【摘要】：目的:1.檢測pannexin1(Panx-1)蛋白在小鼠睪丸癌細(xì)胞敏感株I-10和耐藥株I-10/DDP中的表達(dá)。2.觀察Panx-1通道及其介導(dǎo)的ATP/IP_3(三磷酸肌醇)通路在順鉑誘導(dǎo)小鼠睪丸癌細(xì)胞凋亡中的作用。3.探究睪丸癌細(xì)胞對順鉑產(chǎn)生耐藥性的機(jī)制是否與Panx-1通道有關(guān)。方法:1.Western blotting法檢測I-10和I-10/DDP細(xì)胞總Panx-1蛋白表達(dá)。2.免疫熒光法檢測I-10和I-10/DDP細(xì)胞膜上Panx-1的表達(dá)。3.MTT法檢測細(xì)胞存活率。4.Annexin V/PI雙染法檢測細(xì)胞早期凋亡。5.Hoechst 33258染色法檢測細(xì)胞晚期凋亡。6.化學(xué)發(fā)光法檢測ATP濃度。7.ELISA法檢測IP_3濃度。8.質(zhì)粒sh RNA轉(zhuǎn)染對Panx-1進(jìn)行干擾和過表達(dá),G418篩選培養(yǎng)基建立穩(wěn)轉(zhuǎn)株。9.統(tǒng)計學(xué)方法:實(shí)驗(yàn)數(shù)據(jù)資料分析使用SPSS 16.0軟件,以均數(shù)±標(biāo)準(zhǔn)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(rèn)為差異有統(tǒng)計學(xué)意義。結(jié)果:1.Panx-1在耐藥株中表達(dá)量低于敏感株耐藥細(xì)胞I-10/DDP中Panx-1總蛋白表達(dá)量低于I-10細(xì)胞,P0.01;耐藥細(xì)胞I-10/DDP中Panx-1膜蛋白表達(dá)量明顯低于I-10細(xì)胞。2.Panx-1通道抑制劑CBX減輕順鉑細(xì)胞毒性與單用DDP組相比,CBX與DDP合用組細(xì)胞存活率增加,P0.01。3.Panx-1通道抑制劑CBX抑制順鉑誘導(dǎo)凋亡與單用DDP組相比,CBX與DDP合用組細(xì)胞早期凋亡減少,P0.001;細(xì)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(xì)胞內(nèi)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(dá)Panx-1增加順鉑細(xì)胞毒性和誘導(dǎo)凋亡作用,增強(qiáng)ATP釋放和細(xì)胞內(nèi)IP_3含量與單用DDP組相比,過表達(dá)Panx-1后應(yīng)用DDP組細(xì)胞存活率減少,P0.001;細(xì)胞早期凋亡增加,P0.01;細(xì)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(xì)胞毒性和誘導(dǎo)凋亡作用,減少ATP釋放和細(xì)胞內(nèi)IP_3含量與單用DDP組相比,沉默Panx-1后應(yīng)用DDP組細(xì)胞存活率增加,P0.01;細(xì)胞早期凋亡減少,P0.01;細(xì)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(xì)胞毒性和誘導(dǎo)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(xì)胞生存率提高,P0.01;細(xì)胞早期凋亡率減小,P0.001;細(xì)胞晚期凋亡率減小,P0.01。結(jié)論:1.睪丸癌I-10細(xì)胞對順鉑產(chǎn)生耐藥性時,Panx-1表達(dá)量降低。2.Panx-1通道介導(dǎo)的ATP/IP_3信號通路參與了順鉑誘導(dǎo)睪丸癌細(xì)胞的凋亡。3.上調(diào)Panx-1通道可以增強(qiáng)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(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 趙世巧;陳忠余;鄧瑋;胡仁智;黎敏;;eIF4E、VEGF-A和VEGF-C在胃癌組織中的表達(dá)及其與淋巴結(jié)侵襲轉(zhuǎn)移的相關(guān)性[J];國際檢驗(yàn)醫(yī)學(xué)雜志;2015年20期

2 鄭霽;方強(qiáng);陳志文;陳志朋;何鵬;鄧國賢;代林勇;潘進(jìn)洪;;IP3R介導(dǎo)的鈣釋放在DI發(fā)生中的作用研究[J];西部醫(yī)學(xué);2015年02期

3 朱長良;裘偉光;洪濤;;縫隙連接及縫隙連接相關(guān)疾病研究進(jìn)展[J];重慶醫(yī)學(xué);2013年15期

，

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