本文選題：高遷移率族蛋白1　切入點(diǎn)：子宮內(nèi)膜癌　出處：《中南大學(xué)》2014年碩士論文

【摘要】：目的：通過RNA干擾抑制子宮內(nèi)膜癌細(xì)胞中高遷移率族蛋白1(High mobility group box1, HMGB1)的表達(dá),研究HMGB1對(duì)子宮內(nèi)膜癌細(xì)胞的增殖及細(xì)胞周期、細(xì)胞凋亡的影響,并探討其分子機(jī)制。 方法：構(gòu)建靶向HMGB1基因的慢病毒shRNA載體,轉(zhuǎn)染子宮內(nèi)膜癌細(xì)胞株HEC-1A,同時(shí)利用實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)法(Real time-PCR)和免疫印跡法(Western Blot)檢測轉(zhuǎn)染HMGB1-siRNA后細(xì)胞HMGB1mRNA和蛋白表達(dá)水平的變化。MTT法和流式細(xì)胞術(shù)分別檢測轉(zhuǎn)染HMGB1-siRNA后HEC-1A細(xì)胞增殖、細(xì)胞周期和細(xì)胞凋亡的變化。Western Blot法檢測轉(zhuǎn)染HMGB1-siRNA后細(xì)胞AKT、pAKT和CyclinD1蛋白的表達(dá)水平。 結(jié)果： 1、子宮內(nèi)膜癌細(xì)胞株HEC-1A轉(zhuǎn)染慢病毒重組質(zhì)粒后,熒光顯微鏡下可見細(xì)胞表達(dá)較強(qiáng)的熒光信號(hào),轉(zhuǎn)染效率達(dá)80%左右,說明轉(zhuǎn)染成功。 2、子宮內(nèi)膜癌細(xì)胞轉(zhuǎn)染慢病毒72h后,Real time-PCR結(jié)果顯示：實(shí)驗(yàn)組細(xì)胞中HMGB1mRNA表達(dá)較空白對(duì)照組和陰性對(duì)照組降低(P0.05); Western Blot檢測細(xì)胞中HMGB1蛋白表達(dá)顯示：實(shí)驗(yàn)組明顯低于空白對(duì)照組和陰性對(duì)照組(P0.01)。 3、MTT檢測結(jié)果顯示：實(shí)驗(yàn)組細(xì)胞增殖率約為72.03%,較空白對(duì)照組和陰性對(duì)照組顯著降低(P0.01)；流式細(xì)胞術(shù)檢測細(xì)胞周期分布結(jié)果顯示：實(shí)驗(yàn)組G0/G1期細(xì)胞比例較空白對(duì)照組和陰性對(duì)照組明顯增加(P0.01),而實(shí)驗(yàn)組S期和G2/M期細(xì)胞比例均顯著低于空白對(duì)照組和陰性對(duì)照組(P0.01)。 4、Annexin V-FITCPI雙染法檢測細(xì)胞凋亡情況顯示：實(shí)驗(yàn)組早期凋亡率、中晚期細(xì)胞凋亡率以及細(xì)胞總凋亡率均高于空白對(duì)照組和陰性對(duì)照組(P0.01)。 5、三組細(xì)胞中AKT蛋白表達(dá)無明顯差異(P0.05),而實(shí)驗(yàn)組細(xì)胞中pAKT和CyclinD1蛋白表達(dá)水平均較空白對(duì)照組和陰性對(duì)照組降低(P0.01)。 結(jié)論：慢病毒介導(dǎo)的HMGB1shRNA重組質(zhì)�？捎行б种谱訉m內(nèi)膜癌細(xì)胞中HMGB1mRNA和蛋白的表達(dá)；沉默子宮內(nèi)膜癌細(xì)胞中HMGB1基因表達(dá),可抑制細(xì)胞增殖,且使細(xì)胞周期阻滯于G0/G1期,并誘導(dǎo)細(xì)胞凋亡；在子宮內(nèi)膜癌中,HMGB1可能通過PI3K/AKT信號(hào)通路影響細(xì)胞增殖過程。
[Abstract]:Aim: to study the effect of HMGB1 on the proliferation, cell cycle and apoptosis of endometrial carcinoma cells by inhibiting the expression of high mobility group 1(High mobility group box1 (HMGB1) in endometrial carcinoma cells by RNA interference, and to explore its molecular mechanism.Methods: a lentivirus shRNA vector targeting HMGB1 gene was constructed.Transfection of endometrial carcinoma cell line HEC-1A, real-time fluorescent quantitative polymerase chain reaction (Real time-PCR) and Western blotting were used to detect the changes of HMGB1mRNA and protein expression after transfection of HMGB1-siRNA. MTT assay and flow cytometry were used to detect the expression of HMGB1mRNA and protein, respectively.HEC-1A cells proliferated after HMGB1-siRNA staining.The changes of cell cycle and apoptosis. Western Blot assay was used to detect the expression of AKT pAKT and CyclinD1 protein after HMGB1-siRNA transfection.Results:1. After transfection of lentivirus recombinant plasmid into endometrial cancer cell line HEC-1A, strong fluorescent signal could be observed under fluorescence microscope, and the transfection efficiency was about 80%, which indicated that the transfection was successful.2. After 72 hours of lentivirus transfection of endometrial cancer cells, the results of Real time-PCR showed that the expression of HMGB1mRNA in the experimental group was lower than that in the blank control group and the negative control group, and the expression of HMGB1 protein in the experimental group was significantly lower than that in the blank control group and the negative control group.Control group and negative control group (P 0.01).The cell proliferation rate of the experimental group was about 72.03, which was significantly lower than that of the blank control group and the negative control group, while the cell cycle distribution of the experimental group in G0/G1 phase was significantly lower than that of the blank control group and the control group by flow cytometry.The percentage of cells in S phase and G 2 / M phase in the negative control group was significantly lower than that in the blank control group and the negative control group.4Annexin V-FITCPI double staining showed that the early apoptosis rate, the late apoptosis rate and the total apoptosis rate in the experimental group were higher than those in the blank control group and the negative control group (P 0.01).There was no significant difference in the expression of AKT protein among the three groups, but the expression levels of pAKT and CyclinD1 in the experimental group were lower than those in the blank control group and the negative control group.Conclusion: lentivirus-mediated HMGB1shRNA recombinant plasmid can effectively inhibit the expression of HMGB1mRNA and protein in endometrial cancer cells, silence the expression of HMGB1 gene in endometrial cancer cells, inhibit cell proliferation, and block cell cycle at G0/G1 stage.In endometrial carcinoma, HMGB1 may affect cell proliferation through PI3K/AKT signaling pathway.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.33

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