本文選題：凋亡 + Eaf2　； 參考：《南方醫(yī)科大學(xué)》2017年博士論文

【摘要】：1前言目前白內(nèi)障患者導(dǎo)致的眼盲占全世界眼盲患者的50%,現(xiàn)在公認(rèn)眼內(nèi)晶狀體受到氧化應(yīng)激的損傷是白內(nèi)障的主要原因。上皮細(xì)胞是氧化應(yīng)激的主要目標(biāo)。研究表明,過(guò)氧化氫誘導(dǎo)的氧化應(yīng)激能夠刺激人晶狀體上皮(HLE)細(xì)胞凋亡,還有研究顯示除了先天性白內(nèi)障以外,其他各種類型的白內(nèi)障發(fā)生的細(xì)胞學(xué)基礎(chǔ)都被認(rèn)為是和細(xì)胞凋亡有關(guān)。因此通過(guò)研究人HLE細(xì)胞凋亡的機(jī)制可能會(huì)為白內(nèi)障防治提供更多理論依據(jù)。2目的研究在氧化應(yīng)激誘導(dǎo)的HLE細(xì)胞的凋亡中Eaf2基因所起的作用和相關(guān)分子機(jī)制,并進(jìn)一步探討Eaf2基因如何通過(guò)抑制caspase-3活性、激活Wnt信號(hào)通路保護(hù)HLE細(xì)胞抑制過(guò)氧化氫導(dǎo)致的凋亡。3方法3.1細(xì)胞培養(yǎng):在含20%FBS的MEM培養(yǎng)基中培養(yǎng)HLE-B3細(xì)胞。培養(yǎng)條件:5%CO2、飽和濕度、37℃。3.2過(guò)氧化氫誘導(dǎo)HLE-B3細(xì)胞凋亡:將HLE-B3細(xì)胞用50μM過(guò)氧化氫別處理 4h、8h和 12h。3.3載體構(gòu)建和細(xì)胞轉(zhuǎn)染:構(gòu)建Eaf2的過(guò)表達(dá),Eaf2基因的CDS全長(zhǎng)被克隆并連接到pcDNA3.0載體;用Wnt3a干擾載體(Wnt3ashRNA)敲低Wnt3a,Wnt3ashRNA購(gòu)自SantaCruz生物,同時(shí)匹配其NC,shCon(干擾載體空載)。確認(rèn)Eaf2過(guò)表達(dá)的細(xì)胞,同時(shí)轉(zhuǎn)染shWnt3a/shCon。3.4檢測(cè)分析細(xì)胞凋亡:將過(guò)氧化氫處理HLEC-B3細(xì)胞后,用流式儀檢測(cè)細(xì)胞,計(jì)算凋亡率。3.5 RNA提取和RT-PCR分析:采用TRIZOL法從細(xì)胞中提取總RNA,將RNA逆轉(zhuǎn)成cDNA,用2-(?)Ct計(jì)算方法來(lái)比較各組間β-catenin,caspase 3,Eaf2,and Wnt3a mRNA的表達(dá)水平。3.6 免疫印跡分析:采用 western blot 檢測(cè) β-catenin,caspase 3,Eaf2,and Wnt3a 的蛋白水平。3.7免疫細(xì)胞化學(xué):免疫熒光檢測(cè)Eaf2和Wnt3a在細(xì)胞中的定位情況。4結(jié)果4.1.構(gòu)建H202誘導(dǎo)的HLE細(xì)胞凋亡的模型。分別用50μM H2O2處理HLE細(xì)胞4、8、12小時(shí)后,凋亡細(xì)胞的比率均顯著高于對(duì)照組(無(wú)H2O2處理組),并且凋亡率增加具有時(shí)間依賴性。RT-PCR和westemBlot結(jié)果均顯示當(dāng)細(xì)胞暴露于H2O2后,HLE-B3細(xì)胞中caspase 3mRNA和蛋白水平均明顯上調(diào),并且具有時(shí)間依賴性;Eaf2mRNA和蛋白水平顯著下調(diào),并且具有時(shí)間依賴性。4.2 Eaf2過(guò)表達(dá)抑制H2O2誘導(dǎo)的HLE細(xì)胞凋亡。Eaf2高水平過(guò)表達(dá)下,凋亡細(xì)胞的比例顯著降低(p0.001),同時(shí)LDH釋放量顯著降低(p0.05)。RT-PCR和western blot分析顯示Eaf2過(guò)表達(dá)的細(xì)胞中,凋亡相關(guān)蛋白caspase 3、Bax的mRNA和蛋白水平顯著下調(diào);Bcl-2的mRNA和蛋白達(dá)水平顯著上調(diào),說(shuō)明Eaf2基因通過(guò)調(diào)節(jié)凋亡相關(guān)蛋白的表達(dá)而抑制過(guò)氧化氫誘導(dǎo)晶狀體上皮細(xì)胞的凋亡。Eaf2過(guò)表達(dá)的細(xì)胞中Wnt3a和Wnt通路的關(guān)鍵蛋白β-catenin的mRNA和蛋白達(dá)水平均顯著上調(diào);Wnt通路中的抑制劑GSK3β蛋白水平明顯下調(diào),其磷酸化物p-GSK3β蛋白水平明顯上調(diào),提示Eaf2 and Wnt3a信號(hào)通路有密切的關(guān)系。免疫熒光也顯示Eaf2過(guò)表達(dá)細(xì)胞中Eaf2和Wnt3a明顯上調(diào)。4.3 Eaf2通過(guò)調(diào)控Wnt3a抑制HLE細(xì)胞中H202誘導(dǎo)的凋亡轉(zhuǎn)染shWnt3a質(zhì)粒干擾Wnt3a表達(dá)后,即使在Eaf2過(guò)表達(dá)情況下,HLE-B3細(xì)胞凋亡總比率仍顯著增加,同時(shí)使已經(jīng)被壓抑的LDH釋放量增加。RT-PCR和western blot顯示W(wǎng)nt3a敲低后caspase 3、Bax的mRNA和蛋白水平明顯上調(diào);Bcl-2的mRNA和蛋白水平明顯下調(diào),β-catenin、p-GSK3β的蛋白水平明顯下調(diào);GSK3β蛋白水平明顯上調(diào)。說(shuō)明Wnt3a被敲低后會(huì)影響到Eaf2對(duì)于HLE細(xì)胞的保護(hù)作用,Eaf2是通過(guò)調(diào)控Wnt3a抑制HLE細(xì)胞中H202誘導(dǎo)的凋亡。5.結(jié)論5.1 H2O2可以誘導(dǎo)晶狀體上皮細(xì)胞發(fā)生凋亡。5.2在晶狀體上皮細(xì)胞中可有效地轉(zhuǎn)染Eaf20E質(zhì)粒及shWnt3a質(zhì)粒。5.3 Eaf2蛋白對(duì)過(guò)氧化氫誘導(dǎo)的晶狀體上皮細(xì)胞的凋亡具有抑制作用。5.4第一次證明Eaf2基因轉(zhuǎn)錄產(chǎn)物可以保護(hù)HLE細(xì)胞免受暴露于過(guò)氧化氫導(dǎo)致的氧化應(yīng)激損害。5.5 Eaf2通過(guò)調(diào)節(jié)凋亡相關(guān)蛋白(caspase 3、Bax、Bcl-2)的表達(dá)保護(hù)HLE細(xì)胞,抑制過(guò)氧化氫誘導(dǎo)的凋亡。6、Eaf2通過(guò)對(duì)Wnt3a信號(hào)通路的作用抑制過(guò)氧化氫導(dǎo)致的氧化應(yīng)激損害。
[Abstract]:1 the eye blindness caused by cataract patients accounts for 50% of the patients with blind eye blindness at present. It is now recognized that the main cause of cataract is the damage of oxidative stress in the intraocular lens. The epithelial cells are the main target of oxidative stress. The study shows that oxidative stress induced by hydrogen peroxide can stimulate apoptosis of human lens epithelial (HLE) cells, and the results suggest that the oxidative stress induced by hydrogen peroxide can induce apoptosis of human lens epithelial cells. In addition to congenital cataracts, the cytological basis of various types of cataracts is considered to be associated with cell apoptosis. Therefore, the mechanism of apoptosis in human HLE cells may provide more theoretical basis for the prevention and treatment of cataracts based on.2 to study the Eaf2 gene in the apoptosis of HLE cells induced by oxidative stress. The role and molecular mechanism of the Eaf2 gene, and further explore how the Eaf2 gene can activate the Wnt signaling pathway to protect HLE cells by inhibiting the activity of Caspase-3, to inhibit the culture of.3 cell apoptosis induced by hydrogen peroxide: the culture of HLE-B3 cells in the MEM medium containing 20%FBS. Culture conditions: 5%CO2, saturated humidity, and 37 C.3.2 hydrogen peroxide induced HLE-B3 cell apoptosis: HLE-B3 cells were treated with 50 u M hydrogen peroxide with 4h, 8h and 12h.3.3 vector construction and cell transfection: the overexpression of Eaf2 was constructed. The CDS full length of the Eaf2 gene was cloned and connected to the pcDNA3.0 vector; Confirm Eaf2 overexpressed cells and transfect shWnt3a/shCon.3.4 to detect cell apoptosis: after treating HLEC-B3 cells with hydrogen peroxide, the cells were detected by flow cytometry,.3.5 RNA extraction and RT-PCR analysis were calculated: TRIZOL method was used to extract total RNA, RNA was reversed into cDNA, and 2- (?) Ct calculation method was used to compare each other. The expression level of beta -catenin, caspase 3, Eaf2, and Wnt3a mRNA was analyzed by.3.6 Western blot analysis, and Western blot was used to detect beta -catenin, caspase 3, Eaf2, and immunocytochemistry. The percentage of apoptotic cells was significantly higher than that of the control group (no H2O2 treatment group) after HLE cells treated with 50 M H2O2 respectively. The increase of apoptosis rate had time dependent.RT-PCR and westemBlot results all showed that when the cells were exposed to H2O2, caspase 3mRNA and protein levels in HLE-B3 cells were obviously up-regulated and time dependent. The Eaf2mRNA and protein levels were significantly down, and the time dependent.4.2 Eaf2 overexpressed H2O2 induced HLE cell apoptosis.Eaf2 high level overexpression, the percentage of apoptotic cells decreased significantly (p0.001), and the release of LDH decreased significantly (P0.05).RT-PCR and Western blot analysis showed the apoptotic phase in the expressed cells. The level of mRNA and protein of Bax, caspase 3, and protein level of Bcl-2 are significantly down regulated, and the level of mRNA and protein content of Bcl-2 is significantly up-regulated, indicating that the Eaf2 gene inhibits the mRNA and protein reaches of the key protein beta -catenin of the Wnt3a and Wnt pathway in the cells of the lens epithelial cells by the regulation of the expression of apoptosis related proteins. The level of GSK3 beta protein in the Wnt pathway was obviously down, and the level of the phosphorylated p-GSK3 beta protein was obviously up-regulated, suggesting a close relationship between the Eaf2 and Wnt3a signaling pathway. The immunofluorescence also showed that Eaf2 and Wnt3a obviously up.4.3 Eaf2 in the Eaf2 overexpressed cells were induced by regulating the induction of Wnt3a inhibition cells. After the apoptosis transfected shWnt3a plasmid interfered with Wnt3a expression, the total apoptosis ratio of HLE-B3 cells increased significantly even in the Eaf2 overexpression, and the increased LDH release of the already suppressed.RT-PCR and Western blot showed caspase 3 after Wnt3a knocking down, Bax mRNA and protein levels were obviously up-regulated. The protein level of beta -catenin, p-GSK3 beta was obviously down, and the level of GSK3 beta protein was obviously up-regulated. It indicated that Wnt3a was knocked down to affect the protective effect of Eaf2 on HLE cells. Eaf2 was the conclusion that Wnt3a inhibited the.5. apoptosis of H202 induced H202 in HLE cells, and the apoptosis of lens epithelial cells could be induced in lens epithelial cells. The Eaf20E plasmid and shWnt3a plasmid.5.3 Eaf2 protein could be effectively transfected to the apoptosis of the lens epithelial cells induced by hydrogen peroxide..5.4 first demonstrated that the Eaf2 gene transcription products could protect HLE cells from oxidative stress damage caused by hydrogen peroxide, and.5.5 Eaf2 by regulating apoptosis related proteins (caspase) (caspase) The expression of 3, Bax, Bcl-2) protects HLE cells and inhibits the apoptotic.6 induced by hydrogen peroxide. Eaf2 inhibits oxidative stress induced by hydrogen peroxide through the role of Wnt3a signaling pathway.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R776.1

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7 袁媛;K562細(xì)胞中Wnt5a介導(dǎo)不同受體途徑激活經(jīng)典或非經(jīng)典Wnt信號(hào)通路及其機(jī)制的研究[D];第三軍醫(yī)大學(xué);2011年

8 劉娜;炎癥微環(huán)境影響下Wnt信號(hào)通路對(duì)牙周膜干細(xì)胞骨向分化的調(diào)控機(jī)制研究[D];第三軍醫(yī)大學(xué);2011年

9 徐揚(yáng);R-spondin及其受體在Wnt信號(hào)通路中相互作用的研究[D];吉林大學(xué);2013年

10 華軍益;Wnt信號(hào)通路與動(dòng)脈損傷后再狹窄的關(guān)系研究[D];浙江大學(xué);2012年

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