本文選題：椎間盤　切入點(diǎn)：髓核干細(xì)胞　出處：《蘇州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分人退變髓核組織來源間充質(zhì)干細(xì)胞的分離、培養(yǎng)及鑒定目的目的:體外分離、培養(yǎng)及鑒定人退變髓核組織來源的間充質(zhì)干細(xì)胞(NP-MSCs)并觀察其生物學(xué)特性。方法法:收集10例腰椎間盤退行性疾病患者的髓核手術(shù)標(biāo)本,采用0.05%II型膠原酶消化加貼壁法分離髓核間充質(zhì)干細(xì)胞并體外培養(yǎng)和擴(kuò)增。用倒置相差顯微鏡觀察人髓核間充質(zhì)干細(xì)胞形態(tài)、流式細(xì)胞儀檢測干細(xì)胞表型分子、定向誘導(dǎo)人髓核間充質(zhì)干細(xì)胞成脂、成骨及成軟骨分化。結(jié)果果:采用0.05%II型膠原酶37℃消化4小時(h)即可從組織標(biāo)本中分離出人髓核間充質(zhì)干細(xì)胞。人髓核間充質(zhì)干細(xì)胞原代培養(yǎng)3天可見細(xì)胞貼壁,30天左右達(dá)到80%融合,傳至第3代后,細(xì)胞形態(tài)轉(zhuǎn)變成均一的長梭形或紡錘形,呈漩渦狀排列。流式細(xì)胞儀檢測人髓核間充質(zhì)干細(xì)胞高表達(dá)CD29和CD105,不表達(dá)或低表達(dá)CD34、CD45及HLA-DR。多向誘導(dǎo)21天(d)后,成骨誘導(dǎo)組Alizareen染色和成軟骨誘導(dǎo)組Safranin’O染色呈陽性,成脂誘導(dǎo)組油紅O染色陰性。結(jié)論論:采用0.05%II型膠原酶消化結(jié)合貼壁法可從人退變髓核組織中提取具有多向分化潛能的間充質(zhì)干細(xì)胞。第二部分低氧對人髓核組織來源間充質(zhì)干細(xì)胞向類髓核細(xì)胞分化的生物學(xué)誘導(dǎo)效應(yīng)目的目的:探索低氧對人退變髓核組織來源的間充質(zhì)干細(xì)胞(NP-MSCs)向類髓核細(xì)胞定向分化的生物學(xué)效應(yīng)。方法法:在5%氧濃度下,用含轉(zhuǎn)化生長因子-β3(TGF-β3)誘導(dǎo)培養(yǎng)基培養(yǎng)人髓核間充質(zhì)干細(xì)胞,同時設(shè)立在21%氧濃度下誘導(dǎo)培養(yǎng)為對照組。誘導(dǎo)培養(yǎng)第7天(d)和14d,實(shí)時熒光定量PCR(Realtime-PCR)檢測低氧誘導(dǎo)因子-1α(HIF-1α)、SOX-9、Aggrecan和II型膠原基因的表達(dá),誘導(dǎo)培養(yǎng)14d,采用免疫熒光法檢測低氧誘導(dǎo)組與常氧誘導(dǎo)組細(xì)胞II型膠原的表達(dá)。組間比較采用獨(dú)立樣本t檢驗(yàn)。結(jié)果果:NP-MSCs誘導(dǎo)培養(yǎng)第7d時低氧組HIF-1α與SOX-9基因的相對表達(dá)量高于常氧組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),但Aggrecan和II型膠原基因相對表達(dá)量無顯著性差異(P0.05)。在誘導(dǎo)培養(yǎng)14d時,低氧組HIF-1α、SOX-9、Aggrecan和II型膠原基因相對表達(dá)量均高于常氧組,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),免疫熒光法檢測兩組細(xì)胞均合成II型膠原且低氧組熒光染色較對照組深。結(jié)論論:人髓核間充質(zhì)干細(xì)胞可定向分化成類髓核細(xì)胞,低氧環(huán)境有利于該分化過程。
[Abstract]:The first part: isolation, culture and identification of mesenchymal stem cells derived from human degenerative nucleus pulposus objective: to isolate in vitro, NP-MSCs derived from degenerative nucleus pulposus were cultured and identified and their biological characteristics were observed. Methods: ten specimens of nucleus pulposus were collected from 10 patients with degenerative lumbar intervertebral disc disease. Mesenchymal stem cells of nucleus pulposus were isolated and cultured and expanded by the method of type 鈪，

本文編號：1606906