發(fā)布時間：2018-07-29 10:35

【摘要】：目的:觀察巨噬細(xì)胞移動抑制因子(macrophage migration inhibitory factor,MIF)在肝癌細(xì)胞系中的表達情況,探討siRNA介導(dǎo)的MIF基因沉默后對肝癌細(xì)胞系生物學(xué)特性的影響及潛在的分子機制。方法:采用實時熒光定量RT-PCR(RT-qPCR)和Western blot技術(shù)檢測MIF在正常肝細(xì)胞系L-O2與肝癌細(xì)胞系Huh7、Hep3B、BEL7402、PLC、HepG2、SMMC-7721中的mRNA及蛋白表達情況。采用脂質(zhì)體法將化學(xué)合成的MIF-siRNA干擾序列轉(zhuǎn)染肝癌細(xì)胞系SMMC-7721和HepG2,以MIF-siRNA轉(zhuǎn)染的細(xì)胞系為實驗組,以Con-siRNA轉(zhuǎn)染的細(xì)胞系為對照組,采用CCK-8及EdU熒光法檢測肝癌細(xì)胞系增殖情況;流式細(xì)胞術(shù)檢測細(xì)胞周期和細(xì)胞凋亡情況;Transwell小室法檢測肝癌細(xì)胞系的遷移能力;采用Western blot法檢測MIF及凋亡相關(guān)蛋白Bcl-2、Bax、p53以及ERK/RSK2信號通路相關(guān)蛋白的水平。結(jié)果:RT-qPCR及Western blot結(jié)果顯示,MIF在正常肝細(xì)胞系與肝癌細(xì)胞系中均表達,尤其在HepG2和SMMC-7721細(xì)胞中的相對表達量最高。CCK-8及EdU實驗結(jié)果顯示,與對照組相比,MIF基因沉默后可明顯抑制肝癌細(xì)胞系的增殖;流式細(xì)胞術(shù)結(jié)果顯示,實驗組G0/G1期細(xì)胞所占百分比明顯增加且凋亡率明顯高于對照組;Transwell實驗結(jié)果顯示,MIF基因沉默后減弱了肝癌細(xì)胞系的遷移能力;Western blot結(jié)果顯示,實驗組細(xì)胞中Bcl-2表達水平下降,Bax及p53表達水平升高,實驗組GSK-3β表達水平升高,p-ERK、p-RSK2、p-Bad和p-GSK-3β水平顯著下調(diào),而ERK、RSK2、Bad表達水平無顯著變化。結(jié)論:MIF在肝癌細(xì)胞系中均呈高表達。siRNA介導(dǎo)的MIF基因沉默后,肝癌細(xì)胞系的增殖和遷移能力減弱,細(xì)胞周期被阻滯在G0/G1期,細(xì)胞凋亡率增加,抗凋亡蛋白Bcl-2減少,促凋亡蛋白p53和Bax增加,ERK/RSK2信號通路的相關(guān)蛋白水平發(fā)生明顯變化。初步推測MIF基因沉默抑制肝癌細(xì)胞系增殖并促進凋亡可能通過調(diào)節(jié)ERK/RSK2信號通路實現(xiàn)的。
[Abstract]:Aim: to investigate the expression of macrophage migration inhibitory factor (macrophage migration inhibitory factor-MIF) in hepatocellular carcinoma (HCC) cell lines, and to investigate the effect of MIF gene silencing mediated by siRNA on the biological characteristics of HCC cell lines and its possible molecular mechanism. Methods: real-time fluorescence quantitative RT-PCR (RT-qPCR) and Western blot techniques were used to detect the expression of mRNA and protein in the normal liver cell line L-O2 and the hepatoma cell line Huh7BUE BEL7402PLC+ HepG2SMMC-7721. The chemically synthesized MIF-siRNA interference sequences were transfected into hepatoma cell lines SMMC-7721 and HepG2 by liposome method. MIF-siRNA transfected cell lines were used as experimental group and Con-siRNA transfected cell lines as control group. The proliferation of HCC cell lines was detected by CCK-8 and EdU fluorescence methods. Flow cytometry was used to detect cell cycle and apoptosis. Transwell chamber assay was used to detect the migration ability of hepatoma cell lines, and Western blot assay was used to detect the levels of MIF, Bcl-2Bax-p53 and ERK/RSK2 signaling pathway related proteins. Results the results of 1: RT-qPCR and Western blot showed that both of them were expressed in normal liver cell line and hepatoma cell line, especially in HepG2 and SMMC-7721 cells. The results of CCK-8 and EdU experiments showed that the expression was the highest in the normal liver cell line and the hepatoma cell line, especially in the HepG2 and SMMC-7721 cells. Compared with the control group, the silencing of MIF gene could significantly inhibit the proliferation of hepatoma cell line. The percentage of G0/G1 phase cells in the experimental group was significantly increased and the apoptosis rate was significantly higher than that in the control group. The results showed that the silencing of the G0/G1 gene decreased the migration ability of the hepatoma cell line. Western blot showed that the apoptosis rate was significantly higher in the experimental group than in the control group. In the experimental group, the expression of Bcl-2 decreased and the expression of Bax and p53 increased. The expression of GSK-3 尾 in the experimental group increased and the levels of p-ERKP- RSK2, p-Bad and p-GSK-3 尾 were down-regulated, but the expression of ERK-RSK2Bad did not change significantly. Conclusion after the silencing of MIF gene mediated by .siRNA, the proliferation and migration ability of HCC cell line was decreased, the cell cycle was blocked in G0/G1 phase, the apoptosis rate was increased, and the anti-apoptotic protein Bcl-2 was decreased. Apoptotic protein p53 and Bax increased the level of ERK / RSK2 signaling pathway. It is suggested that MIF gene silencing inhibits the proliferation of HCC cell lines and promotes apoptosis by regulating the ERK/RSK2 signaling pathway.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

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