人脂肪组织来源内皮祖细胞的分离、培养及鉴定

发布时间：2018-03-10 05:18

本文选题：人脂肪组织　切入点：内皮祖细胞　出处：《青岛大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的探讨人脂肪组织来源的内皮祖细胞(endothelial progenitor cells,EPCs)的分离、培养及鉴定方法,并评价EPCs的增殖特性。方法脂肪抽吸术获取人体脂肪组织,消化法得到的细胞接种在FN包被(Fibronectin coating)的培养皿内,用含2%胎牛血清(FBS)的DMEM培养,传至第二代,分诱导组(EGM-2,2%FBS,VEGF,bFGF等)和非诱导组(DMEM,2%FBS)进行培养。免疫细胞荧光分别检测诱导组和非诱导组的CD34、vWF和PECAM-1表达;流式细胞仪(FACS)分别检测诱导组和非诱导组的CD34、CD45、CD133和PECAM-1表达率;荧光显微镜观察细胞摄取DiI-ac-LDL的功能;诱导组细胞接种于甲基纤维素半固体培养基进行三维培养,观察血管样结构形成情况。取原代EPCs分别于培养后第2,4,6,8,10,12,14,16天予以细胞计数,将EPCs传代至P9,分别用细胞计数及MTT法观察P1、P5、P7、P9细胞生长情况。将P2、P5、P7、P9代EPCs用EGM-2诱导培养后,用FACS分别检测各代CD34、CD45,CD133,PECAM-1的表达率。结果诱导组细胞12天后呈现内皮细胞典型的铺路石样形态,未诱导组细胞未观察到这种变化;免疫细胞荧光显示诱导组vWF、PECAM-1表达阳性,未诱导组细胞CD34表达阳性;流式细胞仪检测显示诱导组PECAM-1阳性率为67.41±13.35%,非诱导组6.73±2.21%(p0.01);非诱导组CD34阳性率为72.39±13.45%,诱导组为16.06±3.86%
[Abstract]:Objective to investigate the isolation, culture and identification of endothelial progenitor cells derived from human adipose tissue, and to evaluate the proliferative characteristics of EPCs. Methods Human adipose tissue was obtained by liposuction. The cells obtained by digestion were inoculated in a petri dish coated with fibronectin. The cells were cultured with DMEM containing 2% fetal bovine serum (FBS) and transferred to the second generation. The expression of CD34VWF and PECAM-1 were detected by immunofluorescence and flow cytometry (FACS). The expression rates of CD34CD45-CD133 and PECAM-1 in induced group and non-induced group were detected by flow cytometry (FCM) and non-induction group (DMEM, 2S), respectively, and the expression rates of CD34VWF and PECAM-1 were detected by flow cytometry. The expression rates of CD34, CD45, CD133 and PECAM-1 were detected by flow cytometry (FCM), and the expression rates of CD34, CD45, CD133 and PECAM-1 were detected by flow cytometry (FCM). The function of uptake of DiI-ac-LDL was observed by fluorescence microscope, and the formation of vascular like structure was observed by inoculation of cells in methylcellulose semisolid medium. The primary EPCs cells were counted on the 2nd day after culture, and then passed to P9. The growth of P1P5P5 P7P9 cells was observed by cell count and MTT method. The expression rate of CD34 CD45-CD133PECAM-1 was detected by FACS after P2P5P5 P7P9 passage EPCs was cultured with EGM-2. Results after 12 days, the cells in the induction group showed typical paving stone morphology of endothelial cells, but no such changes were observed in the cells of the non-induced group, and the expression of vWFnPECAM-1 was positive in the induced group by immunofluorescence, but the expression of CD34 was positive in the uninduced group. Flow cytometry showed that the positive rate of PECAM-1 was 67.41 卤13.35 in the induced group, 6.73 卤2.21 in the non-induced group, 72.39 卤13.45 in the non-induced group and 16.06 卤3.86% in the induced group.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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