721细胞凋亡
发布时间：2018-07-15 17:54
【摘要】：目的探讨AQP9基因过表达对顺铂诱导肝癌SMMC-7721细胞凋亡的影响及其可能的机制。方法将重组慢病毒LV-AQP9(以LV-GFP作为对照)导入SMMC-7721细胞中,构建SMMC-7721/LV-AQP9重组细胞。激光共聚焦显微镜观察重组慢病毒LV-AQP9在SMMC-7721中的感染效率,实时荧光定量PCR和蛋白质印迹法检测AQP9在SMMC-7721中表达情况。CCK-8法检测不同浓度顺铂(DDP)处理肝癌SMMC-7721细胞后24h、36h、48h细胞的增殖抑制率并选取最佳顺铂处理浓度和时间。顺铂分别处理转染前后的肝癌SMMC-7721细胞。实验分组:GFP组为空载体慢病毒转染的SMMC-7721细胞;AQP9组为含过表达AQP9基因慢病毒载体转染的SMMC-7721细胞;GFP+DDP组为顺铂处理的空载体慢病毒转染的SMMC-7721细胞;AQP9+DDP组为顺铂处理的含过表达AQP9基因慢病毒载体转染的SMMC-7721细胞。采用AnnexinⅤ/7AAD双标流式细胞术和DAPI染色检测各组细胞的凋亡情况。用蛋白质印迹法检测AQP9,GRP78以及Caspase-12的蛋白表达水平。结果激光共聚焦显微镜观察到重组慢病毒LV-AQP9在SMMC-7721细胞中转染效率达90%以上,且AQP9 mRNA和AQP9蛋白表达水平在SMMC-7721细胞中均显著增加,差异均有统计学意义(P0.01)。顺铂作用SMMC-7721细胞24h后的半数抑制率(IC50)为5ug/mL。四组细胞的凋亡率之间都有显著差异,差异有统计学意义(P0.05)。采用多因素方差分析AQP9和顺铂对人肝癌细胞对凋亡的影响,其结果显示:AQP9过表达明显促进细胞凋亡(FAQP9=71.391.780,P=0.000);DDP显著诱导肝癌细胞凋亡(FCDDP=361.682,P=0.000);AQP9可显著增强顺铂诱导肝癌细胞凋亡,具有协同作用(FAQP9×CDDP=26.681,P=0.001)。AQP9+DDP组细胞GRP78和cleaved Caspase12蛋白表达显著高于GFP组、AQP9组、GFP+DDP组,差异有统计学意义(P值均0.01),而GFP组和AQP9组间无明显统计学差异。结论AQP9过表达能够增强DDP诱导肝癌SMMC-7721细胞发生凋亡,增强其对化疗的敏感性。其机制可能是AQP9通过内质网应激信号通路细胞凋亡途径,发挥与DDP的协同作用促进凋亡的发生。
[Abstract]:Objective to investigate the effect of AQP9 gene overexpression on cisplatin induced apoptosis of hepatoma SMMC-7721 cells and its possible mechanism. Methods Recombinant lentivirus LV-AQP9 (LV-GFP as control) was transfected into SMMC-7721 cells to construct SMMC-7721 / LV-AQP9 recombinant cells. Laser confocal microscopy was used to observe the infection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721. The expression of AQP9 in SMMC-7721 was detected by real-time fluorescence quantitative PCR and Western blot. CCK-8 assay was used to detect the proliferation inhibition rate of SMMC-7721 cells treated with different concentrations of cisplatin (DDP) for 24 h or 36 h or 48 h, and the optimal concentration and time of cisplatin treatment were selected. Cisplatin was used to treat SMMC-7721 cells before and after transfection. SMMC-7721 cells transfected with empty vector lentivirus were divided into two groups: SMMC-7721 cells transfected with empty vector lentivirus and SMMC-7721 cells transfected with lentivirus vector containing overexpression of AQP9 gene. The GFP DDP group was treated with cisplatin and treated with cisplatin in SMMC-7721 cells transfected with empty vector lentivirus-transfected SMMC-7721 cells. SMMC-7721 cells transfected with lentivirus vector containing overexpression of AQP9 gene. Apoptosis was detected by Annexin V / 7 AAD double flow cytometry and DAPI staining. The protein expression levels of AQP9, GRP78 and Caspase-12 were detected by Western blot. Results the transfection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721 cells was more than 90%, and the expression of AQP9 mRNA and AQP9 protein were significantly increased in SMMC-7721 cells (P0.01). The half inhibition rate (IC50) of SMMC-7721 cells treated with cisplatin for 24 hours was 5ug- mL. There were significant differences in apoptosis rate among the four groups (P0.05). The effects of AQP9 and cisplatin on apoptosis of human hepatoma cells were analyzed by multivariate variance analysis. The results showed that the overexpression of AQP9 significantly promoted apoptosis (FAQP9 71.391.780 P0. 000) DDP significantly induced apoptosis of hepatoma cells (FCDDP 361.682P0.000) AQP9 significantly enhanced cisplatin induced apoptosis in hepatoma cells. The expression of GRP78 and cleaved Caspase12 protein in AQP9 DDP group was significantly higher than that in GFP group (P < 0.01), but there was no significant difference between GFP group and AQP9 group. Conclusion the overexpression of AQP9 can enhance the apoptosis of SMMC-7721 cells induced by DDP and enhance its sensitivity to chemotherapy. The mechanism may be that AQP9 plays a synergistic role with DDP in promoting apoptosis through endoplasmic reticulum stress signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
本文编号：2124891
[Abstract]:Objective to investigate the effect of AQP9 gene overexpression on cisplatin induced apoptosis of hepatoma SMMC-7721 cells and its possible mechanism. Methods Recombinant lentivirus LV-AQP9 (LV-GFP as control) was transfected into SMMC-7721 cells to construct SMMC-7721 / LV-AQP9 recombinant cells. Laser confocal microscopy was used to observe the infection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721. The expression of AQP9 in SMMC-7721 was detected by real-time fluorescence quantitative PCR and Western blot. CCK-8 assay was used to detect the proliferation inhibition rate of SMMC-7721 cells treated with different concentrations of cisplatin (DDP) for 24 h or 36 h or 48 h, and the optimal concentration and time of cisplatin treatment were selected. Cisplatin was used to treat SMMC-7721 cells before and after transfection. SMMC-7721 cells transfected with empty vector lentivirus were divided into two groups: SMMC-7721 cells transfected with empty vector lentivirus and SMMC-7721 cells transfected with lentivirus vector containing overexpression of AQP9 gene. The GFP DDP group was treated with cisplatin and treated with cisplatin in SMMC-7721 cells transfected with empty vector lentivirus-transfected SMMC-7721 cells. SMMC-7721 cells transfected with lentivirus vector containing overexpression of AQP9 gene. Apoptosis was detected by Annexin V / 7 AAD double flow cytometry and DAPI staining. The protein expression levels of AQP9, GRP78 and Caspase-12 were detected by Western blot. Results the transfection efficiency of recombinant lentivirus LV-AQP9 in SMMC-7721 cells was more than 90%, and the expression of AQP9 mRNA and AQP9 protein were significantly increased in SMMC-7721 cells (P0.01). The half inhibition rate (IC50) of SMMC-7721 cells treated with cisplatin for 24 hours was 5ug- mL. There were significant differences in apoptosis rate among the four groups (P0.05). The effects of AQP9 and cisplatin on apoptosis of human hepatoma cells were analyzed by multivariate variance analysis. The results showed that the overexpression of AQP9 significantly promoted apoptosis (FAQP9 71.391.780 P0. 000) DDP significantly induced apoptosis of hepatoma cells (FCDDP 361.682P0.000) AQP9 significantly enhanced cisplatin induced apoptosis in hepatoma cells. The expression of GRP78 and cleaved Caspase12 protein in AQP9 DDP group was significantly higher than that in GFP group (P < 0.01), but there was no significant difference between GFP group and AQP9 group. Conclusion the overexpression of AQP9 can enhance the apoptosis of SMMC-7721 cells induced by DDP and enhance its sensitivity to chemotherapy. The mechanism may be that AQP9 plays a synergistic role with DDP in promoting apoptosis through endoplasmic reticulum stress signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
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