microRNA在TNF-α诱导的人脐静脉内皮细胞凋亡中作用及机制的研究
发布时间：2018-07-25 09:17
【摘要】： 研究目的(1)建立原代人脐静脉内皮细胞(HUVEC)体外培养方法并探讨TNF-α对HUVEC凋亡程度的影响,筛选出TNF-αt诱导凋亡的最佳作用浓度和时间；(2)探讨TNF-α诱导凋亡的HUVEC microRNA的差异表达谱,验证在TNF-α诱导HUVEC凋亡中显著差异表达的microRNA;(3)根据筛选结果,探讨microRNA-23a在HUVEC凋亡中的调节功能。(4)研究microRNA-23a在TNF-α诱导的内皮细胞凋亡中的作用机制,为对抗内皮细胞凋亡的治疗途径提供理论和实验依据。 研究方法实验分为四个部分：(1)原代细胞培养参照文献报道方法并加以改进,通过形态学观察和免疫细胞化学法对细胞进行鉴定；然后用不同浓度TNF-α(0、1ng/ml、10ng/ml、40ng/ml、100ng/ml)处理内皮细胞不同时间(0、12h、24h、48h),通过电镜,Hoechst 33258荧光染色,TUNEL法,实时荧光定量PCR及western blot检测HUVEC的凋亡程度,确定TNF-α能够诱导内皮细胞凋亡的最佳作用浓度和时间；(2)采用μParafloTM microRNA芯片技术,将4例空白对照标本与4例TNF-α(10ng/ml,24h)诱导凋亡的HUVEC标本的microRNA进行比对,根据统计学的有关方法分析芯片实验数据,筛选出共同差异表达的候选microRNA;并采用荧光实时定量PCR的方法,分别验证这些候选microRNA的差异表达情况。将芯片和荧光实时定量PCR两种方法结论一致的候选microRNA确定为有意义的共同差异表达microRNA;(3)采用microRNA-23a抑制剂(LNA-anti-miR-23a,50nmol/L)和microRNA-23a前体(Pre-miR-23a,50nmol/L)瞬时转染HUVEC使内皮细胞抑制或过表达microRNA-23a,设立空白对照组,用实时荧光定量PCR法检测转染效率。随后用TNF-α干预转染后的HUVEC,通过Hoechst 33258荧光染色,TUNEL染色,实时荧光定量PCR及western blot等方法检测不同转染组与对照组HUVEC的凋亡程度改变；(4)使用miRanda, picTar和Targetscan软件分析,结合文献查询和基因芯片研究结果,预测与凋亡相关的microRNA-23a的靶基因；用microRNA抑制剂转染HUVEC使microRNA-23a表达水平下调,并采用实时荧光定量PCR法和Western blot法观察其是否引起靶基因表达增多。 研究结果(1)经相差显微镜观察以及Ⅷ因子染色证实培养的细胞(97.5%)为HUVEC,台盼蓝染色显示95%以上细胞存活良好；TNF-α呈浓度依赖性和时间依赖性引起HUVEC凋亡；(2)TNF-α(10ng/ml)处理HUVEC 24h后,芯片结果示microRNA表达谱有明显变化,其中有12个microRNA表达上调,9个microRNA表达下调；实时荧光定量PCR结果证实miRNA-23a, miRNA-126表达显著减少；(3)Hoechst 33258荧光染色,TUNEL染色,实时荧光定量PCR及western blot结果显示：microRNA-23a抑制剂使TNF-α诱导的HUVEC凋亡数量明显增多；microRNA-23a过表达使得TNF-α诱导的HUVEC凋亡数量明显减少。(4)经过生物信息学分析预测APAF-1, caspase-7和STK4可能是microR-23a的3个候选靶基因,并通过实时荧光定量PCR和Western blot证实microRNA-23a抑制剂组该3个靶基因的表达有显著性升高。 研究结论(1)本研究在成功建立HUVEC体外培养方法的基础上,建立了TNF-α诱导HUVEC凋亡的模型,确立TNF-α能够诱导内皮细胞凋亡的最佳作用浓度为10ng/ml,时间为24h；(2)运用microRNA芯片技术,首次发现TNF-α诱导凋亡的内皮细胞中microRNA-23a表达明显减少,microRNA-126表达也有明显下调。这为进一步研究microRNA在TNF-α引起的血管内皮疾病中的功能创造了条件。(3) microRNA-23a能明显抑制TNF-α介导的HUVEC凋亡,促进细胞生存,起保护样的作用。(4) microRNA-23a抑制TNF-α介导的HUVEC凋亡作用可能是通过转录后水平抑制其靶基因APAF-1、Caspase-7和STK4的表达而介导的。
[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.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346
本文编号：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.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R346
【引证文献】
相关期刊论文 前1条
1 崔世红;张婷;韩笑;程国梅;杨健丽;赵岚岚;;肿瘤坏死因子α、X-连锁凋亡抑制蛋白在特发性胎儿生长受限中的表达[J];实用儿科临床杂志;2012年02期
，本文编号：2143354
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