骨髓间充质干细胞定向诱导前体骨细胞治疗兔骨损伤的研究

发布时间：2018-03-01 18:21

本文关键词： 兔骨髓间充质干细胞诱导分化移植骨折　出处：《昆明医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：[目的]：了解兔骨髓间充质干细胞(Bone mesenchymal stem cells,BMSCs)在成骨诱导剂的作用下定向分化为不同分化状态的细胞(包括多能干细胞、专能干细胞或前体骨细胞、成体骨细胞多个阶段),然后将各个阶段细胞移植到兔骨折模型后,观察不同分化阶段细胞对组织修复能力的差异及是否已归巢,以寻找出最适宜移植的细胞时间和状态。 [方法]：体外获得BMSCs,用GFP转基因标记后,向前体骨细胞定向诱导,分别在诱导第0、3、7、14、21、28d检测ALP活性。将实验兔随机分为5组：BMSCs移植组5只；成骨诱导3天移植组10只；成骨诱导7天移植组10只；成骨诱导21天移植组10只；模型对照组5只。建立兔尺骨骨折动物模型。造模成功24h后,将带GFP的各组细胞按照3×106cells/kg经尺骨骨折处局部注射,分别于第2、4和6周进行X射线检查,第6周全部处死后,取兔尺骨组织制作切片,观察不同诱导阶段的BMSCs移植后对骨折组织治疗的效果差异。 [结果]：①BMSCs能够正常贴壁成长并具有相应的分化能力,通过慢病毒转染GFP基因后不影响细胞正常生长及分化能力,抗原表达为CD34-/CD45-/CD73+/CD105+。②成骨诱导过程中进行碱性磷酸酶染色,成骨诱导0d组细胞染色为阴性；诱导3天组部分细胞染色,出现少量棕红色；诱导第7大组,大部分染色为强阳性,14、21、28天组和7天组对比阳性细胞数无明显变化。③移植后在动物的组织切片中发现骨组织中有绿色荧光存在,说明前体骨细胞已经发生归巢,并参与了机体重建。④分别于2、4、6周行X射线检查：5组参数总评分,BMSCs组和诱导7、21天组比较差异无显著性意义(P0.05),3天组和模型对照组比较差异有非常显著性意义(P0.01),3天组和BMSCs组.7、21天组比较差异有显著性意义(P0.05)。组织形态学分析：3天组缺损区骨端连接紧密,连接区有大量小梁状新骨形成,排列较规则,骨髓腔再通：BMSCs组和7、21天组断端内、外骨痂已相连,骨髓腔未再通：模型对照组骨缺损区未建立连接,骨断端之间骨小梁较细且排列紊乱,骨髓腔未再通。BMSCs和诱导分化的前体骨细胞在骨损伤的治疗上都发挥了作用,但是BMSCs(?)骨诱导3天组疗效更具有显著差异。 [结论]：①建立了BMSCs的体外分离、培养、鉴定及GFP标记的技术平台。②成功建立了兔尺骨骨折模型。③开展了BMSCs诱导分化的不同阶段细胞移植.对组织损伤修复的疗效差异的研究。BMSCs成骨诱导3天移植组的各项检查指标：X射线.病理染色、扫描电镜较其它组有明显改善,结果提示诱导3天组是比较适宜移植的细胞状态。
[Abstract]:Objective: to investigate the differentiation of bone mesenchymal stem cells from rabbit bone marrow mesenchymal stem cells (BMSCs) into differentiated cells (including pluripotent stem cells, specialized stem cells or precursor bone cells) under the action of osteogenic inducer. Adult bone cells were transplanted into the rabbit fracture model at different stages. The difference of the ability of tissue repair and the homing of the cells in different differentiation stages were observed in order to find out the most suitable time and state of transplantation. [methods]: BMSCs were obtained in vitro and labeled with GFP gene. The ALP activity was detected on the 0th day after induction by GFP transgenic cells. The experimental rabbits were randomly divided into 5 groups (n = 5): BMSCs transplantation group (n = 5), osteogenic induction group (n = 10) and osteogenic induction group (n = 10). After 7 days of osteogenic induction, 10 rats in the transplantation group, 10 rats in the 21 day group and 5 rats in the model control group, the animal model of ulna fracture was established. After 24 hours of success, the cells with GFP were injected locally at the ulna fracture according to 3 脳 106 cells / kg. X-ray examination was performed at the 4th and 6th weeks, and the ulnar tissues were taken to make sections after 6 weeks of death. The effect of BMSCs transplantation in different induction stages on the treatment of fracture tissue was observed. [results] BMSCs could grow normally and have the corresponding differentiation ability. The normal growth and differentiation of GFP cells were not affected by lentivirus transfection, and the antigen was expressed as alkaline phosphatase staining during osteogenesis induction of CD34-/CD45-/CD73 / CD105. 2. The staining of cells was negative in the group of 0 d after osteogenesis induction, a small amount of brown red was observed in some cells in the group of 3 days of induction, and the seventh largest group was induced in group 7. There was no significant change in the number of positive cells in most of the groups. 3. After transplantation, green fluorescence was found in the bone tissue, indicating that the precursor bone cells had homing. There was no significant difference between the BMSCs group and the 71-day induction group. There was no significant difference between the P0.05D group and the model control group. There was a significant difference between the 3 day group and the model control group (P 0.01) and the model control group (P 0.01) and the model control group (P < 0.05) and the model control group (P < 0.05). The difference was not significant between the BMSCs group and the 721 day group. There was a significant difference between the three groups and the model control group. There was a significant difference between the BMSCs group and the 21 day group (P 0.05). Histomorphology analysis showed that the osseous ends of the defect area in the 3 days group were closely connected. A large number of trabecular-shaped new bone formed in the junction area, arranged regularly. In the broken end of BMSCs group and 721 day group, the external callus was connected, and the medullary cavity was not re-opened. No connection was established in the bone defect area of the model control group. The trabecular meshwork between the broken ends of bone is fine and disordered. Both BMSCs and osteoblasts that induce differentiation play a role in the treatment of bone injury, but BMSCs have no effect on the treatment of bone injury, but BMSCs and BMSCs play an important role in the treatment of bone injury. There was significant difference in the effect of bone induction for 3 days. [conclusion] BMSCs was isolated and cultured in vitro. A rabbit model of ulnar fracture was established successfully by identifying and labeling the technique platform of GFP. 3. Cell transplantation was carried out in different stages of differentiation induced by BMSCs. Study on the difference of curative effect of tissue injury and repair. The three days of osteogenic induction of rabbit ulnar fracture. Item examination measure: X ray. Pathological staining. The results showed that the 3 day induction group was more suitable for transplantation than other groups.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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