体外联合培养体系中HUVECs对hBMSCs成骨分化的影响及其机制研究

发布时间：2019-07-02 14:24

【摘要】：[研究背景及目的] 不同原因所致各种类型骨缺损在临床上十分常见,颌骨缺损的修复问题一直是临床上难以解决的问题。组织工程学的出现和发展为骨缺损修复带来了机遇,其最终目标是将功能细胞与可降解三维生物支架材料在体外联合培养,构建成为有活性的组织或器官,然后植入体内,替代病损的组织,恢复其形态、结构和功能。目前组织工程骨构建方法多用单纯种子细胞接种,虽然能够成骨,但存在组织工程骨血管化缓慢,新骨生长迟缓等问题。骨髓间充质干细胞(bone marrow Mesenchymal stem cells, BMSCs)具有多向分化的潜能,在适当条件下不仅可以分化为脂肪细胞、骨细胞、软骨细胞、心肌细胞、神经元细胞、成肌细胞、肌腱细胞及星形胶质细胞等。目前关于诱导单纯BMSCs向成骨细胞分化的研究取得了较大的进展,然而诱导单纯BMSCs向成骨细胞分化存在成骨周期长、成骨效率低、细胞易老化等缺点。近年来,研究人员发现内皮细胞可以分泌骨形态发生蛋白(Bone morphogenetic protein, BMP),促进成骨分化的同时刺激成骨细胞及其前体细胞分泌血管内皮生长因子(vascular endothelial growth factor, VEGF),而VEGF在血管发生和形成过程中发挥着重要作用,可以促进内皮细胞增殖。然而目前血管内皮细胞对骨髓间充质干细胞成骨分化作用的具体机制还不清楚,尚缺乏从基因水平验证血管内皮细胞对骨髓间充质干细胞成骨分化的作用。为此,本课题着重探讨人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)在联合培养体系中对人骨髓间充质干细胞(human bone marrow Mesenchymal stem cells, hBMSCs)的形态、生长、细胞分化及其Bmi-1基因和Runx2基因表达的影响,从基因水平验证血管内皮细胞对骨髓间充质干细胞成骨分化的作用。为脐静脉内皮细胞和骨髓间充质干细胞联合培养作为骨组织工程的种子细胞提供理论基础。 [方法] (1)抽取志愿者骨髓液,使用密度梯度离心法分离骨髓单个核细胞,并借助MSCs黏附于塑料瓶底这一特性进行纯化,相差显微镜观察形态变化。将MSCs传代扩增培养至第三代,流式细胞仪检测CD34.CD29.CD44表面抗原表达,鉴定MSCs; (2)将订购的HUVECs用ECM+10%新生胎牛血清扩增至第三代后与第三代HUVECs按1：1比例建立以DMEM+10%胎牛血清为培养基的联合培养体系。以单独培养的hBMSCs组及hUVECs组作为阴性对照组,分别于第4、6、8、10天相差显微镜观察形态变化,用计数板计数各组hBMSCs数量; (3)分别于第4、6、8、10天每组每个时间点取6孔检测三组培养体系中碱性磷酸酶(Alkaline phosphatase,ALP)及骨钙素(Osteocalin,OC)含量。用SPSS17.0软件对各项检测值进行统计学分析； (4)采用实时荧光定量PCR(fluorescence quantitative PCR,FQ-PCR)检测第4、6、8、10天单独培养的hBMSCs组及联合培养组中hBMSCs的Bmi-1和Runx2基因表达的情况,每组每个时间点取6孔。用SPSS17.0软件对各项检测值进行统计学分析。 [结果] (1)采用Ficoll液密度梯度离心法分离、提纯hBMSCs可达到较高的纯度。用流式细胞仪对第3代hMSCs进行细胞表型分析鉴定,CD34低表达,CD29.CD44高表达； (2)新生胎牛血清分离培养的人骨髓间充质干细胞成细长梭形,细胞较细小,原代培养4至5天就可见成团生长的细胞；第三代骨髓间充质干细胞形态单一,成梭形,呈旋涡状分布,没有细胞重叠现象。hBMSCs在4-6天呈对数生长,8-10天后生长进入平台期。HUVECs成单层生长,形态成多角形、鹅卵石状镶嵌排列,边界清楚,胞浆丰富,胞核呈圆形或椭圆形,偶见双核,第5d融合成片,第一代至第四代生长速度较快,2-3天即可传代； (3)各组碱性磷酸酶检测量随时间延长先增高后降低,各时间联合培养组ALP较高,8天时联合培养组碱性磷酸酶最高；骨髓间充质干细胞组和脐静脉内皮细胞组ALP基本没有变化；联合培养组和其它各组之间两两比较均有显著统计学意义(P0.01)；各组骨钙素检测量随时间延长先增高后降低；8天时联合培养组骨钙素最高；联合培养组和其它各组之间两两比较均有显著统计学意义(P0.01)； (4)联合培养组Bmi-1、Runx2基因检测量随时间延长逐渐增高,各时间联合培养组Runx2基因表达较高；骨髓间充质干细胞组Bmi-1基因随时间延长略有增高,Runx2基因表达基本没有变化；8th天时联合培养组Bmi-1、Runx2基因检测量最高；联合培养组和骨髓间充质干细胞组之间比较有显著统计学意义(P0.01)。 [结论] (1)采用Ficoll液密度梯度离心法分离、提纯的hBMSCs,经流式细胞仪鉴定为较高纯度的细胞； (2)骨髓间充质干细胞与脐静脉内皮细胞联合培养相容性良好,脐静脉内皮细胞对体外联合培养体系中骨髓间充质干细胞具有促进增殖的作用； (3)在体外联合培养体系中,脐静脉内皮细胞能促进骨髓间充质干细胞Bmi-1及Runx2基因的表达,诱导其向成骨细胞方向分化。
[Abstract]:[Background and purpose of the study] The various types of bone defects due to different causes are very common in the clinic, and the problem of the repair of the defect of the jaw is always difficult to solve. Problems. The emergence and development of tissue engineering brings an opportunity for the repair of bone defects. The ultimate goal is to combine functional cells and degradable three-dimensional biological scaffold materials in vitro, to construct an active tissue or organ, Form, structure and The present tissue engineering bone construction method is multi-purpose simple seed cell inoculation, although it can be formed, the tissue engineering bone vascularization is slow, the growth of the new bone is slow, and the like. The bone marrow mesenchymal stem cells (BMSCs) have the potential of multi-directional differentiation, and can not only be differentiated into adipocytes, osteocytes, chondrocytes, cardiac muscle cells, neuron cells, myoblasts, tendon cells and star-like cells under appropriate conditions. At present, it has made great progress in the study of inducing the differentiation of only BMSCs to the osteoblast, but the induction of the differentiation of the BMSCs to the osteoblast is long, the osteogenic efficiency is low, and the cells are easy to use. In recent years, the researchers have found that endothelial cells can secrete bone morphogenetic protein (BMP), promote osteogenic differentiation, and stimulate the secretion of vascular endothelial growth factor (VEGF) in the osteoblast and its precursor cells. or, VEGF), and VEGF plays an important role in angiogenesis and formation, and can To promote the proliferation of the endothelial cells, the specific mechanism of the current vascular endothelial cells to the osteogenic differentiation of the bone marrow-derived mesenchymal stem cells is not clear. To study the effects of human umbilical vein endothelial cells (HUVECs) on human bone marrow mesenchymal stem cells (hBMSCs), the morphology, growth, cell differentiation and Bmi-1 gene and R of human bone marrow-derived mesenchymal stem cells (hBMSCs) in human umbilical vein endothelial cells (HUVECs) were discussed in this paper. The effect of unx 2 gene expression on the expression of bone marrow from vascular endothelial cells from the gene level The role of the osteogenic differentiation of the mesenchymal stem cells is the combination of the umbilical vein endothelial cells and the bone marrow mesenchymal stem cells as bone tissue engineering. fine seed The cell provides a theoretical basis.[Method] (1) The bone marrow liquid of the volunteers is extracted, and the bone marrow mononuclear cells are separated by using a density gradient centrifugation method, and the bone marrow mononuclear cells are adhered to the bottom of the plastic bottle by means of MSCs. The culture of MSCs was cultured to the third generation, and the CD34. CD29 was detected by flow cytometry. The expression of the surface antigen of CD44 was identified and the MSCs were identified; (2) the newly-ordered HUVECs was expanded to the third generation with the ECM + 10% of the new fetal calf serum, and the second generation HUVECs was set up in a 1:1 ratio with the third generation HUVECs for DME The combined culture system of M + 10% fetal calf serum was used as the negative control group for the cultured hBMSCs group and the hUVECs group. the number of hBMSCs in each group is counted by using a counting plate, and (3) the number of each group of hBMSCs is detected by a counting plate; and (3) six holes are taken at each time point of each group in groups 4,6,8 and 10 respectively to detect the alkaline in three groups of culture systems Phosphatase, AL P) and the content of Osteocin (OC). (4) Bmi of hBMSCs and hBMSCs in group 4,6,8 and 10 were detected by real-time fluorescence quantitative PCR (FQ-PCR). -1 and Runx2 gene expression,6 for each group of time points per group Hole. S The PSS17.0 software performs a statistical analysis of the detection values.[Results] (1) Use F The purification of hBMSCs can achieve higher purity by the method of density gradient centrifugation, and the third generation hMSCs were determined by flow cytometry. The analysis and identification of the cell phenotype, the low expression of CD34, the high expression of CD29. CD44, and (2) the isolation and culture of the new fetal bovine serum. the mesenchymal stem cells are in an elongated shuttle shape, the cells are small, and the cells growing in a cluster can be seen in the primary culture for 4 to 5 days; The third generation of the bone marrow-derived mesenchymal stem cells has a single form, a spindle shape, a vortex-like distribution, no cells, the hBMSCs are logarithmic growth on 4-6 days and grow into the platform stage after 8-10 days; the HUVECs are single-layer growth, the shape is polygonal, the cobblestone is embedded and arranged, the boundary is clear, the cytoplasm is rich, the nucleus is circular or elliptical, 2-3 days after the first-generation to fourth-generation growth, and 2-3 days of passage; (3) the amount of alkaline phosphatase in each group was decreased with the increase of time, the ALP of the combined culture group was higher, and the combination of 8 days was combined. The alkaline phosphatase of the culture group was the highest, and the ALP of the bone marrow mesenchymal stem cell group and the umbilical vein endothelial cell group did not change basically, and there was a significant difference between the combined culture group and the other groups (P0 .01). The amount of bone-bone detection in each group was decreased with the increase of time, and the combined culture group was cultured on 8 days. There was a significant difference between the two groups in the combined culture group and the other groups (P0.01), and (4) the combined culture group Bmi- 1. The amount of the Runx2 gene was increased with the time, and the expression of the Runx2 gene in the combined culture group was high; the Bmi-1 gene of the bone marrow-derived mesenchymal stem cell group increased slightly with the time, and the expression of the Runx2 gene was basically unchanged; the Bmi-1 and Runx2 groups in the combined culture group at the 8th day due to the amount of detection There was a significant difference between the combination culture group and the bone marrow mesenchymal stem cell group (P0.01).[Conclusion] (Conclusion] ( 1) Separation and purification of hBMSC by Ficoll density gradient centrifugation (2) bone marrow mesenchymal stem cells and umbilicus; the combined culture of the vein endothelial cells is good, the umbilical vein endothelial cells have the effect of promoting the proliferation of the bone marrow mesenchymal stem cells in the in vitro combined culture system, and (3) in vitro combination
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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