發(fā)布時間：2018-03-14 05:26

  本文選題：脂肪間充質(zhì)干細(xì)胞　切入點：髓核細(xì)胞　出處：《第四軍醫(yī)大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 椎間盤退變是一類嚴(yán)重影響人們生活質(zhì)量的常見病,為腰背痛主要原因之一,其退變主要是由于椎間盤內(nèi)髓核細(xì)胞減少和細(xì)胞外基質(zhì)成份的改變。近年椎間盤組織工程研究中,利用種子細(xì)胞移植修復(fù)組織損害和促進(jìn)功能恢復(fù)作用的治療方法成為目前醫(yī)學(xué)領(lǐng)域中最引人關(guān)注的熱點之一。在諸多種子細(xì)胞的研究中,脂肪組織中的脂肪干細(xì)胞具有含量多、位置表淺易取材及增殖能力強的優(yōu)點,是一種具有多分化潛能的成體干細(xì)胞,很多實驗已經(jīng)證明脂肪間充質(zhì)干細(xì)胞能分化為髓核樣軟骨細(xì)胞。細(xì)胞的微球培養(yǎng)技術(shù)在組織工程中應(yīng)用表明,在細(xì)胞微球的中心由于缺氧和低營養(yǎng)狀態(tài),這與體內(nèi)的髓核細(xì)胞的生存環(huán)境相類似,更利于種子細(xì)胞向髓核樣軟骨細(xì)胞分化�；谡T導(dǎo)脂肪間充質(zhì)干細(xì)胞向髓核樣軟骨細(xì)胞分化的各項研究,本實驗旨在通過與髓核細(xì)胞共培養(yǎng)和細(xì)胞因子兩種誘導(dǎo)方式做比較,探索體外細(xì)胞誘導(dǎo)分化的最佳方法,為今后種子細(xì)胞移植治療椎間盤退變性疾病提供實驗依據(jù)。 方法 1.取新西蘭大白兔腹股溝、腋下脂肪,0.3%的Ⅱ型膠元酶膠原酶溶液中下剪碎并消化40 min (37℃),所得的消化液過濾、離心獲取脂肪間充質(zhì)干細(xì)胞。取新西蘭大白兔整段脊柱,切開纖維環(huán)取髓核組織,0.3%的Ⅱ型膠元酶膠原酶溶液中下剪碎并消化30 min (37℃),所得的消化液過濾、離心獲取髓核細(xì)胞。所收集的脂肪間充質(zhì)干細(xì)胞和髓核細(xì)胞用DMEM/F12(1:1)培養(yǎng)液分別單層培養(yǎng)。 2.脂肪干間充質(zhì)干細(xì)胞培養(yǎng)至3代經(jīng)鑒定后,按5×106個髓核細(xì)胞和脂肪間充質(zhì)干細(xì)胞分別置于Eppendorf管中離心,培養(yǎng)3-4天制成細(xì)胞微球。 3.將脂肪間充質(zhì)干細(xì)胞微球置于Transwell下層培養(yǎng),分別通過5×106個髓核細(xì)胞微球及TGF-β1/IGF-1誘導(dǎo);于誘導(dǎo)前、誘導(dǎo)7、14天,觀察細(xì)胞形態(tài)變化,并通過RT-PCR對Ⅱ型膠原、蛋白多糖含量進(jìn)行測定。 結(jié)果 在體外誘導(dǎo)7、14天,兩組脂肪間充質(zhì)干細(xì)胞微團(tuán)體積、形態(tài)無明區(qū)別,RT-PCR檢測結(jié)果顯示7天TGF-β1/IGF-1組與髓核誘導(dǎo)組均有Ⅱ型膠原及蛋白多糖mRNA表達(dá),但TGF-β1/IGF-1組表達(dá)更強;誘導(dǎo)14天髓核誘導(dǎo)組Ⅱ型膠原及蛋白多糖mRNA表達(dá)明顯增高,優(yōu)于但TGF-β1/IGF-1組。 結(jié)論 在體外NPCs和TGF-β1/IGF-1對ADSCs進(jìn)行誘導(dǎo)均有促進(jìn)其向NPCs分化作用,而TGF-β1、IGF-1為正常髓核細(xì)胞分泌重要因子,本實驗結(jié)果說明NPCs對ADSCs的誘導(dǎo)之間除細(xì)胞因子作用外,尚存在相互促進(jìn)增殖分化作用,為更好向髓核樣細(xì)胞分化仍其它誘導(dǎo)條件存在。 髓核細(xì)胞與脂肪間充質(zhì)干細(xì)胞的三維共同培養(yǎng)較細(xì)胞因子誘導(dǎo)經(jīng)濟(jì)有效,不但能更好促進(jìn)種子細(xì)胞向目的細(xì)胞分化,而且能模擬椎間盤內(nèi)環(huán)境以利于今后的體內(nèi)研究。
[Abstract]:Intervertebral disc degeneration is a common disease that seriously affects people's quality of life and is one of the main causes of low back pain. The degeneration is mainly due to the decrease of nucleus pulposus cells and the changes of extracellular matrix in intervertebral disc. The treatment of repairing tissue damage and promoting functional recovery by seed cell transplantation has become one of the most attractive topics in the field of medicine. In many seed cell studies, adipose stem cells are abundant in adipose tissue. The position surface is a kind of adult stem cells with multiple differentiation potential because of its advantages of easy access to materials and strong proliferative ability. Many experiments have shown that adipose mesenchymal stem cells can differentiate into nucleus pulposus like chondrocytes. This is similar to the living environment of nucleus pulposus cells in vivo and is more conducive to the differentiation of seed cells into nucleus pulposus chondrocytes, based on studies on inducing adipose mesenchymal stem cells to differentiate into nucleus pulposus chondrocytes. The purpose of this study was to explore the best method of cell differentiation in vitro by comparing with the co-culture of nucleus pulposus cells and cytokines in order to provide experimental evidence for the treatment of degenerative disc diseases by seed cell transplantation in the future. Method. 1. Adipose mesenchymal stem cells were obtained from the inguinal region of New Zealand white rabbits and 0.3% of type 鈪，

本文編號：1609793