人退变椎间盘髓核组织来源干细胞表面标志物的低表达对其生物学特性的影响

发布时间：2018-06-27 14:46

本文选题：退变椎间盘 + 髓核　；参考：《暨南大学》2017年硕士论文

【摘要】：目的:运用组织块贴壁法从脊柱内镜微创手术摘除的退变椎间盘髓核组织中高效分离培养间充质干细胞样细胞,并从细胞形态、细胞表型、增殖能力、三系分化潜能、多能性基因和髓核细胞标志基因表达等方面对从不同个体退变椎间盘髓核组织样本来源的髓核干细胞(nucleus pulposus derived stem cells,NPSCs)进行深入研究。方法:组织块贴壁法高效分离人退变椎间盘髓核组织来源干细胞。将获得的细胞通过传代进行纯化和扩增培养,进行形态学观察,取第2代生长良好的干细胞,流式细胞术检测NPSCs表面标志物的表达情况,并进行成骨成脂分化潜能鉴定及成软骨分化潜能鉴定。分别取生长良好的第2代的不同个体退变椎间盘髓核组织来源的干细胞,利用流式细胞术检测间充质干细胞表面标志物、髓核细胞表面标志物CD24的表达,以及细胞周期和细胞凋亡情况。利用CCK8试剂盒(Cell Counting Kit,CCK-8)检测比较细胞活力,细胞计数法检测比较细胞的倍增时间,并绘制细胞生长曲线。qRT-PCR检测比较髓核细胞相关标志基因COL2A1、Aggrecan和SOX9,髓核干细胞标志基因Tie2,以及多能性基因NANOG和OCT4的表达水平。茜素红S染色法鉴定比较成骨分化潜能,油红O染色法鉴定比较成脂分化潜能。阿利新蓝染色法鉴定比较成软骨分化潜能。使用Image J软件计算向成骨成脂成软骨诱导分化后茜素红S、油红O和阿利新蓝染色定量分析。结果:组织块贴壁法分离的NPSCs,形态多为典型梭形细胞,少数为多边形或圆形;流式细胞术检测细胞表面标志物CD29、CD44、CD73、CD90和CD105呈阳性表达,细胞表面标志物CD11b、CD14、CD34、CD45和HLA-DR呈阴性表达;经过21天的成骨成脂成软诱导分化后,茜素红染色、阿利新蓝染色及油红O染色均呈阳性,说明NPSCs具有向成骨、成软骨和成脂肪诱导分化的能力。进一步对不同个体退变椎间盘髓核组织来源的干细胞比较研究发现,根据MSC特异性表面标志物的表达水平可分为两组:MSC表面标志物高表达组(H-NPSCs)和MSC表面标志物低表达组(L-NPSCs),流式细胞术检测结果显示CD29、CD44、CD73、CD90和CD105表面标志物表达率均大于95%,与之相应的是其传代后的细胞形态均一,呈成纤维细胞样的长梭形。而L-NPSCs的MSC特异性表面标志物CD29和CD105表达率显著低于H-NPSCs,CD29的表达率为36.52±0.14%,CD105的表达率为46.58±0.15%,细胞形态表现多样性,除典型梭形细胞外,可见多边形或圆形出现。两组都不表达CD11b、CD14、CD24、CD34、CD45和HLA-DR(表达率低于1%)。CCK8检测的细胞活性和细胞周期结果表明:与L-NPSCs相比,H-NPSCs具有更强的增殖能力。与细胞周期结果分析一致,L-NPSCs中G0/G1和G2/M期停滞增加,S期的进入量低于H-NPSCs,细胞凋亡、细胞倍增时间结果分析表明H-NPSCs的凋亡率和倍增时间都要低于L-NPSCs。qRT-PCR检测这三种细胞髓核细胞标志基因COL2A1、Aggrecan、SOX9的表达情况显示:L-NPSCs的三种标志基因的表达量都显著高于H-NPSCs的表达量。髓核干细胞标志基因Tie2的表达情况显示:H-NPSCs的表达量的显著高于L-NPSCs的表达量。而多能性基因NANOG和OCT4的表达情况显示:L-NPSCs的表达量显著高于H-NPSCs的表达量。诱导分化后特异性染色检测显示,L-NPSCs的多向分化能力明显弱于H-NPSCs。结论:1.组织块贴壁法可从人退变椎间盘髓核组织中分离出间充质干细胞样细胞,表达间充质干细胞表面标志物CD29、CD44、CD73、CD90和CD105,不表达造血干细胞表面标志物CD11b、CD14、CD34、CD45和HLA-DR,并具有向成骨成脂及成软骨多向分化潜能。2.退变椎间盘髓核组织来源的NPSCs可根据其间充质干细胞表面标志物的表达水平分为高表达组(H-NPSCs)与低表达组(L-NPSCs)。与H-NPSCs相比,L-NPSCs的增殖能力、细胞活力和多向分化潜能明显减弱,而L-NPSCs的髓核细胞标志基因表达水平显著高于H-NPSCs。髓核干细胞的MSC表面标志物的表达水平与细胞增殖能力、多向分化潜能和髓核细胞特异性标志基因表达水平相关。
[Abstract]:Objective: to isolate and culture mesenchymal stem cell like cells from degenerative intervertebral discs removed from spinal endoscope minimally invasive surgery by tissue block adherence, and to degenerate intervertebral discs from different individuals in terms of cell morphology, cell phenotype, proliferation, three lineage differentiation potential, pluripotent gene and gene expression of nucleus pulposus cells. Nucleus pulposus derived stem cells (NPSCs) of nucleus pulposus samples. Methods: the tissue block adherence method was used to efficiently separate the stem cells from the human degeneration intervertebral disc nucleus. The obtained cells were purified and expanded through the passage, and the morphological observation was carried out for the second generations of fine stem cells. Cell, flow cytometry was used to detect the expression of NPSCs surface markers, identification of osteogenic differentiation potential and identification of chondrogenic differentiation potential. Stem cells from different individuals with good growth of second generations of degenerative intervertebral disc nucleus were taken respectively. Flow cytometry was used to detect the surface markers of mesenchymal stem cells and the cell surface of nucleus pulposus cells. The expression of CD24, cell cycle and cell apoptosis. The cell viability was compared with the CCK8 Kit (Cell Counting Kit, CCK-8). Cell counting method was used to detect the doubling time of the cells, and the cell growth curve.QRT-PCR was plotted and compared with the nucleus pulposus related marker gene COL2A1, Aggrecan and SOX9, and the nucleus pulposus stem cells. The marker gene Tie2, as well as the expression level of the pluripotent gene NANOG and OCT4. The alizarin red S staining was used to identify the osteogenic differentiation potential. The oil red O staining method was used to identify the lipid differentiation potential. Alizarin red S and oil red after differentiation of osteogenic adipogenic into cartilage by Image J software were used to calculate the differentiation of alizarin red S and oil red. Quantitative analysis of O and alicaln blue staining. Results: the morphology of NPSCs separated by tissue block adherence was mostly spindle cells with a few polygons or circles; flow cytometry was used to detect the cell surface markers CD29, CD44, CD73, CD90 and CD105, and the cell surface markers CD11b, CD14, CD34, CD45 and HLA-DR were negative expression; after 21 days After a soft induction of osteogenesis, alizarin red staining, alizarin blue staining and oil red O staining were positive, indicating that NPSCs has the ability to induce differentiation into osteogenesis, cartilage and adipose tissue. Further research on stem cells from different individual degenerative intervertebral discs has been found to be based on the expression of specific surface markers of MSC. The level can be divided into two groups: MSC surface markers high expression group (H-NPSCs) and low expression group (L-NPSCs) of MSC surface markers. Flow cytometry results showed that the expression rates of CD29, CD44, CD73, CD90 and CD105 surface markers were more than 95%, corresponding to the cell morphology of the cells after its passage, and the long spindle shape of fibroblast like, and L-NPSCs. The expression rate of MSC specific surface markers, CD29 and CD105, was significantly lower than that of H-NPSCs, the expression rate of CD29 was 36.52 + 0.14%, the expression rate of CD105 was 46.58 + 0.15%, and the cell morphology was varied. The polygon or round appearance was visible except for the typical spindle cells. The two groups did not express CD11b, CD14, CD24, CD34, CD45 and HLA-DR (the expression rate under 1%). The results of cell activity and cell cycle test showed that compared with L-NPSCs, H-NPSCs had stronger proliferation ability. In accordance with the analysis of cell cycle results, the stagnation of G0/G1 and G2/M in L-NPSCs increased, and the entry of S stage was lower than H-NPSCs, cell apoptosis, and the result of cell doubling time results showed that the apoptosis rate and doubling time of H-NPSCs were lower than L-NP. The expression of the three cell marker genes COL2A1, Aggrecan, and SOX9 showed that the expression of the three markers of L-NPSCs was significantly higher than the expression of H-NPSCs. The expression of Tie2 in the stem cell marker gene of the nucleus pulposus showed that the expression of H-NPSCs was significantly higher than the expression of L-NPSCs, while the pluripotent gene N was expressed as N. The expression of ANOG and OCT4 showed that the expression of L-NPSCs was significantly higher than that of H-NPSCs. After induction of differentiation, specific staining showed that the multidirectional differentiation ability of L-NPSCs was significantly weaker than that of H-NPSCs. conclusion: 1. tissue block adherent method can separate mesenchymal stem like cells from the nucleus of human degeneration intervertebral disc and Express mesenchymal stem cells. Cell surface markers, CD29, CD44, CD73, CD90 and CD105, do not express the surface markers of hematopoietic stem cells, CD11b, CD14, CD34, CD45 and HLA-DR, and NPSCs can be divided into high expression groups according to the expression level of the surface markers of mesenchymal stem cells. Compared with the low expression group (L-NPSCs), the proliferation ability, cell viability and pluripotent differentiation potential of L-NPSCs were significantly lower than that of H-NPSCs, while the expression level of L-NPSCs was significantly higher than the expression level and proliferation ability of MSC surface markers in H-NPSCs. nucleus pulposus stem cells, the pluripotent differentiation potential and the specific markers of nucleus pulposus cells. The expression level of the chronicles is correlated.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R681.5

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