大鼠骨髓基质细胞体外培养的生物学特性

发布时间：2018-01-19 06:12

本文关键词： 骨髓基质细胞成骨潜能细胞培养生物学特性　出处：《山西医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的:骨创伤后的再生能力提示有能够自我复制并能多向分化的干细胞存在。一般认为,干细胞存在于骨、骨膜、骨髓及其他由中胚层发育来的组织中,在出生后仍然有少量干细胞继续存在,在机体损伤时参与组织的修复。组织工程的基础是有功能的细胞,骨组织工程需要大量的成骨细胞。十多年来,国内外学者和临床工作者对各种组织的细胞成骨能力进行了大量的研究,提出了定向性骨祖细胞、诱导性骨祖细胞和基质干细胞的概念。目前,在研究中使用的作为种子细胞的成骨细胞来源有以下几种:骨、软骨、骨外膜、骨髓和骨外组织。越来越多的证据表明,骨髓基质干细胞(BMSCs)有多分化潜能,移植人体内后可分化为骨、软骨、肌肉、脂肪、血管内皮、肝脏、神经等多种细胞。能够作为组织工程中不同细胞的来源;在体内正常循环中能够分布于多种组织和器官,可以用于细胞和基因治疗。而且取材、分离培养、扩增以及导入外源基因也相对方便。BMSC。通过抽取自体骨髓得到,对病人造成创伤较少,且无免疫排斥反应。因此,BMSCs在实际应用时将具有潜在的优势。木试验目的是建立一种简单有效的骨髓基质细胞的体外培养及成骨诱导方法,并探讨在体外培养中其增殖和分化的相互关系,总结骨髓基质细胞体外培养的生物学规律,为将来在体外分离、培养、扩增出大量的有成骨活性的细胞以满足再造骨组织的需要提供了美好的前景。方法:冲洗法获取Weistar大鼠骨髓,用贴壁法分离提纯骨髓基质细胞(Bone MarrowStromal Cells BMSCs),传至第3代细胞后分组:1.分别用普通培养基和诱导培养基(DMEM加入地米松10~(-7)mmol/L,β-甘油磷酸钠10mmol/L,维生素C50mg/L)培养10天,每3天换液1次。绘制各自细胞生长曲线。2.分别用普通培养基和诱导培养基培养15天,每3天换液1次,于第4、7、10、13、16天,检测两组的碱性磷酸酶表达量。3.以5×10~4/ml密度接种于6孔培养板各1板,各孔放置载玻片,分别加入基础培养液和诱导培养液培养,每3天换液,20天后分别取出各自细胞爬片行Von kossa染色。结果:1.普通培养基中细胞的生长速度明显快于诱导培养基中的细胞。2.细胞碱性磷酸酶表达量两组有显著性差异(P＜0.05),普通培养基组明显低于诱导组。3.对照组细胞Vonkossa染色阳性,试验组为阴性。结论:1.贴壁筛选法是一种简单有效的骨髓基质细胞的分离纯化方法。2.骨髓基质细胞在体外可诱导分化为成骨细胞,可作为骨组织工程中种子细胞的来源。3.骨髓基质细胞的增殖和分化存在相互抑制的关系。
[Abstract]:Objective: the ability of regeneration after bone trauma suggests the existence of stem cells capable of self-replicating and multidifferentiation. Stem cells are generally thought to exist in bone, periosteum, bone marrow and other tissues developed from mesoderm. After birth, a small number of stem cells continue to exist, participate in the repair of tissue when the body is damaged. Tissue engineering is based on functional cells, bone tissue engineering requires a large number of osteoblasts for more than a decade. Scholars and clinical workers at home and abroad have done a lot of research on the osteogenic ability of various tissues and put forward the concepts of directional bone progenitor cells induced bone progenitor cells and stromal stem cells. The following sources of osteoblasts are used as seed cells in the study: bone, cartilage, periosteum, bone marrow and extraosseous tissue. Bone marrow stromal cells (BMSCs) can differentiate into bone, cartilage, muscle, fat, vascular endothelium and liver after transplantation. Nerve and other cells. Can be used as a source of different cells in tissue engineering; In the normal circulation in the body can be distributed in a variety of tissues and organs, can be used in cell and gene therapy. Amplification and introduction of foreign genes is also relatively convenient .BMSC.Through extraction of autologous bone marrow, the trauma to patients is less and there is no immune rejection. BMSCs will have potential advantages in practical application. The purpose of wood test is to establish a simple and effective method of bone marrow stromal cell culture and osteogenesis induction in vitro. The relationship between proliferation and differentiation of bone marrow stromal cells in vitro was discussed, and the biological rules of bone marrow stromal cells culture in vitro were summarized in order to isolate and culture bone marrow stromal cells in vitro. The amplification of a large number of osteogenic cells to meet the needs of bone tissue reconstruction provides a bright prospect. Methods: bone MarrowStromal Cells BMSCs of Weistar rats were isolated and purified by adherent method. The cells were transferred to the third passage and divided into two groups: 1. DMEM and DMEM were added to DMEM and 10 mmol / L 尾 -glycerophosphate (10 mmol / L, 10 mmol / L), respectively. Vitamin C 50 mg / L) was cultured for 10 days and changed solution once every 3 days. The cell growth curve was drawn. The cells were cultured in normal medium and induction medium for 15 days and once every 3 days. The expression of alkaline phosphatase (ALP) in the two groups was detected for 16 days. At the density of 5 脳 10 ~ (4) / ml, the two groups were inoculated with 6 hole culture plates (1 plate each), and the slides were placed in each hole. The cells were cultured in basic culture medium and induction medium respectively. After 20 days of culture, the cells were removed for Von kossa staining. Results 1. The growth rate of cells in the ordinary medium was significantly faster than that in the induction medium. There was a significant difference in the expression of alkaline phosphatase between the two groups (P < 0.05). The normal medium group was significantly lower than the induction group. 3. The Vonkossa staining was positive in the control group and negative in the experimental group. Conclusion the adherent screening method is a simple and effective method for the isolation and purification of bone marrow stromal cells. Bone marrow stromal cells can be induced to differentiate into osteoblasts in vitro. It can be used as a source of seed cells in bone tissue engineering. 3. The proliferation and differentiation of bone marrow stromal cells can be inhibited by each other.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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