人臍帶MSCs源microvesicles的分離及特異microRNAs檢測
發(fā)布時間：2018-11-09 15:12
【摘要】：背景微粒體(Microvesicles, MVs)是多種細(xì)胞釋放在微環(huán)境中的質(zhì)膜囊泡。近年來人們關(guān)注到細(xì)胞源MVs作為細(xì)胞間一個新的分子通訊介質(zhì),能夠轉(zhuǎn)運(yùn)大量生物活性分子。 目的分離人臍帶間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)源MVs,檢測特異性microRNAs表達(dá),探討MSCs潛在的細(xì)胞通訊方式。 方法 1.組織塊培養(yǎng)法分離培養(yǎng)人臍帶MSCs,進(jìn)行傳代擴(kuò)增；從人臍帶MSCs條件培養(yǎng)基(conditioned medium, CM)中提取MVs。 2.對第3代MSCs細(xì)胞標(biāo)記CD29-PE、CD44-APC、CD34-FITC、CD45-PerCP,流式細(xì)胞儀分析鑒定。 3.TRIzol法提取細(xì)胞人臍帶間充質(zhì)干細(xì)胞及MVs中總RNA,采用實時熒光定量PCR技術(shù)檢測miR-100和miR-21的表達(dá)水平,以U6作為內(nèi)參對照。 結(jié)果 1.細(xì)胞形態(tài)呈長梭形,原代培養(yǎng)12d、傳代培養(yǎng)4d細(xì)胞融合度達(dá)90%,細(xì)胞排列成漩渦狀。 2.人臍帶間充質(zhì)干細(xì)胞不表達(dá)造血細(xì)胞標(biāo)志CD34(0.87%)、CD45(1.28%),強(qiáng)表達(dá)β1-整合素CD29(96.68%)、基質(zhì)受體CD44(92.78%)。 3.miR-100在正常培養(yǎng)MSCs、MVs、凍存MSCs中均能檢測到表達(dá)。其中miR-100在MVs和正常培養(yǎng)MSCs中表達(dá)量無明顯差異(P0.05),miR-100在凍存MSCs中表達(dá)量顯著低于正常培養(yǎng)MSCs (P0.05). 4.miR-21在正常培養(yǎng)MSCs、MVs、凍存MSCs中均能檢測到表達(dá)。其中miR-21在MVs中表達(dá)量較正常培養(yǎng)MSCs中明顯提高(P0.05),miR-21在凍存MSCs中表達(dá)量顯著低于正常培養(yǎng)MSCs (P0.05)。 結(jié)論 1.從人臍帶組織中成功分離培養(yǎng)MSCs,收獲大量經(jīng)過生物學(xué)鑒定的MSCs。 2.從臍帶MSC-CM中分離提取出MVs,在MVs中可以檢測到miR-100、miR-21特異表達(dá)。 3.在MVs中microRNAs呈現(xiàn)特異性富集；凍存MSCs較正常培養(yǎng)MSCs的microRNAs含量顯著降低。
[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.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：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.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329
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