【摘要】：目的:探討SIRT3對(duì)肝癌細(xì)胞化療藥物敏感性影響的分子機(jī)制。方法:1通過(guò)Western blot和q PCR檢測(cè)不同化療藥物作用下,SIRT3在肝癌細(xì)胞和永生化肝細(xì)胞中的表達(dá)水平。2通過(guò)MTS實(shí)驗(yàn)檢測(cè)在化療藥物不同濃度作用下,過(guò)表達(dá)SIRT3對(duì)肝癌細(xì)胞SMMC-7721、Huh-7和PLC/PRF/5細(xì)胞活率的影響。3通過(guò)流式細(xì)胞術(shù)檢測(cè)過(guò)表達(dá)SIRT3后,化療藥物處理組和對(duì)照組中肝癌細(xì)胞的凋亡比例;通過(guò)Western blot檢測(cè)凋亡相關(guān)蛋白PARP和Caspase9的表達(dá)水平。4通過(guò)MTS和流式細(xì)胞術(shù)分別檢測(cè)在化療藥物的作用下,沉默SIRT3對(duì)肝癌細(xì)胞SMMC-7721和Huh-7細(xì)胞活率和凋亡比例的影響。5通過(guò)Western blot檢測(cè)不同濃度索拉菲尼對(duì)SIRT3在肝癌細(xì)胞中表達(dá)水平的影響,MTS和流式細(xì)胞術(shù)分別檢測(cè)過(guò)表達(dá)SIRT3對(duì)肝癌細(xì)胞SMMC-7721、Huh-7和PLC/PRF/5細(xì)胞活率和凋亡比例的影響。6通過(guò)Western blot和q PCR分別檢測(cè)在化療藥物處理組和對(duì)照組中,過(guò)表達(dá)SIRT3對(duì)肝癌細(xì)胞中GSTP1蛋白水平和m RNA水平的影響;以及磷酸化蛋白p-JNK、總蛋白JNK、磷酸化蛋白p-c-Jun、總蛋白c-Jun蛋白的影響;進(jìn)一步通過(guò)IP實(shí)驗(yàn)檢測(cè)過(guò)表達(dá)SIRT3對(duì)肝癌細(xì)胞SMMC-7721中GSTP1與SIRT3結(jié)合的影響。7通過(guò)流式細(xì)胞術(shù)和Western blot檢測(cè)在不同化療藥物作用下,過(guò)表達(dá)GSTP1或?qū)Ψ�(wěn)定表達(dá)SIRT3的肝癌細(xì)胞凋亡比例和凋亡相關(guān)蛋白PARP的影響。通過(guò)流式細(xì)胞術(shù)檢測(cè)JNK抑制劑SP600125對(duì)HCC細(xì)胞凋亡的影響。結(jié)果:1在三種肝癌細(xì)胞SMMC-7721、Huh-7和PLC/PRF/5中,SIRT3的蛋白水平和m RNA水平明顯低于永生化肝細(xì)胞MIHA;并隨著化療藥物濃度的增加,SIRT3在肝癌細(xì)胞中的表達(dá)逐漸降低。2過(guò)表達(dá)SIRT3可以促進(jìn)肝癌細(xì)胞SMMC-7721、Huh-7和PLC/PRF/5對(duì)三種化療藥物的敏感性。3在不同化療藥物作用下,過(guò)表達(dá)SIRT3可以增加肝癌細(xì)胞SMMC-7721、Huh-7和PLC/PRF/5的凋亡比例,并促進(jìn)凋亡相關(guān)蛋白PARP和Caspase9的剪切。4 SIRT3基因沉默可以降低肝癌細(xì)胞SMMC-7721和Huh-7對(duì)化療藥物的敏感性。5在索拉菲尼的作用下,不同藥物濃度可以降低肝癌細(xì)胞中SIRT3的蛋白表達(dá)水平,并且SIRT3過(guò)表達(dá)可以促進(jìn)肝癌細(xì)胞SMMC-7721和Huh-7對(duì)索拉菲尼的敏感性。6在不同化療藥物作用下,過(guò)表達(dá)SIRT3可以下調(diào)肝癌細(xì)胞中GSTP1蛋白水平和m RNA水平,Western blot證明JNK和c-Jun蛋白磷酸化水平增高;進(jìn)一步,IP實(shí)驗(yàn)證實(shí)GSTP1與JNK的結(jié)合減少。7在不同化療藥物作用下,過(guò)表達(dá)GSTP1或JNK抑制劑SP600125可以拮抗SIRT3過(guò)表達(dá)對(duì)化療藥物處理下肝癌細(xì)胞凋亡的促進(jìn)作用。結(jié)論:SIRT3通過(guò)GSTP1/JNK信號(hào)通路促進(jìn)肝癌細(xì)胞對(duì)化療藥物的敏感性。
[Abstract]:Objective: to investigate the molecular mechanism of the effect of SIRT3 on chemotherapeutic drug sensitivity of hepatoma cells. Methods Western blot and Q PCR were used to detect the expression of SIRT3 in hepatoma cells and immortalized hepatocytes. 2. The expression of SIRT3 in hepatoma cells and immortalized hepatocytes was detected by MTS assay. The effect of overexpression of SIRT3 on the viability of SMMC-7721,Huh-7 and PLC/PRF/5 cells. 3 after overexpression of SIRT3 by flow cytometry, the apoptotic ratio of HCC cells in chemotherapeutic treatment group and control group was detected. Expression of apoptosis-related proteins PARP and Caspase9 were detected by Western blot. MTS and flow cytometry were used to detect the expression of apoptosis-related proteins under the action of chemotherapeutic drugs, respectively. Effects of silencing SIRT3 on the viability and apoptosis of SMMC-7721 and Huh-7 cells the effect of different concentrations of Solafenil on the expression of SIRT3 in Hepatocellular carcinoma cells by Western blot Assay and flow Cytometry The effect of SMMC-7721,Huh-7 and PLC/PRF/5 on cell viability and apoptosis in cancer cells. 6. Western blot and Q PCR were used to detect the cell viability and apoptosis in chemotherapeutic and control groups, respectively. The effects of overexpression of SIRT3 on the levels of GSTP1 protein and m RNA, and the effects of phosphorylated protein p-JNK, total protein JNK, phosphorylated protein p-c-Junand total protein c-Jun protein on the levels of GSTP1 protein and m RNA in hepatoma cells. Furthermore, the effect of overexpression of SIRT3 on the binding of GSTP1 to SIRT3 in hepatocellular carcinoma cell SMMC-7721 was detected by IP assay. 7 under different chemotherapeutic agents, flow cytometry and Western blot were used. Overexpression of GSTP1 or effect on apoptosis rate and apoptosis-related protein PARP of HCC cells stably expressing SIRT3. The effect of JNK inhibitor SP600125 on apoptosis of HCC cells was detected by flow cytometry. Results the protein level and m RNA level of sir3 in SMMC-7721,Huh-7 and PLC/PRF/5 were significantly lower than those in immortalized hepatocytes MIHA;. With the increase of chemotherapeutic drug concentration, the expression of SIRT3 in HCC cells decreased gradually, and the overexpression of SIRT3 could be promoted. The sensitivity of SMMC-7721,Huh-7 and PLC/PRF/5 to three kinds of chemotherapeutic drugs in hepatoma cells was different. Overexpression of SIRT3 could increase the apoptotic ratio of SMMC-7721,Huh-7 and PLC/PRF/5, and promote the silencing of apoptosis related protein PARP and Caspase9. 4 SIRT3 gene silencing could reduce the sensitivity of SMMC-7721 and Huh-7 to chemotherapeutic drugs. Different concentrations of drugs decreased the expression of SIRT3 protein in hepatoma cells, and the overexpression of SIRT3 could promote the sensitivity of SMMC-7721 and Huh-7 to Solafenib in different chemotherapeutic agents. Overexpression of SIRT3 could down-regulate the levels of GSTP1 protein and m RNA in hepatocellular carcinoma cells. Western blot showed that the phosphorylation of JNK and c-Jun protein was increased, and further IP assay showed that the combination of GSTP1 and JNK decreased by 7. 7 in different chemotherapeutic agents. Overexpression of GSTP1 or JNK inhibitor SP600125 could antagonize the effect of overexpression of SIRT3 on apoptosis of hepatoma cells treated with chemotherapeutic drugs. Conclusion: SIRT3 promotes the sensitivity of hepatocellular carcinoma cells to chemotherapeutic drugs through GSTP1/JNK signaling pathway.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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相關(guān)期刊論文 前2條

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本文編號(hào)：2239144