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

发布时间：2018-04-30 01:19

本文选题：椎间盘退变 + 髓核间充质干细胞　；参考：《中国脊柱脊髓杂志》2014年05期

【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(P0.05)。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(P0.05)。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。
[Abstract]:Aim: to compare the cellular metabolic activity and dry gene expression of nucleus pulposus mesenchymal stem cells (NPMSCs) from normal and degenerated nucleus pulposus. Methods: the degenerative nucleus pulposus tissues were collected from 6 patients with non-degenerative nucleus pulposus (normal group) and 6 patients with lumbar disc herniation (degenerative group). Standard MSC culture medium was used for cell culture and cell morphology was observed. Flow cytometry was used to detect the expression of CD90, CD105, CD73, CD45, CD34 and HLA-DR, and to induce differentiation of mesenchymal stem cells by osteogenesis, adipogenesis and chondrogenesis. After 28 days of induction, alizarin red staining was used to identify the osteogenic ability of the cells, oil red O staining was used to identify the lipophilic ability of the cells, and toluidine blue staining was used to identify the cartilage differentiation ability of the cells. According to the criteria of mesenchymal stem cells proposed by the International Stem Cell Therapy Association (ISCT), the isolated cells were comprehensively evaluated and identified. The metabolic activity of P2 generation NPMSCs was detected by CCK-8. The total RNAs of P2 generation cells were extracted from two groups, and the expression of Oct4 and Nanog related to dryness maintenance of P2 generation cells was detected by RT-PCR. Results: the P 0 passage cells of the two groups were adherent to the wall, and there was no significant difference in morphology between the two groups. Immunophenotypic identification showed that the expression of CD90, CD105, CD73 on the surface of mesenchymal stem cells in normal group and degenerative group was above 9698,95%, respectively. Both groups expressed low expression of hematopoietic marker CD45, CD34, HLA-DR3 (all lower than 4%). Alizarin red staining, oil red O staining and toluidine blue staining confirmed that cells in normal group and degenerative group could differentiate into bone, fat and chondrocytes. The above results confirmed that the isolated cells were NPMSCs. The cell metabolic activity of P2 generation cells was higher than that of the degenerative group on the 5th day, 7d, 9d, 9d and 13d after culture, and there was a significant difference between the two groups (P 0.05). The expression of Oct4 and Nanog in the normal group was 4.63 卤1.177.36 卤1.19 times of that in the degenerative group, which was significantly higher than that in the degenerative group (P 0.05). Conclusion: NPMSCs exist in both normal and degenerative nucleus pulposus, but NPMSCs derived from normal intervertebral disc has stronger cellular metabolic activity and stronger gene expression of "dry maintenance" than that of "dry maintenance".
【作者单位】：南方医科大学第三临床医学院附属海军总医院骨科;
【基金】：国家自然科学基金面上项目(编号:81171740)
【分类号】：R363

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