Pannexin1通道在睾丸癌I-10细胞株对顺铂耐药性中的作用及可能机制
发布时间：2018-11-06 17:12
【摘要】：目的:1.检测pannexin1(Panx-1)蛋白在小鼠睾丸癌细胞敏感株I-10和耐药株I-10/DDP中的表达。2.观察Panx-1通道及其介导的ATP/IP_3(三磷酸肌醇)通路在顺铂诱导小鼠睾丸癌细胞凋亡中的作用。3.探究睾丸癌细胞对顺铂产生耐药性的机制是否与Panx-1通道有关。方法:1.Western blotting法检测I-10和I-10/DDP细胞总Panx-1蛋白表达。2.免疫荧光法检测I-10和I-10/DDP细胞膜上Panx-1的表达。3.MTT法检测细胞存活率。4.Annexin V/PI双染法检测细胞早期凋亡。5.Hoechst 33258染色法检测细胞晚期凋亡。6.化学发光法检测ATP浓度。7.ELISA法检测IP_3浓度。8.质粒sh RNA转染对Panx-1进行干扰和过表达,G418筛选培养基建立稳转株。9.统计学方法:实验数据资料分析使用SPSS 16.0软件,以均数±标准差表示,多组之间的比较采用方差分析。统计图表采用Sigma Plot 10.0绘制,P0.05认为差异有统计学意义。结果:1.Panx-1在耐药株中表达量低于敏感株耐药细胞I-10/DDP中Panx-1总蛋白表达量低于I-10细胞,P0.01;耐药细胞I-10/DDP中Panx-1膜蛋白表达量明显低于I-10细胞。2.Panx-1通道抑制剂CBX减轻顺铂细胞毒性与单用DDP组相比,CBX与DDP合用组细胞存活率增加,P0.01。3.Panx-1通道抑制剂CBX抑制顺铂诱导凋亡与单用DDP组相比,CBX与DDP合用组细胞早期凋亡减少,P0.001;细胞晚期凋亡减少,P0.01。4.Panx-1通道抑制剂CBX减少ATP释放和细胞内IP_3含量与单用DDP组相比,CBX与DDP合用组ATP浓度减小,P0.05;IP_3含量减小,P0.05。5.过表达Panx-1增加顺铂细胞毒性和诱导凋亡作用,增强ATP释放和细胞内IP_3含量与单用DDP组相比,过表达Panx-1后应用DDP组细胞存活率减少,P0.001;细胞早期凋亡增加,P0.01;细胞晚期凋亡增加,P0.001;ATP浓度增加,P0.01;IP_3含量增加,P0.01。6.沉默Panx-1降低顺铂细胞毒性和诱导凋亡作用,减少ATP释放和细胞内IP_3含量与单用DDP组相比,沉默Panx-1后应用DDP组细胞存活率增加,P0.01;细胞早期凋亡减少,P0.01;细胞晚期凋亡减少,P0.001;ATP浓度减小,P0.05。;IP_3含量减小,P0.01。7.ATP酶和光溜海绵素C降低顺铂细胞毒性和诱导凋亡作用与单用DDP组相比,ATP酶与DDP合用组ATP浓度减小,P0.05;IP_3含量减小,P0.01。与单用DDP组相比,ATP酶或光溜海绵素C与DDP合用组细胞生存率提高,P0.01;细胞早期凋亡率减小,P0.001;细胞晚期凋亡率减小,P0.01。结论:1.睾丸癌I-10细胞对顺铂产生耐药性时,Panx-1表达量降低。2.Panx-1通道介导的ATP/IP_3信号通路参与了顺铂诱导睾丸癌细胞的凋亡。3.上调Panx-1通道可以增强ATP/IP_3信号通路,提高顺铂的抗肿瘤作用。
[Abstract]:Objective: 1. To detect the expression of pannexin1 (Panx-1) protein in mouse testicular cancer cell line I-10 and I-10/DDP. 2. To observe the role of Panx-1 channel and ATP/IP_3 (inositol triphosphate) pathway in cisplatin induced apoptosis of mouse testicular cancer cells. To investigate whether the mechanism of resistance of testicular cancer cells to cisplatin is related to Panx-1 channels. Methods: the expression of total Panx-1 protein in I-10 and I-10/DDP cells was detected by 1.Western blotting assay. Immunofluorescence assay was used to detect the expression of Panx-1 on cell membrane of I-10 and I-10/DDP. 3.MTT method was used to detect cell survival rate. 4.Annexin V/PI double staining method was used to detect early apoptosis. 5.Hoechst 33258 staining method was used to detect late apoptosis. The concentration of ATP was detected by chemiluminescence and the concentration of IP_3 by 7.ELISA. Plasmid sh RNA transfection interfered with and overexpression of Panx-1. G418 screening medium was used to establish a stable transgenic strain. 9. 9. Statistical method: the experimental data were analyzed by SPSS 16.0 software, expressed as mean 卤standard deviation, and compared with each other by ANOVA. The statistical chart was drawn by Sigma Plot 10.0, and the difference was statistically significant (P0.05). Results: the expression of 1.Panx-1 in drug resistant cell line was lower than that in sensitive cell line I-10/DDP and the expression of Panx-1 total protein was lower than that in I-10 cell line (P0.01). The expression of Panx-1 membrane protein in I-10/DDP cells was significantly lower than that in I-10 cells. CBX, an inhibitor of 2.Panx-1 channel, reduced the cytotoxicity of cisplatin cells. Compared with DDP alone, the survival rate of CBX combined with DDP increased. CBX, a P0.01.3.Panx-1 channel inhibitor, inhibited cisplatin induced apoptosis. Compared with DDP alone, the combination of CBX and DDP decreased the early apoptosis of cells (P0.001). Late apoptosis was decreased, CBX, a P0.01.4.Panx-1 channel inhibitor, decreased ATP release and intracellular IP_3 content. Compared with DDP group, the ATP concentration of CBX combined with DDP group was decreased, and the content of P0.05 / IP3 and P0.05.5in CBX / DDP group. Overexpression of Panx-1 increased the cytotoxicity and apoptosis of cisplatin cells, enhanced the release of ATP and the content of IP_3 in cells. Compared with the group of DDP alone, the survival rate of DDP group was decreased and the cell survival rate was decreased (P0.001) after overexpression of Panx-1. Apoptosis increased in the early stage and increased in the late stage, the concentration of P0.001 + ATP increased, the content of P0.01 + IP3 increased, and the content of P0.01.6 increased. Silencing Panx-1 decreased the cytotoxicity and apoptosis of cisplatin cells, decreased the release of ATP and the content of IP_3 in cells, compared with the group of DDP alone, the survival rate of DDP group was increased after Panx-1 silencing, and the cell survival rate was increased (P0.01). Early apoptosis decreased, P0.01, late apoptosis decreased, P0.001ATP concentration decreased, P0.05. Compared with DDP group, the concentration of ATP, the content of P0.05 / IP _ 3 and the content of P0.01in the combination of ATP enzyme and DDP decreased. Compared with the group of DDP alone, the survival rate of cells in the combination of ATP and DDP increased (P0.01), the early apoptosis rate decreased (P0.001), and the late apoptosis rate decreased (P0.01). Conclusion: 1. The expression of Panx-1 decreased when I-10 cells were resistant to cisplatin. The ATP/IP_3 signaling pathway mediated by 2.Panx-1 channel was involved in cisplatin induced apoptosis of testicular cancer cells. Upregulation of Panx-1 channel can enhance ATP/IP_3 signaling pathway and enhance the anti-tumor effect of cisplatin.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
本文编号：2314925
[Abstract]:Objective: 1. To detect the expression of pannexin1 (Panx-1) protein in mouse testicular cancer cell line I-10 and I-10/DDP. 2. To observe the role of Panx-1 channel and ATP/IP_3 (inositol triphosphate) pathway in cisplatin induced apoptosis of mouse testicular cancer cells. To investigate whether the mechanism of resistance of testicular cancer cells to cisplatin is related to Panx-1 channels. Methods: the expression of total Panx-1 protein in I-10 and I-10/DDP cells was detected by 1.Western blotting assay. Immunofluorescence assay was used to detect the expression of Panx-1 on cell membrane of I-10 and I-10/DDP. 3.MTT method was used to detect cell survival rate. 4.Annexin V/PI double staining method was used to detect early apoptosis. 5.Hoechst 33258 staining method was used to detect late apoptosis. The concentration of ATP was detected by chemiluminescence and the concentration of IP_3 by 7.ELISA. Plasmid sh RNA transfection interfered with and overexpression of Panx-1. G418 screening medium was used to establish a stable transgenic strain. 9. 9. Statistical method: the experimental data were analyzed by SPSS 16.0 software, expressed as mean 卤standard deviation, and compared with each other by ANOVA. The statistical chart was drawn by Sigma Plot 10.0, and the difference was statistically significant (P0.05). Results: the expression of 1.Panx-1 in drug resistant cell line was lower than that in sensitive cell line I-10/DDP and the expression of Panx-1 total protein was lower than that in I-10 cell line (P0.01). The expression of Panx-1 membrane protein in I-10/DDP cells was significantly lower than that in I-10 cells. CBX, an inhibitor of 2.Panx-1 channel, reduced the cytotoxicity of cisplatin cells. Compared with DDP alone, the survival rate of CBX combined with DDP increased. CBX, a P0.01.3.Panx-1 channel inhibitor, inhibited cisplatin induced apoptosis. Compared with DDP alone, the combination of CBX and DDP decreased the early apoptosis of cells (P0.001). Late apoptosis was decreased, CBX, a P0.01.4.Panx-1 channel inhibitor, decreased ATP release and intracellular IP_3 content. Compared with DDP group, the ATP concentration of CBX combined with DDP group was decreased, and the content of P0.05 / IP3 and P0.05.5in CBX / DDP group. Overexpression of Panx-1 increased the cytotoxicity and apoptosis of cisplatin cells, enhanced the release of ATP and the content of IP_3 in cells. Compared with the group of DDP alone, the survival rate of DDP group was decreased and the cell survival rate was decreased (P0.001) after overexpression of Panx-1. Apoptosis increased in the early stage and increased in the late stage, the concentration of P0.001 + ATP increased, the content of P0.01 + IP3 increased, and the content of P0.01.6 increased. Silencing Panx-1 decreased the cytotoxicity and apoptosis of cisplatin cells, decreased the release of ATP and the content of IP_3 in cells, compared with the group of DDP alone, the survival rate of DDP group was increased after Panx-1 silencing, and the cell survival rate was increased (P0.01). Early apoptosis decreased, P0.01, late apoptosis decreased, P0.001ATP concentration decreased, P0.05. Compared with DDP group, the concentration of ATP, the content of P0.05 / IP _ 3 and the content of P0.01in the combination of ATP enzyme and DDP decreased. Compared with the group of DDP alone, the survival rate of cells in the combination of ATP and DDP increased (P0.01), the early apoptosis rate decreased (P0.001), and the late apoptosis rate decreased (P0.01). Conclusion: 1. The expression of Panx-1 decreased when I-10 cells were resistant to cisplatin. The ATP/IP_3 signaling pathway mediated by 2.Panx-1 channel was involved in cisplatin induced apoptosis of testicular cancer cells. Upregulation of Panx-1 channel can enhance ATP/IP_3 signaling pathway and enhance the anti-tumor effect of cisplatin.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
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