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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。
OBJECTIVE: To compare the cellular metabolic activities and the expression of dry genes in mesenchymal stem cells (NPMSCs) derived from normal and degenerated nuclei. Methods: Degenerative nucleus pulposus (degeneration group) was collected from 6 cases of non-degenerative patients with nucleus pulposus (normal group) and 6 cases of lumbar disc herniation. The cells were isolated by enzyme digestion and cultured with standard mesenchymal stem cells Cells were cultured in a standard MSC culture medium and cell morphology was observed. One group of isolated cells were taken from each group to detect the expression of CD90, CD105, CD73, CD45, CD34 and HLA-DR on mesenchymal stem cells by flow cytometry. , Adipocytes and chondrocytes were induced to differentiate. After induced for 28 days, alizarin red staining was used to identify cell osteogenic ability, oil red O staining to identify adipogenic capacity of cells, toluidine blue staining to identify cells into cartilage differentiation ability, and in accordance with international stem cell therapy Association (ISCT) proposed criteria for mesenchymal stem cells, isolated cells were evaluated comprehensively identified. CCK-8 was used to detect the metabolic activity of P2 generation NPMSCs. The total RNA was extracted from each group of P2 generation cells in each group. The expression of Oct4 and Nanog gene in P2 generation cells ”dry maintenance“ was detected by RT-PCR. Results: The two groups of P0 generation cells adherent growth, morphology and no significant difference between the two groups. Immunophenotyping showed that the expression rates of CD90, CD105 and CD73 on the surface of MSCs were up to 96%, 98% and 95%, respectively. The hematopoietic cell markers CD45, CD34, HLA-DR (all less than 4%). Alizarin red staining, oil red O staining and toluidine blue staining confirmed that both normal and degenerated cells could differentiate into bone, fat and chondrocytes in three lines. The above results confirmed that the isolated cells, NPMSCs. The cell metabolic activity assay showed that the cell viability of P2 generation cells was significantly higher than that of degeneration group on the 5th, 7th, 9th, 11th and 13th day after culture. The cell viability was significantly different between the two groups (P <0.05). The expression levels of Oct4 and Nanog in normal group, ”dry maintenance“, were 4.63 ± 1.17 and 7.36 ± 1.19 times higher in degenerative group than in degenerative group (P <0.05). CONCLUSION: NPMSCs exist in both normal and degenerative nucleus pulposus tissues. However, NPMSCs derived from normal disc tissue have stronger cellular metabolic activity than that of ”strong“ dry maintenance ”gene expression.