正常与退变髓核的髓核间充质干细胞代谢活性及干性基因表达的比较

Cell activity and stem cell-related genes expression of mesenchymal stromal cells from non-degenerative and degenerative nucleus pulposus: a comparative study

中文关键词:  椎间盘退变  髓核间充质干细胞  干性基因  细胞活性

英文关键词:Intervertebral disc degeneration  Nucleus pulposus mesenchymal stem cells  Stem cell-related genes  Cell activity

中文摘要:

  【摘要】 目的：比较来源于正常与退变髓核的髓核间充质干细胞（NPMSCs）的细胞代谢活性及干性基因表达情况。方法：收集6例非退变患者髓核组织（正常组）与6例腰椎间盘突出症患者的退变髓核组织（退变组），采用酶消化法分离细胞，应用标准间充质干细胞培养基（standard MSC culture medium）进行细胞培养并观察细胞形态。两组内各取1例分离得到的细胞进行流式细胞仪检测间充质干细胞表面蛋白分子标记CD90、CD105、CD73、CD45、CD34及人类白细胞抗原（HLA）-DR表达情况；并进行成骨、成脂及成软骨诱导分化，诱导28d后分别应用茜素红染色鉴定细胞成骨能力、油红O染色鉴定细胞成脂能力、甲苯胺蓝染色鉴定细胞成软骨分化能力，并按照国际干细胞治疗协会（ISCT）提出的间充质干细胞的判定标准，对分离得到的细胞进行综合评估鉴定。采用CCK-8检测两组P2代NPMSCs的代谢活力。提取两组每例P2代细胞总RNA，行RT-PCR检测P2代细胞“干性维持”相关基因Oct4及Nanog表达情况。结果：两组P0代细胞均贴壁生长，形态学方面两组并无明显差异。免疫表型鉴定显示正常组和退变组间充质干细胞表面分子标记CD90、CD105、CD73表达比例分别高达96％、98％、95%以上，两组均低表达造血细胞标志物CD45、CD34、HLA-DR（均低于4%）。茜素红染色、油红O染色及甲苯胺蓝染色分别证实正常组与退变组细胞均可向骨、脂肪及软骨细胞三系诱导分化。上述结果证实分离得到的细胞即NPMSCs。细胞代谢活性测定示P2代细胞在培养后5d、7d、9d、11d、13d正常组细胞活性均强于退变组，两组细胞活性有统计学差异（P<0.05）。正常组“干性维持”相关基因Oct4及Nanog表达量分别为退变组的4.63±1.17、7.36±1.19倍，正常组均明显高于退变组（P<0.05）。结论：正常与退变髓核组织内均存在NPMSCs，但正常椎间盘来源的NPMSCs具有较强的细胞代谢活性，较”强的“干性维持”基因表达。

英文摘要:

  【Abstract】 Objectives： To compare the cell activity and stem cell-related genes expression of mesenchymal stem cells(MSCs) in nucleus pulposus(NP) from non-degenerative and degenerative disc． Methods: The human nucleus pulposus mesenchymal stem cells(NPMSCs) were isolated by using standard MSC culture medium as suitable for MSCs culture by Cyagen from 6 non-degenerative patients of either scoliosis or burst fracture and 6 patients with degenerative intervertebral disc separately． One case of cells was selected from each group, respectively, and immunophenotype(CD90/CD105/CD73/CD45/CD34 and HLA-DR) and multilineage(Osteogenic, chondrogenic and adipogenic) differentiation potential were analyzed under the criteria to define MSCs, which was stated by the International Society for Cellular Therapry(ISCT). The cell activity of second passage from each group was analyzed by using cell counting kit-8(CCK-8). The total RNA of both non-degenerative and degenerative group were extracted, then the Real-Time PCR was used to detect the relative expressions of stem cell-related genes Oct4 and Nanog. Results: The original cells from both non-degenerative and degenerative group showed adherent growth, cell morphology of two groups showed no significant difference. The immune phenotype showed both normal and degenerative cells highly expressed the mesenchymal stem cell surface markers CD90, CD105, CD73(expression ratio as high as 96%, 98%, 95%, respectively). And hematopoietic markers CD45, CD34, HLA-DR were lower expressions(all less than 4%). Multilineage differentiation results showed cells obtained from both non-degenerative and degenerative NP developed red stained calcium salts, and adipocyte-like cells which were stained red by Oil red O, and chondrocyte-like cells stained blue by toluidine blue, after Osteogenic, chondrogenic and adipogenic differentiation for 28 days. Together with the immunophenotype, cells obtained from both non-degenerative and degenerative NP fulfilled the criteria to define MSCs stated by ISCT. Cell viability assay by CCK-8 showed that cell activity in normal group was stronger than that in the degeneration group at 5, 7, 9, 11, 13 days, there was significant difference between two groups(P<0.05). The Real-Time PCR results showed that Stemness maintenance related gene Oct4 and Nanog expression in normal group was 4.63±1.17, 7.36±1.19 times respectively than degeneration group. The Stemness maintenance related gene expression in normal group was significantly higher than that in the degeneration group(P<0.05). Conclusions: Both the non-degenerative and degenerative NP contain NPMSCs． Moreover, the non-degenerative NPMSCs show stronger ability of cell metabolic activity and stem cell-related genes expression.