microRNA在TNF-α誘導(dǎo)的人臍靜脈內(nèi)皮細(xì)胞凋亡中作用及機(jī)制的研究
發(fā)布時間：2018-07-25 09:17
【摘要】： 研究目的(1)建立原代人臍靜脈內(nèi)皮細(xì)胞(HUVEC)體外培養(yǎng)方法并探討TNF-α對HUVEC凋亡程度的影響,篩選出TNF-αt誘導(dǎo)凋亡的最佳作用濃度和時間；(2)探討TNF-α誘導(dǎo)凋亡的HUVEC microRNA的差異表達(dá)譜,驗證在TNF-α誘導(dǎo)HUVEC凋亡中顯著差異表達(dá)的microRNA;(3)根據(jù)篩選結(jié)果,探討microRNA-23a在HUVEC凋亡中的調(diào)節(jié)功能。(4)研究microRNA-23a在TNF-α誘導(dǎo)的內(nèi)皮細(xì)胞凋亡中的作用機(jī)制,為對抗內(nèi)皮細(xì)胞凋亡的治療途徑提供理論和實驗依據(jù)。 研究方法實驗分為四個部分：(1)原代細(xì)胞培養(yǎng)參照文獻(xiàn)報道方法并加以改進(jìn),通過形態(tài)學(xué)觀察和免疫細(xì)胞化學(xué)法對細(xì)胞進(jìn)行鑒定；然后用不同濃度TNF-α(0、1ng/ml、10ng/ml、40ng/ml、100ng/ml)處理內(nèi)皮細(xì)胞不同時間(0、12h、24h、48h),通過電鏡,Hoechst 33258熒光染色,TUNEL法,實時熒光定量PCR及western blot檢測HUVEC的凋亡程度,確定TNF-α能夠誘導(dǎo)內(nèi)皮細(xì)胞凋亡的最佳作用濃度和時間；(2)采用μParafloTM microRNA芯片技術(shù),將4例空白對照標(biāo)本與4例TNF-α(10ng/ml,24h)誘導(dǎo)凋亡的HUVEC標(biāo)本的microRNA進(jìn)行比對,根據(jù)統(tǒng)計學(xué)的有關(guān)方法分析芯片實驗數(shù)據(jù),篩選出共同差異表達(dá)的候選microRNA;并采用熒光實時定量PCR的方法,分別驗證這些候選microRNA的差異表達(dá)情況。將芯片和熒光實時定量PCR兩種方法結(jié)論一致的候選microRNA確定為有意義的共同差異表達(dá)microRNA;(3)采用microRNA-23a抑制劑(LNA-anti-miR-23a,50nmol/L)和microRNA-23a前體(Pre-miR-23a,50nmol/L)瞬時轉(zhuǎn)染HUVEC使內(nèi)皮細(xì)胞抑制或過表達(dá)microRNA-23a,設(shè)立空白對照組,用實時熒光定量PCR法檢測轉(zhuǎn)染效率。隨后用TNF-α干預(yù)轉(zhuǎn)染后的HUVEC,通過Hoechst 33258熒光染色,TUNEL染色,實時熒光定量PCR及western blot等方法檢測不同轉(zhuǎn)染組與對照組HUVEC的凋亡程度改變；(4)使用miRanda, picTar和Targetscan軟件分析,結(jié)合文獻(xiàn)查詢和基因芯片研究結(jié)果,預(yù)測與凋亡相關(guān)的microRNA-23a的靶基因；用microRNA抑制劑轉(zhuǎn)染HUVEC使microRNA-23a表達(dá)水平下調(diào),并采用實時熒光定量PCR法和Western blot法觀察其是否引起靶基因表達(dá)增多。 研究結(jié)果(1)經(jīng)相差顯微鏡觀察以及Ⅷ因子染色證實培養(yǎng)的細(xì)胞(97.5%)為HUVEC,臺盼藍(lán)染色顯示95%以上細(xì)胞存活良好；TNF-α呈濃度依賴性和時間依賴性引起HUVEC凋亡；(2)TNF-α(10ng/ml)處理HUVEC 24h后,芯片結(jié)果示microRNA表達(dá)譜有明顯變化,其中有12個microRNA表達(dá)上調(diào),9個microRNA表達(dá)下調(diào)；實時熒光定量PCR結(jié)果證實miRNA-23a, miRNA-126表達(dá)顯著減少；(3)Hoechst 33258熒光染色,TUNEL染色,實時熒光定量PCR及western blot結(jié)果顯示：microRNA-23a抑制劑使TNF-α誘導(dǎo)的HUVEC凋亡數(shù)量明顯增多；microRNA-23a過表達(dá)使得TNF-α誘導(dǎo)的HUVEC凋亡數(shù)量明顯減少。(4)經(jīng)過生物信息學(xué)分析預(yù)測APAF-1, caspase-7和STK4可能是microR-23a的3個候選靶基因,并通過實時熒光定量PCR和Western blot證實microRNA-23a抑制劑組該3個靶基因的表達(dá)有顯著性升高。 研究結(jié)論(1)本研究在成功建立HUVEC體外培養(yǎng)方法的基礎(chǔ)上,建立了TNF-α誘導(dǎo)HUVEC凋亡的模型,確立TNF-α能夠誘導(dǎo)內(nèi)皮細(xì)胞凋亡的最佳作用濃度為10ng/ml,時間為24h；(2)運(yùn)用microRNA芯片技術(shù),首次發(fā)現(xiàn)TNF-α誘導(dǎo)凋亡的內(nèi)皮細(xì)胞中microRNA-23a表達(dá)明顯減少,microRNA-126表達(dá)也有明顯下調(diào)。這為進(jìn)一步研究microRNA在TNF-α引起的血管內(nèi)皮疾病中的功能創(chuàng)造了條件。(3) microRNA-23a能明顯抑制TNF-α介導(dǎo)的HUVEC凋亡,促進(jìn)細(xì)胞生存,起保護(hù)樣的作用。(4) microRNA-23a抑制TNF-α介導(dǎo)的HUVEC凋亡作用可能是通過轉(zhuǎn)錄后水平抑制其靶基因APAF-1、Caspase-7和STK4的表達(dá)而介導(dǎo)的。
[Abstract]:Objective (1) to establish the culture method of human umbilical vein endothelial cells (HUVEC) in vitro and to explore the effect of TNF- alpha on the apoptosis of HUVEC, to screen out the optimal concentration and time of TNF- alpha t induced apoptosis, and (2) to explore the differential expression profiles of HUVEC microRNA induced by TNF- alpha, and to verify the significant difference in the expression of TNF- alpha induced HUVEC apoptosis. MicroRNA (3) (3) according to the screening results, the regulatory function of microRNA-23a in the apoptosis of HUVEC was investigated. (4) the mechanism of microRNA-23a in the apoptosis of endothelial cells induced by TNF- a was studied to provide theoretical and experimental basis for the treatment of endothelial cell apoptosis.
The research method experiment was divided into four parts: (1) the primary cell culture was reported by reference literature and improved. The cells were identified by morphological observation and immunocytochemical method. Then different concentrations of TNF- alpha (0,1ng/ml, 10ng/ml, 40ng/ml, 100ng/ml) were used to treat endothelial cells at different time (0,12h, 24h, 48h), through electron microscopy, Hoechst 3 3258 fluorescence staining, TUNEL method, real-time fluorescence quantitative PCR and Western blot were used to detect the degree of apoptosis of HUVEC, and determine the optimum concentration and time of TNF- a to induce endothelial cell apoptosis. (2) 4 cases of blank control specimens and 4 cases of TNF- alpha (10ng/ml, 24h) were used to induce apoptotic HUVEC specimens. According to the method of statistical analysis, the experimental data of the chip are analyzed and the candidate microRNA of the common differential expression is screened. The differential expression of these candidate microRNA is verified by the method of real time fluorescence quantitative PCR, and the candidate microRNA of the two formula of the chip and real-time quantitative PCR is determined to be meaningful. (3) (3) transient transfection of HUVEC by microRNA-23a inhibitor (LNA-anti-miR-23a, 50nmol/L) and microRNA-23a precursor (Pre-miR-23a, 50nmol/L) temporarily transfected HUVEC to inhibit or overexpress microRNA-23a in endothelial cells, set up a blank control group and detect transfection efficiency by real-time fluorescent quantitative PCR. Subsequently, TNF- alpha was used to interfere with the transfected HUVEC, Hoechst 33258 fluorescence staining, TUNEL staining, real-time fluorescence quantitative PCR and Western blot were used to detect the changes in the degree of apoptosis of HUVEC in different transfected groups and control groups. (4) using miRanda, picTar and Targetscan software analysis, combined with literature query and gene chip research, the target genes related to apoptosis were predicted. The expression level of microRNA-23a was reduced by transfection of microRNA inhibitor to HUVEC, and the increase of target gene expression was observed by real-time fluorescence quantitative PCR method and Western blot method.
Results (1) the cells (97.5%) cultured in phase contrast microscope and factor VIII staining (97.5%) were HUVEC, trypan blue staining showed that more than 95% cells survived well; TNF- alpha was dependent on the concentration and time dependence to induce HUVEC apoptosis; (2) TNF- a (10ng/ml) treated HUVEC 24h, and the chip results showed that microRNA expression profiles had obvious changes. 12 microRNA expressions were up-regulated and 9 microRNA expressions were downregulated; real-time fluorescence quantitative PCR results confirmed that miRNA-23a, miRNA-126 expression decreased significantly; (3) Hoechst 33258 fluorescence staining, TUNEL staining, real-time fluorescent quantitative PCR and Western blot results showed that microRNA-23a inhibitor made the number of apoptotic apoptosis induced by TNF- alpha significantly increased; The overexpression of roRNA-23a resulted in a significant decrease in the number of apoptotic HUVEC induced by TNF- alpha. (4) through bioinformatics analysis and prediction of APAF-1, caspase-7 and STK4 may be the 3 candidate genes for microR-23a, and the expression of the 3 target genes in the microRNA-23a inhibitor group was confirmed by real-time quantitative PCR and Western blot.
Conclusions (1) on the basis of the successful establishment of HUVEC in vitro culture method, the model of TNF- alpha induced HUVEC apoptosis was established. The optimum concentration of TNF- alpha to induce endothelial cell apoptosis was 10ng/ml, time was 24h. (2) the microRNA-23a table in the endothelial cells induced by TNF- alpha induced apoptosis was first detected by microRNA chip technology. There is a significant decrease in the expression of microRNA-126. This provides a further study of the function of microRNA in the vascular endothelial disease induced by TNF- alpha. (3) microRNA-23a can obviously inhibit the apoptosis of HUVEC mediated by TNF- a, promote cell survival, and play a protective role. (4) microRNA-23a inhibits the HUVEC apoptosis mediated by TNF- a It may be mediated by the inhibition of the expression of APAF-1, Caspase-7 and STK4 after target transcription.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346
本文編號：2143354
[Abstract]:Objective (1) to establish the culture method of human umbilical vein endothelial cells (HUVEC) in vitro and to explore the effect of TNF- alpha on the apoptosis of HUVEC, to screen out the optimal concentration and time of TNF- alpha t induced apoptosis, and (2) to explore the differential expression profiles of HUVEC microRNA induced by TNF- alpha, and to verify the significant difference in the expression of TNF- alpha induced HUVEC apoptosis. MicroRNA (3) (3) according to the screening results, the regulatory function of microRNA-23a in the apoptosis of HUVEC was investigated. (4) the mechanism of microRNA-23a in the apoptosis of endothelial cells induced by TNF- a was studied to provide theoretical and experimental basis for the treatment of endothelial cell apoptosis.
The research method experiment was divided into four parts: (1) the primary cell culture was reported by reference literature and improved. The cells were identified by morphological observation and immunocytochemical method. Then different concentrations of TNF- alpha (0,1ng/ml, 10ng/ml, 40ng/ml, 100ng/ml) were used to treat endothelial cells at different time (0,12h, 24h, 48h), through electron microscopy, Hoechst 3 3258 fluorescence staining, TUNEL method, real-time fluorescence quantitative PCR and Western blot were used to detect the degree of apoptosis of HUVEC, and determine the optimum concentration and time of TNF- a to induce endothelial cell apoptosis. (2) 4 cases of blank control specimens and 4 cases of TNF- alpha (10ng/ml, 24h) were used to induce apoptotic HUVEC specimens. According to the method of statistical analysis, the experimental data of the chip are analyzed and the candidate microRNA of the common differential expression is screened. The differential expression of these candidate microRNA is verified by the method of real time fluorescence quantitative PCR, and the candidate microRNA of the two formula of the chip and real-time quantitative PCR is determined to be meaningful. (3) (3) transient transfection of HUVEC by microRNA-23a inhibitor (LNA-anti-miR-23a, 50nmol/L) and microRNA-23a precursor (Pre-miR-23a, 50nmol/L) temporarily transfected HUVEC to inhibit or overexpress microRNA-23a in endothelial cells, set up a blank control group and detect transfection efficiency by real-time fluorescent quantitative PCR. Subsequently, TNF- alpha was used to interfere with the transfected HUVEC, Hoechst 33258 fluorescence staining, TUNEL staining, real-time fluorescence quantitative PCR and Western blot were used to detect the changes in the degree of apoptosis of HUVEC in different transfected groups and control groups. (4) using miRanda, picTar and Targetscan software analysis, combined with literature query and gene chip research, the target genes related to apoptosis were predicted. The expression level of microRNA-23a was reduced by transfection of microRNA inhibitor to HUVEC, and the increase of target gene expression was observed by real-time fluorescence quantitative PCR method and Western blot method.
Results (1) the cells (97.5%) cultured in phase contrast microscope and factor VIII staining (97.5%) were HUVEC, trypan blue staining showed that more than 95% cells survived well; TNF- alpha was dependent on the concentration and time dependence to induce HUVEC apoptosis; (2) TNF- a (10ng/ml) treated HUVEC 24h, and the chip results showed that microRNA expression profiles had obvious changes. 12 microRNA expressions were up-regulated and 9 microRNA expressions were downregulated; real-time fluorescence quantitative PCR results confirmed that miRNA-23a, miRNA-126 expression decreased significantly; (3) Hoechst 33258 fluorescence staining, TUNEL staining, real-time fluorescent quantitative PCR and Western blot results showed that microRNA-23a inhibitor made the number of apoptotic apoptosis induced by TNF- alpha significantly increased; The overexpression of roRNA-23a resulted in a significant decrease in the number of apoptotic HUVEC induced by TNF- alpha. (4) through bioinformatics analysis and prediction of APAF-1, caspase-7 and STK4 may be the 3 candidate genes for microR-23a, and the expression of the 3 target genes in the microRNA-23a inhibitor group was confirmed by real-time quantitative PCR and Western blot.
Conclusions (1) on the basis of the successful establishment of HUVEC in vitro culture method, the model of TNF- alpha induced HUVEC apoptosis was established. The optimum concentration of TNF- alpha to induce endothelial cell apoptosis was 10ng/ml, time was 24h. (2) the microRNA-23a table in the endothelial cells induced by TNF- alpha induced apoptosis was first detected by microRNA chip technology. There is a significant decrease in the expression of microRNA-126. This provides a further study of the function of microRNA in the vascular endothelial disease induced by TNF- alpha. (3) microRNA-23a can obviously inhibit the apoptosis of HUVEC mediated by TNF- a, promote cell survival, and play a protective role. (4) microRNA-23a inhibits the HUVEC apoptosis mediated by TNF- a It may be mediated by the inhibition of the expression of APAF-1, Caspase-7 and STK4 after target transcription.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 崔世紅;張婷;韓笑;程國梅;楊健麗;趙嵐嵐;;腫瘤壞死因子α、X-連鎖凋亡抑制蛋白在特發(fā)性胎兒生長受限中的表達(dá)[J];實用兒科臨床雜志;2012年02期
，本文編號：2143354
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