【作者】 王庆德；

【导师】 杨述华；

【作者基本信息】 华中科技大学 ， 骨外科， 2008， 博士

【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(P<0.05);低氧组中PCNA染色细胞增多,高于正常氧组(P<0.05);低氧组中凋亡细胞减少,抗凋亡蛋白bcl-2表达增多(P<0.05);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(P<0.01);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(P<0.01);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(P<0.05),CollagenⅠ蛋白表达无显著差异(P<0.05)。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(P<0.05) ,而n组和nos组,HIF-1αmRNA表达始终处于较低的水平(P<0.05),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(P<0.05),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(P<0.05)。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(P<0.05),nos组7天后SDF-1蛋白表达量明显增加(P<0.05),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。更多还原

【Abstract】 Part One Proliferation and identification of human bone marrow mesenchymal stem cells in vitroObjective To supply seed cells for tissue engineering, the human bone marrow- derived mesenchymal stem cells (hMSCs) were isolated from adult human whose bone marrow is normal and cultured in vitro.Methods Bone marrow was obtained by aspirated in the posterior superior iliac spine from three adult patients whose marrow is normal. Cells were isolated by gradient centrifugation,.the cell morphologic features and growth status were observed by lightmicroscope. The cell surface antigen and osteo-differntiation were used to identify hMSCs.Results Isolated cells grow adherence,and the cells exhibited dominant growth of spiral type. Immunocytochemistry analysis demonstrated hMSCs were uniformly positive for CD44 and CD71,while negative for CD34 and CD45,which indicated no contamination of hematopoietic cells,the cell surface marker is consistent with character of mesenchymal stem cells.Isolated cells were induced by bone formation condition medium,2 weeks later cells alkali phosphatase were stained positively,4 weeks later calcium nodule could be found,all of these data showed that isolated cells were human mesenchymal stem cells.Conclusion Cells were isolated by human lymphocyte separating medium,could harvest more purity of hMSCs,which supply stable separation method for hMSCs. Part Two Effection of hypoxia on biological behavior of human bone marrow mesenchymal stem cellsObjective To establish a model of hypoxia culture and osteoblast differentiation in vitro, and investigate the biological characteristics of cultured adult mesenchymal stem cells(MSCs) in hypoxia. That will be experimental basis of MSCs used as the functional cells in bone tissue engineering.Methods Bone marrow was obtained by aspirated in the posterior superior iliac spine from three adult patients whose marrow is normal. Cells were isolated by gradient centrifugation and grown to confluence.The second passages According to the oxygen concentrations and nutrient mediums, was categorized into four groups: normoxic group ( 20 % O2, DMEM-LG), hypoxic group(1%O2, DMEM-LG), normoxic osteoblast induction group(20%O2 , conditioned medium), hypoxic osteoblast induction group(1%O2, conditioned medium).Proliferations and apoptosis of all the cultured cells were observed by an inverted phase contrast microscope,growth curve,detection of PCNA,apoptosis proteins and Colony forming unit-fibroblast (CFU-F); effection of osteo-differentiation of hMSCs were observed by real-time RT-PCR、activities of alkaline phosphatase and Alizarin red staining;Detected expression of Fibronectin、Laminin and CollagenⅠproteins,was used to view extracellular matrix secretary in low oxygen.Results the cells cultured in hypoxic group proliferated continuously throughout the culture period, while maintaining significantly higher colony-forming unit capabilities than hMSC cultured at normoxic oxygen(P<0.05);In low oxygen groups,apoptosis cells decreased,and anti-apoptosis protein bcl-1 increased(P<0.05). Upon induction, hMSCs of hypoxic group expressed less levels of osteoblast than normoxic osteoblast induction (P<0.01); Immunocytochemistry and Western blot detected,expression of FN and LA proteins increased(P<0.05),no signify -cant difference in expression of CollagenⅠ(P>0.05)Conclusion hMSCs maintained the ability to thrive in prolonged hypoxic conditions suggesting that hypoxia may be an essential element of the in vivo hMSCs niche.chronic hypoxia is a key parameter that infuences the in vitro biological behavior of hMSCs.Part Three Effects of chronic hypoxia on the expression of HIF-1α、SDF-1 and VEGF165 in human bone marrow mesenchymal stem cellsObjective To establish a model of chronic hypoxia in vitro culture , and observe the epression of HIF-1α、SDF-1 and VEGF165 in cultured adult human bone marrow stromal cells(hMSCs) ,That will be experimental basis of MSCs used as the functional cells for hypoxic disease therapy;by comparision of expression of hypoxia-inducible factor-1α(HIF-1α)、stomal cell- derived factor (SDF-1) and Vascular Endothelial Growth Factor 165(VEGF165 ) proteins,to document mechanism of hMSCs osteo-differentiation.Methods Bone marrow was obtained by aspirated in the posterior superior iliac spine from three adult patients whose marrow is normal. Cells were isolated by gradient centrifugation and grown to confluence.The second passages According to the oxygen concentrations and nutrient mediums, was categorized into four groups: normoxic group ( 20 % O2, DMEM-LG), hypoxic group(1%O2, DMEM-LG), normoxic osteoblast induction group(20%O2 , conditioned medium), hypoxic osteoblast induction group(1%O2, conditioned medium ) .HIF-1α、SDF-1 and VEGF proteins were measured by immuocytochemistry,Western-blot was used to detect HIF-1αand SDF-1 protein. HIF-1α、SDF-1 and VEGF165 mRNA were detected by real-time RT-PCR.Results cytoplasm of hMSCs of n group and nos group were stained by HIF-1αpositive,and strong positive staining were found in cytoplasm and cell nuclei of hMSCs in h group and hos group. Under chronic hypoxic condition,hMSC expressed mRNA of HIF-1α、SDF-1 and VEGF165 more strongly than that under normal oxygen condition(P<0.05),expression of HIF-1αmRNA in n group and nos group were in low level(P<0.05), Quantity of SDF-1 mRNA and VEGF165 mRNA increased with time,reached the peak maximium value at fourteenth days, then decreaed quickly(P<0.05).Western blot detected that,expression of HIF-1αand SDF-1 proteins in h and hos group were more than proteins in n and nos group(P<0.05),SDF-1 proteins in nos group increased greatly 7 days later(P<0.05),then decresed after 14 days.Conclusions in normoxia condition,SDF-1 and VEGF play a role in osteogenic capacity of hMSCs,although HIF-1 stimulate SDF-1 and VEGF secretion, inhibit function of SDF-1 and VEGF in osteo-differentiatio of hMSCs, perhaps it has another pathway for regulation of SDF-1 and VEGF secretion.更多还原