Pannexin1通道在睪丸癌I-10細胞株對順鉑耐藥性中的作用及可能機制
發(fā)布時間：2018-11-06 17:12
【摘要】：目的:1.檢測pannexin1(Panx-1)蛋白在小鼠睪丸癌細胞敏感株I-10和耐藥株I-10/DDP中的表達。2.觀察Panx-1通道及其介導的ATP/IP_3(三磷酸肌醇)通路在順鉑誘導小鼠睪丸癌細胞凋亡中的作用。3.探究睪丸癌細胞對順鉑產(chǎn)生耐藥性的機制是否與Panx-1通道有關。方法:1.Western blotting法檢測I-10和I-10/DDP細胞總Panx-1蛋白表達。2.免疫熒光法檢測I-10和I-10/DDP細胞膜上Panx-1的表達。3.MTT法檢測細胞存活率。4.Annexin V/PI雙染法檢測細胞早期凋亡。5.Hoechst 33258染色法檢測細胞晚期凋亡。6.化學發(fā)光法檢測ATP濃度。7.ELISA法檢測IP_3濃度。8.質粒sh RNA轉染對Panx-1進行干擾和過表達,G418篩選培養(yǎng)基建立穩(wěn)轉株。9.統(tǒng)計學方法:實驗數(shù)據(jù)資料分析使用SPSS 16.0軟件,以均數(shù)±標準差表示,多組之間的比較采用方差分析。統(tǒng)計圖表采用Sigma Plot 10.0繪制,P0.05認為差異有統(tǒng)計學意義。結果:1.Panx-1在耐藥株中表達量低于敏感株耐藥細胞I-10/DDP中Panx-1總蛋白表達量低于I-10細胞,P0.01;耐藥細胞I-10/DDP中Panx-1膜蛋白表達量明顯低于I-10細胞。2.Panx-1通道抑制劑CBX減輕順鉑細胞毒性與單用DDP組相比,CBX與DDP合用組細胞存活率增加,P0.01。3.Panx-1通道抑制劑CBX抑制順鉑誘導凋亡與單用DDP組相比,CBX與DDP合用組細胞早期凋亡減少,P0.001;細胞晚期凋亡減少,P0.01。4.Panx-1通道抑制劑CBX減少ATP釋放和細胞內IP_3含量與單用DDP組相比,CBX與DDP合用組ATP濃度減小,P0.05;IP_3含量減小,P0.05。5.過表達Panx-1增加順鉑細胞毒性和誘導凋亡作用,增強ATP釋放和細胞內IP_3含量與單用DDP組相比,過表達Panx-1后應用DDP組細胞存活率減少,P0.001;細胞早期凋亡增加,P0.01;細胞晚期凋亡增加,P0.001;ATP濃度增加,P0.01;IP_3含量增加,P0.01。6.沉默Panx-1降低順鉑細胞毒性和誘導凋亡作用,減少ATP釋放和細胞內IP_3含量與單用DDP組相比,沉默Panx-1后應用DDP組細胞存活率增加,P0.01;細胞早期凋亡減少,P0.01;細胞晚期凋亡減少,P0.001;ATP濃度減小,P0.05。;IP_3含量減小,P0.01。7.ATP酶和光溜海綿素C降低順鉑細胞毒性和誘導凋亡作用與單用DDP組相比,ATP酶與DDP合用組ATP濃度減小,P0.05;IP_3含量減小,P0.01。與單用DDP組相比,ATP酶或光溜海綿素C與DDP合用組細胞生存率提高,P0.01;細胞早期凋亡率減小,P0.001;細胞晚期凋亡率減小,P0.01。結論:1.睪丸癌I-10細胞對順鉑產(chǎn)生耐藥性時,Panx-1表達量降低。2.Panx-1通道介導的ATP/IP_3信號通路參與了順鉑誘導睪丸癌細胞的凋亡。3.上調Panx-1通道可以增強ATP/IP_3信號通路,提高順鉑的抗腫瘤作用。
[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 鹵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.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96
本文編號：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 鹵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.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R96
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