本文選題：接觸式共培養(yǎng) + 間充質(zhì)干細胞��； 參考：《中國脊柱脊髓雜志》2014年06期

【摘要】：目的:比較兩種人間充質(zhì)干細胞在接觸式共培養(yǎng)體系下對退變髓核細胞生物學(xué)功能的影響。方法:取同一例腰椎間盤突出癥患者的退變髓核組織、脂肪組織和骨髓組織,分別分離培養(yǎng)退變髓核細胞(NPCs)、脂肪間充質(zhì)干細胞(ADSCs)和骨髓間充質(zhì)干細胞(BMSCs)。取3種細胞傳代至第3代的細胞進行接觸式共培養(yǎng)。共培養(yǎng)前,利用PKH26對第3代退變NPCs進行染色標記,然后按1∶1的細胞比例,將NPCs分別與ADSCs和BMSCs在普通6孔板內(nèi)進行接觸式共培養(yǎng)。實驗分為3組,A組為單獨培養(yǎng)的NPCs,為對照組;B組為與ADSCs共培養(yǎng)的NPCs;C組為與BMSCs共培養(yǎng)的NPCs。共培養(yǎng)第7天時,利用MoFlo高速流式細胞分選儀將共培養(yǎng)的細胞進行分選,獲取各組NPCs。然后提取各組NPCs的總RNA,進行反轉(zhuǎn)錄后,再利用Real-Time PCR技術(shù)檢測各組NPCs的Ⅱ型膠原、蛋白多糖和SOX-9基因的相對表達量。將Ⅱ型膠原、蛋白多糖和SOX-9基因的相對表達量按分組進行組間t檢驗,P0.05為有統(tǒng)計學(xué)差異。結(jié)果:從退變髓核組織、骨髓組織和脂肪組織中,均成功分離培養(yǎng)出NPCs、BMSCs和ADSCs。待各細胞傳至第3代時,成功利用PKH26對NPCs進行染色標記。按實驗分組將各細胞進行接觸式共培養(yǎng)后第7天,成功利用MoFlo高速流式細胞分選儀將共培養(yǎng)細胞進行分選,獲取各組NPCs。經(jīng)提取各組NPCs的總RNA,反轉(zhuǎn)錄并進行Real-time PCR后,獲取各組NPCs的Ⅱ型膠原、蛋白多糖和SOX-9基因相對表達量,A組分別為1.03±0.04,1.05±0.07,1.04±0.17;B組分別為5.26±0.24,7.71±0.21,3.84±0.25;C組分別為9.33±0.39,11.07±0.34,5.64±0.26;B、C組NPCs的Ⅱ型膠原、蛋白多糖和SOX-9基因相對表達量與A組比較顯著性增加(P0.05);C組與B組比較,各指標基因相對表達量顯著性增加(P0.05)。結(jié)論:在接觸式共培養(yǎng)條件下,BMSCs和ADSCs對退變NPCs均具有營養(yǎng)激活效應(yīng);BMSCs對退變NPCs的激活效應(yīng)比ADSCs更好,對于椎間盤退行性疾病的生物學(xué)治療可能更具優(yōu)勢。
[Abstract]:Aim: to compare the effects of two human mesenchymal stem cells on the biological function of degenerative nucleus pulposus cells in contact coculture system. Methods: the degenerative nucleus pulposus tissue, adipose tissue and bone marrow tissue were isolated from the same patient with lumbar disc herniation. NPCsm cells, adipose mesenchymal stem cells (ADSCss) and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured respectively. Three kinds of cells were subcultured to the third passage for contact coculture. Before co-culture, the third generation degenerative NPCs was stained with PKH26, and then NPCs and ADSCs and BMSCs were co-cultured in ordinary 6-well plate according to the ratio of 1:1 cells. The experiment was divided into 3 groups: group A was isolated NPCs and group B was co-cultured with ADSCs. Group C was co-cultured with BMSCs. On the 7th day of co-culture, the co-cultured cells were separated by MoFlo high speed flow cytometry to obtain each group of NPCs. Then the total RNAs of each group were extracted, and then the relative expression of type 鈪，

本文編號：1894081