体外低氧环境对人骨髓间充质干细胞成骨诱导分化影响的初步研究

发布时间：2018-06-15 10:07

本文选题：间充质干细胞 + 细胞培养　；参考：《华中科技大学》2008年博士论文

【摘要】： 第一部分人骨髓间充质干细胞的体外培养及鉴定目的体外分离培养人骨髓间充质干细胞(hMSCs)并进行鉴定,为组织工程提供适宜的种子细胞。方法抽取健康成人骨髓,采用人淋巴细胞分离液分离纯化获取hMSCs,观察细胞形态特征、生长状况,从表面抗原表达及成骨分化能力两个方面进行鉴定。结果分离细胞贴壁生长,呈集落样增殖,放射状、涡漩状分布排列。免疫细胞化学检测细胞表达CD44和CD71,CD34、CD45呈阴性表达,细胞表面标志表达与间充质干细胞的特征一致。分离的细胞经过成骨条件培养基诱导分化后2周,细胞碱性磷酸酶染色阳性,4周后可以形成钙结节,证明所培养细胞有向成骨细胞分化的潜能。表明分离细胞为间充质干细胞。结论采用人淋巴细胞分离液分离细胞,能获得较为纯化的hMSCs,可为培养hMSCs提供稳定的分离方法。第二部分低氧环境对人骨髓间充质干细胞生物学行为的影响目的建立人骨髓间充质干细胞(human mesenchymal stem cells ,hMSCs)体外低氧培养及向成骨细胞分化的生物模型,研究其生物学特征,为骨组织工程提供实验依据。方法采用人淋巴细胞分离液分离健康成人骨髓中的间充质干细胞。取第3代细胞,根据培养氧浓度及培养基类型分为4组:正常氧组(20%O2加DMEM-LG)、低氧组(1%O2加DMEM-LG)、正常氧成骨诱导组(20%O2加条件培养基)及低氧成骨诱导组(1%O2加条件培养基),通过细胞计数、细胞增殖测定(MTT法)、增殖细胞核抗原(PCNA)检测、凋亡蛋白及集落形成检测,观察低氧对hMSCs增殖凋亡的影响;RT-PCR检测、ALP活性检测及茜素红染色,研究低氧环境对hMSCs成骨分化的影响;检测Fibronectin、Laminin和CollagenⅠ蛋白表达,观察低氧对hMSCs细胞外基质分泌的影响。结果与正常氧组相比,低氧组中的hMSCs有较高的增殖速度,生长差异显著(P0.05);低氧组中PCNA染色细胞增多,高于正常氧组(P0.05);低氧组中凋亡细胞减少,抗凋亡蛋白bcl-2表达增多(P0.05);低氧组中的hMSCs生成的集落逐渐增多,明显高于正常氧组。低氧成骨诱导组培养的hMSC碱性磷酸酶活性逐渐增高,但明显低于正常氧成骨诱导组,两者有明显的差异(P0.01);定量RT-PCR检测,正常氧成骨诱导组ALP、OC、COL1A2及BSP mRNA表达量明显增加,ALP、OC、COL1A2及BSP mRNA明显增高(P0.01);培养4周后,与其它三组相比,正常氧成骨诱导组可见到明显的钙盐沉积和染成红色的钙结节。免疫组织化学和Western blot检测,低氧组中FN和LA分泌增多(P0.05),CollagenⅠ蛋白表达无显著差异(P0.05)。结论低氧使hMSCs增殖率增加,集落形成能力增强,抑制成骨细胞分化,保持干细胞特征,促进hMSCs分泌细胞外基质。第三部分慢性低氧影响HIF-1α、SDF-1及VEGF165在hMSCs中的表达目的建立体外hMSCs低氧模型,观察慢性低氧对hMSCs表达HIF-1α(低氧诱导因子-1α)、SDF-1(基质细胞衍生因子-1)及VEGF165(血管内皮细胞生长因子)的影响,为hMSCs治疗缺氧性疾病提供实验依据。通过比较HIF-1α、SDF-1及VEGF165,探讨低氧抑制hMSCs成骨分化的机制。方法采用人淋巴细胞分离液分离健康成人骨髓中的间充质干细胞。取第3代细胞,根据培养氧浓度及培养基类型分为4组:正常氧(n组)(20%O2加DMEM-LG)、低氧组(h组)(1%O2加DMEM-LG)、正常氧成骨诱导组(nos组)(20%O2加条件培养基)及低氧成骨诱导组(hos组)(1%O2加条件培养基),运用免疫组织化学检测HIF-1α、SDF-1及VEGF蛋白表达, western blot检测HIF-1α及SDF-1蛋白表达,定量RT-PCR检测HIF-1α、SDF-1及VEGF165 mRNA表达。结果n组和nos组hMSCs细胞质内HIF-1α染色阳性,h组和hos组hMSCs胞质及胞核内均强阳性染色。低氧条件下,SDF-1染色细胞数目明显增多,VEGF染色呈阳性。hMSCs在慢性低氧的条件下,h组和hos组HIF-1α、SDF-1及VEGF165 mRNA表达水平明显增高,与n组相比,两者有显著差异(P0.05) ,而n组和nos组,HIF-1αmRNA表达始终处于较低的水平(P0.05),nos组SDF-1 mRNA表达量持续增高,14天时达到峰值,随后开始降低(P0.05),n组、h组、nos组及hos组随着时间的延长,VEGF165 mRNA表达均逐渐增多,但h组及hos组VEGF mRNA表达始终高于n组和nos组,nos组14天时VEGF1165 mRNA表达量快速增加达到峰值,随后开始降低(P0.05)。western blot检测,h组和hos组可见HIF-1α及SDF-1蛋白表达增多,明显高于n组和nos组(P0.05),nos组7天后SDF-1蛋白表达量明显增加(P0.05),14天后减少。结论SDF-1和VEGF在正常氧条件下有协同成骨的作用,低氧条件下HIF-1虽然刺激SDF-1和VEGF分泌增多,但抑制SDF-1和VEGF协同成骨的作用,SDF-1和VEGF分泌还存在着除HIF-1调控以外的途径。
[Abstract]:Part one: culture and identification of human bone marrow mesenchymal stem cells in vitro
Objective to isolate and culture human bone marrow mesenchymal stem cells (hMSCs) in vitro and identify them, so as to provide suitable seed cells for tissue engineering.
Methods the bone marrow of healthy adults was extracted, and hMSCs was obtained by separation and purification by human lymphocyte separation solution. The morphological characteristics and growth status of the cells were observed. The expression of surface antigen and the ability of osteogenic differentiation were identified in two aspects.
Results the cell proliferation, radiate and whirlpool distribution were arranged in the isolated cells. The expression of CD44 and CD71, CD34, CD45 in immunocytochemical detection cells were negative, and the expression of the cell surface markers was consistent with the characteristics of mesenchymal stem cells. The isolated cells were induced 2 weeks after the differentiation of bone conditioned medium, and the alkaline phosphatase of the cells was induced. Staining positive, calcium nodules can be formed after 4 weeks, indicating that the cultured cells have the potential to differentiate into osteoblasts, indicating that the isolated cells are mesenchymal stem cells.
Conclusion the purified hMSCs can be obtained by separating the cells from human lymphocytes, which can provide a stable separation method for hMSCs culture.
The second part is the effect of hypoxic environment on the biological behavior of human bone marrow mesenchymal stem cells.
Objective to establish a biological model of human bone marrow mesenchymal stem cells (human mesenchymal stem cells (hMSCs) in vitro hypoxia culture and osteoblast differentiation, and to study the biological characteristics of bone marrow mesenchymal stem cells, and to provide experimental basis for bone tissue engineering.
Methods human mesenchymal stem cells were separated from healthy adult bone marrow by human lymphocyte separation solution. Third generation cells were divided into 4 groups according to the oxygen concentration and medium type: normal oxygen group (20%O2 plus DMEM-LG), hypoxia group (1%O2 plus DMEM-LG), normal oxygen induction group (20% O2 plus conditioned medium) and hypoxia induction group (1%O2 plus conditioned culture). Yang Ji), by cell count, cell proliferation assay (MTT), proliferating cell nuclear antigen (PCNA) detection, apoptotic protein and colony formation detection, the effect of hypoxia on hMSCs proliferation and apoptosis was observed. RT-PCR detection, ALP activity detection and alizarin red staining were used to study the effect of hypoxia on the osteogenesis of hMSCs, and Fibronectin, Laminin and Collagen I were detected. Protein expression was observed to observe the effect of hypoxia on the secretion of extracellular matrix in hMSCs cells.
Results compared with the normal oxygen group, the hMSCs in the hypoxia group had a higher proliferation rate and significant difference in growth (P0.05), and the number of PCNA stained cells in the hypoxia group was higher than that in the normal oxygen group (P0.05), the apoptotic cells decreased and the expression of anti apoptotic protein Bcl-2 increased (P0.05) in the hypoxia group, and the colony of hMSCs generated in the hypoxia group increased gradually, obviously higher than that in the normal oxygen group. Oxygen group. The activity of hMSC alkaline phosphatase in the hypoxia induction group increased gradually, but it was significantly lower than that of the normal oxygen induction group (P0.01). Quantitative RT-PCR detection, the expression of ALP, OC, COL1A2 and BSP mRNA in the normal oxygen induction group increased obviously, ALP, OC, COL1A2 and BSP. In the other three groups, there were obvious calcium salts and red calcium nodules in the normal oxygen induction group. Immunohistochemistry and Western blot detection, the increase of FN and LA secretion in the hypoxia group (P0.05), and no significant difference in the expression of Collagen I protein (P0.05).
Conclusion hypoxia can increase the proliferation rate of hMSCs, enhance colony forming ability, inhibit osteoblast differentiation, maintain the characteristics of stem cells, and promote the secretion of extracellular matrix by hMSCs.
The third part of chronic hypoxia affects the expression of HIF-1, SDF-1 and VEGF165 in hMSCs.
Objective to establish an in vitro hMSCs hypoxia model and observe the effect of chronic hypoxia on hMSCs expression of HIF-1 alpha (hypoxia inducible factor -1 alpha), SDF-1 (matrix cell derived factor -1) and VEGF165 (vascular endothelial cell growth factor), and provide experimental basis for the treatment of hypoxia disease by hMSCs. Through comparison of HIF-1 alpha, SDF-1 and VEGF165, hypoxia inhibits hMSCs osteogenesis. The mechanism of differentiation.
Methods human mesenchymal stem cells were separated from healthy adult bone marrow by human lymphocyte separation solution. The third generation cells were divided into 4 groups according to the oxygen concentration and medium type: normal oxygen (group n) (20%O2 plus DMEM-LG), hypoxia group (H Group) (1%O2 plus DMEM-LG), normal oxygen induction group (NOS group) and hypoxia induction group. In group HOS) (1%O2 plus conditioned medium), the expression of HIF-1 alpha, SDF-1 and VEGF protein was detected by immunohistochemistry. The expression of HIF-1 A and SDF-1 protein was detected by Western blot, and HIF-1 alpha, SDF-1 and expression were detected by RT-PCR.
Results in group n and group NOS, HIF-1 alpha staining was positive in hMSCs cytoplasm, and both in group H and hos group were strongly positive in cytoplasm and nucleus of hMSCs. The number of SDF-1 stained cells increased obviously in hypoxia condition, VEGF staining showed positive.HMSCs in chronic hypoxia, H group and hos group were increased obviously, two There were significant differences (P0.05), while the expression of HIF-1 alpha mRNA in group n and NOS was always at a lower level (P0.05). The mRNA expression of SDF-1 in NOS Group continued to increase, reaching the peak at 14 days, then began to decrease (P0.05), N group, H group, group and group gradually increased with time. Higher than group n and group NOS, VEGF1165 mRNA expression increased rapidly at 14 days in group NOS, and then decreased (P0.05).Western blot detection. H group and hos group showed increased expression of HIF-1 alpha and SDF-1 protein, which was significantly higher than that in group and group. After 7 days, the expression of protein was obviously increased, and decreased after 14 days.
Conclusion SDF-1 and VEGF have synergistic osteogenesis in normal oxygen condition, while HIF-1 stimulates the increase of SDF-1 and VEGF secretion in hypoxic conditions, but inhibits the synergistic effect of SDF-1 and VEGF on osteogenesis, and there are other pathways other than HIF-1 regulation in SDF-1 and VEGF secretion.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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