microRNA在TNF-α誘導的人臍靜脈內皮細胞凋亡中作用及機制的研究
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皮細胞(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誘導凋亡的最佳作用濃度和時間；(2)探討TNF-α誘導凋亡的HUVEC microRNA的差異表達譜,驗證在TNF-α誘導HUVEC凋亡中顯著差異表達的microRNA;(3)根據(jù)篩選結果,探討microRNA-23a在HUVEC凋亡中的調節(jié)功能。(4)研究microRNA-23a在TNF-α誘導的內皮細胞凋亡中的作用機制,為對抗內皮細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)原代細胞培養(yǎng)參照文獻報道方法并加以改進,通過形態(tài)學觀察和免疫細胞化學法對細胞進行鑒定；然后用不同濃度TNF-α(0、1ng/ml、10ng/ml、40ng/ml、100ng/ml)處理內皮細胞不同時間(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能夠誘導內皮細胞凋亡的最佳作用濃度和時間；(2)采用μParafloTM microRNA芯片技術,將4例空白對照標本與4例TNF-α(10ng/ml,24h)誘導凋亡的HUVEC標本的microRNA進行比對,根據(jù)統(tǒng)計學的有關方法分析芯片實驗數(shù)據(jù),篩選出共同差異表達的候選microRNA;并采用熒光實時定量PCR的方法,分別驗證這些候選microRNA的差異表達情況。將芯片和熒光實時定量PCR兩種方法結論一致的候選microRNA確定為有意義的共同差異表達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)瞬時轉染HUVEC使內皮細胞抑制或過表達microRNA-23a,設立空白對照組,用實時熒光定量PCR法檢測轉染效率。隨后用TNF-α干預轉染后的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,結合文獻查詢和基因芯片研究結果,預測與凋亡相關的microRNA-23a的靶基因；用microRNA抑制劑轉染HUVEC使microRNA-23a表達水平下調,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增多。 研究結果(1)經相差顯微鏡觀察以及Ⅷ因子染色證實培養(yǎng)的細胞(97.5%)為HUVEC,臺盼藍染色顯示95%以上細胞存活良好；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,芯片結果示microRNA表達譜有明顯變化,其中有12個microRNA表達上調,9個microRNA表達下調；實時熒光定量PCR結果證實miRNA-23a, miRNA-126表達顯著減少；(3)Hoechst 33258熒光染色,TUNEL染色,實時熒光定量PCR及western blot結果顯示：microRNA-23a抑制劑使TNF-α誘導的HUVEC凋亡數(shù)量明顯增多；microRNA-23a過表達使得TNF-α誘導的HUVEC凋亡數(shù)量明顯減少。(4)經過生物信息學分析預測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個靶基因的表達有顯著性升高。 研究結論(1)本研究在成功建立HUVEC體外培養(yǎng)方法的基礎上,建立了TNF-α誘導HUVEC凋亡的模型,確立TNF-α能夠誘導內皮細胞凋亡的最佳作用濃度為10ng/ml,時間為24h；(2)運用microRNA芯片技術,首次發(fā)現(xiàn)TNF-α誘導凋亡的內皮細胞中microRNA-23a表達明顯減少,microRNA-126表達也有明顯下調。這為進一步研究microRNA在TNF-α引起的血管內皮疾病中的功能創(chuàng)造了條件。(3) microRNA-23a能明顯抑制TNF-α介導的HUVEC凋亡,促進細胞生存,起保護樣的作用。(4) microRNA-23a抑制TNF-α介導的HUVEC凋亡作用可能是通過轉錄后水平抑制其靶基因APAF-1、Caspase-7和STK4的表達而介導的。
[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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R346
【引證文獻】
相關期刊論文 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[J];實用兒科臨床雜志;2012年02期
，本文編號：2143354
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