两种同源间充质干细胞在接触式共培养体系下对退变髓核细胞生物学影响的比较

发布时间：2018-05-15 21:40

  本文选题：接触式共培养 + 间充质干细胞　； 参考：《中国脊柱脊髓杂志》2014年06期

【摘要】：目的:比较两种人间充质干细胞在接触式共培养体系下对退变髓核细胞生物学功能的影响。方法:取同一例腰椎间盘突出症患者的退变髓核组织、脂肪组织和骨髓组织,分别分离培养退变髓核细胞(NPCs)、脂肪间充质干细胞(ADSCs)和骨髓间充质干细胞(BMSCs)。取3种细胞传代至第3代的细胞进行接触式共培养。共培养前,利用PKH26对第3代退变NPCs进行染色标记,然后按1∶1的细胞比例,将NPCs分别与ADSCs和BMSCs在普通6孔板内进行接触式共培养。实验分为3组,A组为单独培养的NPCs,为对照组;B组为与ADSCs共培养的NPCs;C组为与BMSCs共培养的NPCs。共培养第7天时,利用MoFlo高速流式细胞分选仪将共培养的细胞进行分选,获取各组NPCs。然后提取各组NPCs的总RNA,进行反转录后,再利用Real-Time PCR技术检测各组NPCs的Ⅱ型胶原、蛋白多糖和SOX-9基因的相对表达量。将Ⅱ型胶原、蛋白多糖和SOX-9基因的相对表达量按分组进行组间t检验,P0.05为有统计学差异。结果:从退变髓核组织、骨髓组织和脂肪组织中,均成功分离培养出NPCs、BMSCs和ADSCs。待各细胞传至第3代时,成功利用PKH26对NPCs进行染色标记。按实验分组将各细胞进行接触式共培养后第7天,成功利用MoFlo高速流式细胞分选仪将共培养细胞进行分选,获取各组NPCs。经提取各组NPCs的总RNA,反转录并进行Real-time PCR后,获取各组NPCs的Ⅱ型胶原、蛋白多糖和SOX-9基因相对表达量,A组分别为1.03±0.04,1.05±0.07,1.04±0.17;B组分别为5.26±0.24,7.71±0.21,3.84±0.25;C组分别为9.33±0.39,11.07±0.34,5.64±0.26;B、C组NPCs的Ⅱ型胶原、蛋白多糖和SOX-9基因相对表达量与A组比较显著性增加(P0.05);C组与B组比较,各指标基因相对表达量显著性增加(P0.05)。结论:在接触式共培养条件下,BMSCs和ADSCs对退变NPCs均具有营养激活效应;BMSCs对退变NPCs的激活效应比ADSCs更好,对于椎间盘退行性疾病的生物学治疗可能更具优势。
[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，

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