人脐带MSCs源microvesicles的分离及特异microRNAs检测
发布时间：2018-11-09 15:12
【摘要】：背景微粒体(Microvesicles, MVs)是多种细胞释放在微环境中的质膜囊泡。近年来人们关注到细胞源MVs作为细胞间一个新的分子通讯介质,能够转运大量生物活性分子。 目的分离人脐带间充质干细胞(mesenchymal stem cells, MSCs)源MVs,检测特异性microRNAs表达,探讨MSCs潜在的细胞通讯方式。 方法 1.组织块培养法分离培养人脐带MSCs,进行传代扩增；从人脐带MSCs条件培养基(conditioned medium, CM)中提取MVs。 2.对第3代MSCs细胞标记CD29-PE、CD44-APC、CD34-FITC、CD45-PerCP,流式细胞仪分析鉴定。 3.TRIzol法提取细胞人脐带间充质干细胞及MVs中总RNA,采用实时荧光定量PCR技术检测miR-100和miR-21的表达水平,以U6作为内参对照。 结果 1.细胞形态呈长梭形,原代培养12d、传代培养4d细胞融合度达90%,细胞排列成漩涡状。 2.人脐带间充质干细胞不表达造血细胞标志CD34(0.87%)、CD45(1.28%),强表达β1-整合素CD29(96.68%)、基质受体CD44(92.78%)。 3.miR-100在正常培养MSCs、MVs、冻存MSCs中均能检测到表达。其中miR-100在MVs和正常培养MSCs中表达量无明显差异(P0.05),miR-100在冻存MSCs中表达量显著低于正常培养MSCs (P0.05). 4.miR-21在正常培养MSCs、MVs、冻存MSCs中均能检测到表达。其中miR-21在MVs中表达量较正常培养MSCs中明显提高(P0.05),miR-21在冻存MSCs中表达量显著低于正常培养MSCs (P0.05)。 结论 1.从人脐带组织中成功分离培养MSCs,收获大量经过生物学鉴定的MSCs。 2.从脐带MSC-CM中分离提取出MVs,在MVs中可以检测到miR-100、miR-21特异表达。 3.在MVs中microRNAs呈现特异性富集；冻存MSCs较正常培养MSCs的microRNAs含量显著降低。
[Abstract]:Background microsomal (Microvesicles, MVs) is a plasma membrane vesicle released by a variety of cells in a microenvironment. In recent years, cell-derived MVs, as a new molecular communication medium between cells, can transport a large number of bioactive molecules. Objective to isolate (mesenchymal stem cells, MSCs) derived MVs, from human umbilical cord mesenchymal stem cells to detect specific microRNAs expression and to explore the potential cellular communication mode of MSCs. Method 1. Isolation and culture of human umbilical cord MSCs, by tissue block culture; extraction of MVs. from human umbilical cord MSCs conditioned medium (conditioned medium, CM) 2. The third passage MSCs cells were identified by CD29-PE,CD44-APC,CD34-FITC,CD45-PerCP, flow cytometry. The expression levels of miR-100 and miR-21 in human umbilical cord mesenchymal stem cells and total RNA, extracted from human umbilical cord mesenchymal stem cells and MVs were detected by real-time fluorescence quantitative PCR. U6 was used as internal control. Result 1. The morphology of the cells was long fusiform, primary culture 12 days, subculture 4 days of cell fusion degree reached 90. The cells arranged in a whirlpool shape. 2. Human umbilical cord mesenchymal stem cells did not express hematopoietic markers CD34 (0.87%), CD45 (1.28%), strongly expressed 尾 1-integrin CD29 (96.68%) and matrix receptor CD44 (92.78%). The expression of 3.miR-100 was detected in the frozen MSCs of normal cultured MSCs,MVs,. There was no significant difference in the expression of miR-100 between MVs and normal cultured MSCs (P0.05), and the expression of miR-100 in frozen MSCs was significantly lower than that in normal MSCs (P0.05). The expression of 4.miR-21 was detected in the frozen MSCs of normal cultured MSCs,MVs,. The expression of miR-21 in MVs was significantly higher than that in normal MSCs (P0.05), and the expression of miR-21 in frozen MSCs was significantly lower than that in normal MSCs (P0.05). Conclusion 1. Successful isolation and culture of MSCs, from human umbilical cord tissue and harvesting of a large number of biologically identified MSCs. 2. The specific expression of miR-100,miR-21 was detected in MVs by isolation and extraction of MVs, from umbilical cord MSC-CM. 3. The concentration of microRNAs in MVs was specific and the content of microRNAs in frozen MSCs was significantly lower than that in normal MSCs.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
[Abstract]:Background microsomal (Microvesicles, MVs) is a plasma membrane vesicle released by a variety of cells in a microenvironment. In recent years, cell-derived MVs, as a new molecular communication medium between cells, can transport a large number of bioactive molecules. Objective to isolate (mesenchymal stem cells, MSCs) derived MVs, from human umbilical cord mesenchymal stem cells to detect specific microRNAs expression and to explore the potential cellular communication mode of MSCs. Method 1. Isolation and culture of human umbilical cord MSCs, by tissue block culture; extraction of MVs. from human umbilical cord MSCs conditioned medium (conditioned medium, CM) 2. The third passage MSCs cells were identified by CD29-PE,CD44-APC,CD34-FITC,CD45-PerCP, flow cytometry. The expression levels of miR-100 and miR-21 in human umbilical cord mesenchymal stem cells and total RNA, extracted from human umbilical cord mesenchymal stem cells and MVs were detected by real-time fluorescence quantitative PCR. U6 was used as internal control. Result 1. The morphology of the cells was long fusiform, primary culture 12 days, subculture 4 days of cell fusion degree reached 90. The cells arranged in a whirlpool shape. 2. Human umbilical cord mesenchymal stem cells did not express hematopoietic markers CD34 (0.87%), CD45 (1.28%), strongly expressed 尾 1-integrin CD29 (96.68%) and matrix receptor CD44 (92.78%). The expression of 3.miR-100 was detected in the frozen MSCs of normal cultured MSCs,MVs,. There was no significant difference in the expression of miR-100 between MVs and normal cultured MSCs (P0.05), and the expression of miR-100 in frozen MSCs was significantly lower than that in normal MSCs (P0.05). The expression of 4.miR-21 was detected in the frozen MSCs of normal cultured MSCs,MVs,. The expression of miR-21 in MVs was significantly higher than that in normal MSCs (P0.05), and the expression of miR-21 in frozen MSCs was significantly lower than that in normal MSCs (P0.05). Conclusion 1. Successful isolation and culture of MSCs, from human umbilical cord tissue and harvesting of a large number of biologically identified MSCs. 2. The specific expression of miR-100,miR-21 was detected in MVs by isolation and extraction of MVs, from umbilical cord MSC-CM. 3. The concentration of microRNAs in MVs was specific and the content of microRNAs in frozen MSCs was significantly lower than that in normal MSCs.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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