【作者】 阮溦；

【导师】 徐军美；

【作者基本信息】 中南大学 ， 麻醉学， 2010， 博士

【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。更多还原

【Abstract】 Objective (1)To establish culturing technique of HUVECs in vitro. To investigate the effects of TNF-a on apoptosis of HUVECs and then to decide the optimal action time course and concentration; (2) To investigate the effects of TNF-a on the differential expression of microRNAs of HUVECs and to establish the relationship between HUVEC apoptosis and the expression of microRNAs; (3) To select predominantly differential expressed microRNAs of HUVECs after treatment of TNF-αand to investigate their effects on TNF-a induced apoptosis of HUVECs; (4) To investigate the action mechanism of microRNA-23a, so as to provide theoretical and experimental evidence for the elucidation of pathogenesis of apoptosis as well as development of the therapeutic strategies.Methods (1) HUVECs were cultured according to references with minor modifications. Endothelial cells were identified by both morpho-logy and immunochemistry response to factorⅧrelated antigen. We measured the extent of TNF-αinduced apoptosis in HUVECs at different concentrations (0,1,10,40,100ng/ml) by different time course (0,12h,24h,48h) by methods of Hoechst 33258 stain、TUNEL stain、qRT-PCR and western blot; (2) RNA was extracted from 4 negative control samples of HUVECs and 4 TNF-a treated samples, and small RNA (200bp) was isolated. Then we detected microRNA profiles in these samples by microRNA microarray (http://microrna.sanger.ac.uk SangermiRNA. May,2009). Meanwhile, we examined the expression of microRNA candidates in these samples by fluorescence based real-time quantitative PCR. Those microRNAs that were identified as differentially expressed by both microRNA microarray and qRT-PCR were considered as significant microRNAs; (3) HUVECs were transfected with LNA-anti-miR-23a (50 nmol/L)/Pre-miR-23a (50 nmol/L) and the transfectional efficacy were measured by qRT-PCR. Following the transfection, HUVECs were treated with TNF-a and the extent of apoptosis was detected by methods of Hoechst 33258 stain、TUNEL stain、qRT-PCR and western blot; (4) The mRNA levels of the potential target genes of microRNA-23a, such as APAF-1, Caspase-7 and STK4 were detected by qRT-PCR and the protein expression of which were examined by Western blot.Results (1) HUVECs present cobblestone-shaped and a positive immunochemistry reaction (97.5%). The TNF-a mediated increase in HUVEC apoptosis is in a time-dependent manner and a dose-dependent manner; (2) There are significant differential expressions of microRNAs after TNF-a treatment (p<0.05),12 microRNAs were up-regulated,9 microRNAs were down-regulated. Among them microRNA-23a and microRNA-126 were significantly down-regulated; (3) Pre-miR-23a at concentration of 50nmol/L decreased TNF-a-induced HUVEC apoptosis as determined by Hoechst stain, TUNEL stain, qRT-PCR and Western blot. In contrast, HUVEC apoptosis was increased after treatment with LNA-anti-miR-23a at concentration of 50nmol/L; (4) In LNA-anti-miR-23a transfected HUVECs, significant changes were noted in the mRNA expression levels of potential target genes APAF-1, Caspase-7 and STK4, and the protein expression levels of Western blot were significantly increased than that of control group.Conclusion (1) The culture of HUVECs was successful. TNF-αwhich is the core factor in systemic inflammatory response is responsible for tissue damage by inducing apoptosis of HUVEC. (2) Our results suggest that the expression of microRNA-23a is up-regulated in TNF-a-mediated apoptosis of HUVEC for the first time. And that of microRNA-126 is also up-regulated which will provide further verification of microRNAs’roles in apoptosis. (3) microRNA-23a had a protective effect against the TNF-a-induced HUVEC apoptosis. (4) APAF-1, Caspase-7 and STK4 may be the potential target genes that are involved in miR-23a-mediated protective effects on TNF-a-induced apoptosis on HUVECs更多还原