【作者】 白燕慧；

【导师】 张明昌；

【作者基本信息】 华中科技大学 ， 眼科， 2012， 博士

【摘要】 目的：(1)建立脐静脉血管内皮细胞(HUVECs)体外培养的方法并探讨姜黄素对HUVECs增殖的影响。(2)探讨姜黄素抑制HUVECs中microRNAs的差异表达谱,验证在姜黄素抑制HUVECs中显著差异表达的microRNAs。方法：(1)采用0.25%胰蛋白酶和0.02%EDTA消化、分离、体外原代培养及消化传代脐静脉血管内皮细胞,简化完全培养液组分(不添加血管内皮细胞生长因子、肝素等辅助因子),当原代培养细胞80%以上汇合,根据细胞特有的形态学特征和Ⅷ因子进行内皮细胞的鉴定。然后用不同浓度姜黄素(20、40、80、160μmol/l)处理内皮细胞不同时间(0、24h、48h、72h、96h),MTT法测定姜黄素对HUVECs增殖的影响。(2)采用GeneChip(?)microRNA 2.0芯片技术,将对照组和姜黄素(40μmol/1)组的microRNAs进行对比,根据统计学的方法分析芯片实验数据,筛选出共同差异表达的microRNAs。采用实时荧光定量PCR的方法,分别验证这些microRNAs的差异表达情况。将芯片和实时荧光定量PCR两种方法一致的microRNAs确定为有意义的共同差异表达的microRNAs,并通过生物信息学方法预测差异显著的microRNA的靶基因。结果：种植在培养瓶中的内皮细胞2h贴壁生长,24h换液后内皮细胞80%融合,细胞状态好,内皮细胞呈单层铺路石样外观,经过镜下观察和Ⅷ因子相关抗原鉴定证明是脐静脉血管内皮细胞。姜黄素呈浓度依赖性和时间依赖性抑制HUVECs的增殖。(2)姜黄素(40μmol/l)处理HUVECs24h后,芯片结果显示和对照组相比,姜黄素药物组表达谱有30个microRNAs明显变化,其中11个microRNA表达上调,19个microRNA表达下调；实时荧光定量PCR证实mi-1275、mi-3127和mi-1246表达显著升高,生物信息学分析结果显示,这些表达差异的microRNAs可以调控与新生血管相关的基因的表达,如mi-1275可能靶向VEGFb等。结论：(1)用胰蛋白酶灌注脐静脉是一种简单、实用的获得人脐静脉血管内皮细胞的方法,可靠性大,成功率高,可以构建体外研究血管内皮细胞的模型。本研究在成功建立HUVECs体外培养方法的基础上,建立了姜黄素抑制HUVEC增殖的模型；(2)运用microRNA芯片技术,首次发现姜黄素抑制血管内皮细胞中mi-1275、mi-3127和mi-1246的表达明显增多,其中mi-1275在姜黄素抑制HUVECs中可能是通过抑制其靶基因的表达而介导的,这为进一步研究microRNA在姜黄素抗角膜新生血管中的作用创造了条件。更多还原

【Abstract】 Objective:(1) To explore how to get and identify human umbilical vein endothelial cell (HUVECs) in vitro. To investigate the effect of curcumin on proliferation of human umbilical vein endothelial cell (HUVECs).(2) To investigate the effect of curcumin on the differential expression of microRNAs of HUVECs and to establish the relationship between HUVEC proliferation and the expression of microRNAs;Methods:(1) 0.25% Trypsin and 0.02%EDTA perfusion method was used to isolate HUVEC from fresh umbilical cord, and the cells were cultured and amplified.The composition of culture medium was simplified by not adding vascular endothelial cell growth factor and heparin.HUVECs were identified by morphological observation and immunofluorescencal method. Then HUVECs proliferation were examined at different curcumin concentrations(0,20,40,80,160 umol/l)by different time course (0,24,48,72,96h) by method of MTT;(2)RNA was extracted from 1 control samples of HUVECs and 1 curcumin (40umol/l) treated sample,and small RNA was isolated.Then we detected microRNA profiles in these samples by microRNA microarray.Meanwhile, examined the expression of microRNAs were examined in these samples by fluorescence based real-time quantitative PCR.Those microRNAs that were identified as differentially expressed by both microRNA microarray and RT-PCR were considered as significant microRNA; Possible target genes of these microRNAs were Predieted through bioinformaties analysis.Results:(1) The endothelial cells spread on the bottom of the dishes in 2h,then coalesced and grew to from confluent monolayers of polygonal cells within 24h.The cultured cells were identified as HUVECs. the effect of curcumin on proliferation of human umbilical vein endothelial cell (HUVECs) is in a time-dependent manner and a dose-dependent manner; (2) After treated with curcumin at a concentration of 40μmol/l for 24h,there are 30 significant differential expressions of microRNAs,11 microRNAs were up-regulated,19 microRNAs were down-regulated;Among them mi-1275,mi-3127 and mi-1246 were significantly up-regulated,Bioinformatie analysis showed that thesemieroRNAs with significantly different expression maybe closely related to many genes regulating conceal neovascularition,sueh as miR-1275 targeting to PEDFb.Conclusion:(1) Trypsin perfusion is a simple and effective method for collection of human umbilical vein endothelial cells. Cells harvest with protocol can be used as models on research of vascular endothelial in vitro.(2)Our results find that the expression of mi-1275,mi-3127,mi-1246 is up-regulated in HUVECs after treated with curcumin at a concentration of 40μmol/l for 24h for the first time,and VEGF may be the potential target genes that are involved in mi-1275-mediated protective effects.This findings will provide further qualification of microRNAs’in neovascularition.更多还原