本文選題：人羊水來源間充質(zhì)干細胞　切入點：軟骨細胞　出處：《復旦大學》2009年碩士論文

【摘要】： 研究目的 探討分離、提純和擴增孕中期羊水來源間充質(zhì)干細胞(AF-MSCs)的方法,以及體外誘導AF-MSCs向軟骨細胞分化的可能性。 研究方法 機械手段挑取孕中期人羊水細胞培養(yǎng)體系中的MSCs集落,培養(yǎng)擴增后,通過流式細胞術對表面抗原表達情況進行鑒定,并通過RT-PCR檢測多能性基因Oct4及Nanog的表達情況。取第3代AF-MSCs,經(jīng)TGF-β1、IGF-1、地塞米松等生長因子向軟骨細胞方向誘導分化。倒置相差顯微鏡觀察誘導后細胞形態(tài)的變化。誘導3周后,免疫熒光染色及RT-PCR分別檢測軟骨特異性基質(zhì)CollengonⅡ、Aggrecan、Chondromudulin-1、S100的蛋白及其基因的表達情況。 研究結果 機械手段分離出的AF-MSCs為CD105、CD166陽性,CD29若陽性,CD34、CD45陰性,符合MSCs特點。AF-MSCs表達多能性基因Oct4,但不表達Nanog。誘導2周后,細胞開始呈多角形樣改變;誘導后3周時,大部分細胞由長梭形轉(zhuǎn)變?yōu)槎嘟切�。免疫熒光及RT-PCR檢測顯示誘導后的AF-MSCs軟骨特異性基質(zhì)CollengonⅡ、Aggrecan、Chondromudulin-1和S100的蛋白和基因呈陽性表達,而未經(jīng)誘導培養(yǎng)的AF-MSCs呈陰性表達。 研究結論 機械分離法可有效獲得孕中期羊水來源間充質(zhì)干細胞,經(jīng)TGF-β1,IGF-1,地塞米松聯(lián)合誘導3周后,AF-MSCs可以分化為軟骨細胞樣細胞。
[Abstract]:Research purpose. To explore the methods of isolation, purification and amplification of AF-MSCs derived from amniotic fluid in the second trimester of pregnancy, and the possibility of inducing AF-MSCs to differentiate into chondrocytes in vitro. Research method. In the second trimester of human amniotic fluid cell culture system, the MSCs colony was selected by mechanical means, and the expression of surface antigen was identified by flow cytometry. The expression of Oct4 and Nanog was detected by RT-PCR. The third generation AF-MSCs were induced to differentiate into chondrocytes by growth factors such as TGF- 尾 1 IGF-1 and dexamethasone. The morphologic changes of the cells were observed by inverted phase contrast microscopy. The expression of Chondromudulin-1 S100 protein and its gene were detected by immunofluorescence staining and RT-PCR, respectively. Research results. The AF-MSCs isolated by mechanical means was CD105, CD166 positive and CD29 positive. If CD34 + CD45 was negative, it was consistent with the characteristics of MSCs. AF-MSCs expressed multipotent gene Oct4, but did not express Nanog.After 2 weeks of induction, the cells began to show polygonal changes, and at 3 weeks after induction, the cells began to show polygonal changes. Immunofluorescence and RT-PCR analysis showed that the AF-MSCs chondromudin-1 and S100 chondromudin-1 and S100 chondromudin-1 were expressed positively, but the uninduced AF-MSCs showed negative expression. Research conclusion. Mesenchymal stem cells derived from amniotic fluid in the second trimester of pregnancy could be obtained by mechanical isolation. AF-MSCs could differentiate into chondrocyte-like cells after induction of TGF- 尾 _ 1 IGF-1 and dexamethasone for 3 weeks.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

【參考文獻】

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1 Patrizia Bossolasco;Tiziana Montemurro;Lidia Cova;Stefano Zangrossi;Cinzia Calzarossa;Simona Buiatiotis;Davide Soligo;Silvano Bosari;Vincenzo Silani;Giorgio Lambertenghi Deliliers;PaoloRebulla;LorenzaLazzari;;Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential[J];Cell Research;2006年04期

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