人脐带间充质干细胞对糖尿病大鼠骨折愈合的影响
发布时间：2018-07-29 11:00
【摘要】：研究背景：糖尿病(Diabetes Mellitus,DM)是由于高血糖导致内分泌代谢紊乱的全身性疾病,它能够造成包括骨骼、神经、肾脏等多器官多系统损伤。糖尿病患者胰岛素相对或绝对缺乏,矿物质组织代谢紊乱。无论是临床还是实验研究均显示,糖尿病能够改变骨的生物力学性能、影响骨折的愈合。许多研究表明,糖尿病骨折愈合时间大约是非糖尿病患者的两倍。每年虽然数以百万计的骨折发生,大多数治愈令人满意,但仍有5%至10%会继续延迟愈合或不愈合,甚至导致假关节形成或骨骼畸形,造成肢体功能障碍,严重影响患者的生活。因此如何治疗糖尿病骨折日益受到人们的重视。人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUCMSCs)和骨髓间充质干细胞(human bone marrow MSCs, hBMSCs)一样是一类具有自我更新和多向分化潜能的成体干细胞。但提取骨髓间充质干细胞时需要有创操作,而且提取数量较少。另外,随着年龄的增大,hBMSCs自我更新和增殖能力降低。最近,人脐带间充质干细胞越来越受到人们的关注,人脐带间充质干细胞可以向骨、软骨、韧带、肌腱、肌肉及脂肪等方向分化。其来源丰富,成本较低,而且无需有创操作。另外,hUCMSCs免疫排斥反应较低,无肿瘤致畸性。因此其在组织工程、干细胞移植以及基因治疗等领域具有广阔的应用前景,成为干细胞研究的热点,也为我们治疗糖尿病骨折愈合提供了新的思路。糖尿病影响骨折愈合的原因较复杂,而细胞因子在骨折愈合过程中的改变是其中重要的因素之一。其中TGF-β1和 BMP-2在骨折愈合过程中具有非常重要的作用。研究表明：TGF-β1和BMP-2是骨重建和骨修复过程中重要的细胞因子。在骨折愈合过程中,TGF-β1促进间充质干细胞和成骨细胞的增值,BMP-2是膜内成骨和软骨内化骨中非常重要的因子,其在骨折愈合过程中的研究较多,但是在糖尿病骨折愈合过程中的TGF-β1l和BMP-2的变化研究相对较少。应用人脐带间充质干细胞治疗糖尿病骨折愈合研究更少。我们应用大鼠制作糖尿病骨折动物模型,研究糖尿病骨折愈合过程中TGF-β1和BMP-2的变化。并应用人脐带间充质干细胞,诱导分化为成骨细胞,然后移植到骨折部位,观察糖尿病骨折愈合过程中TGF-p1和BMP-2的变化。为其临床治疗糖尿病骨折提供理论依据。研究目的：1.人脐带间充质干细胞的培养及成骨分化。2.糖尿病对骨折愈合的影响及TGF-pl和BMP-2在骨折愈合过程中的变化。3.人脐带间充质干细胞注射入骨折部位后,对糖尿病骨折愈合的影响。研究方法：1人脐带间充质干细胞的分离、鉴定及分化。1.1细胞培养人脐带间充质干细胞的分离、培养。获取健康、足月、剖腹产胎儿脐带,剥离脐带wharton'sjelly胶,充分剪碎,酶消化法获得脐带间充质干细胞,体外传代、纯化、扩增,倒置显微镜下观察细胞形态。1.2人脐带间充质干细胞的表面分子标志检测收集培养的第3代hUC-MSCs,加入CD14-PE.CD73.PE、CD105-PE.CD34-FITC..CD44-FITC.CD45-FITC.CD90-FITC.MHCⅡ-FITC单克隆抗体,1gGl-PE. 1gG1-FITC作为同型对照,24小时内应用流式细胞仪对细胞表面的CD分子进行鉴定。1.3人脐带间充质干细胞的成骨分化及成脂分化1.3.1收集第3代hUC-MSCs,以成骨诱导培养基培养,诱导7天后,行碱性磷酸酶染色；诱导分化21天后,进行茜素红染色,检测钙化基质沉淀。1.3.2收集第3代hUC-MSCs,以脂肪诱导培养基培养,诱导分化培养14天后,进行油红0染色。2糖尿病动物骨折模型的制作。2.1糖尿病骨折动物模型的制作及分组。雄性Wister大鼠75只随机分为三组：糖尿病组(n=25)、HUCMSCs组(n=25)、正常对照组(n=25)。糖尿病组及HUCMSCs组大鼠腹腔注射四氧嘧啶(160mg/kg)建立糖尿病动物模型,当血糖浓度血糖大于16.7mmol/l,则为糖尿病大鼠模型诱导成功。中途死亡和造模失败的大鼠再行造模补充,以保证每次每组用于检测的大鼠数量为5只。糖尿病模型制作成功后,在无菌条件下将三组动物均用手术方法于左胫骨上中1/3交界处用线据锯断胫骨人为造成左胫骨骨折。然后复位外固定。HUCMSCs组大鼠在骨折处用注射器穿刺至骨折端注射成骨诱导7天的第三代干细胞1x107个/Kg,正常对照组和糖尿病组注射等量的生理盐水做为对照。2.2骨密度检测对各组实验动物在术后1周、2周、3周、4周、5周,每组随机分别选取5只大鼠,然后使用DEXA骨密度仪扫描测定骨折处新生骨痂BMD,计算机记录数据。检测时骨密度仪设置为实验小动物模式,以新生骨痂为中心由近至远扫描1厘米范围,每组标本测量三次,每次测量前都重置标本,取平均值。2.3组织学及免疫组化学检查手术后第1、2、3、4、5周,每组分别随机抽取动物各5只大鼠,麻醉状态下以骨折端为中心,切取上下各0.5cm长标本,一半取出后立即放入液氮中储存(用于荧光定量RT-PCR检测),一半立即置入10%福尔马林中固定24小时,10%ED' A脱钙、包埋完毕后,石蜡切片机将骨组织切成5μm厚切片。常规进行病理切片的制作,免疫组化检测按说明书进行。Leica-QwinV3图像分析软件检测TGF-β1和BMP-2灰度值的变化。2.4荧光定量RT-PCR检测每组5只胫骨骨痂样本在液氮中取出后,进行荧光定量RT-PCR检测。qRT-PCR采用SYBR绿色PCR试剂盒,不加模板和反转录反应物的做为阴性对照。用GAPDH作为RT-PCR的内参,设定PCR程序。荧光定量PCR仪检测相关数据并进行分析。研究结果：1.采用酶消化法能有效分离纯化hUCMSCs。接种24时后,贴壁细胞形态多为长梭型、多边形或成纤维细胞样形态、大小均一,9天后成旋涡状生长。流式细胞仪分析,第3代细胞高表达CD44、CD73、CD90及CD105,低表达D14、CD34、CD45及MHCIⅠ。经成骨诱导分化后碱性磷酸酶染色呈强阳性,茜素红染色可见明显钙结节,油红O染色可见胞浆中红色的油滴。2.大鼠注射四氧嘧啶后,大部分在24-48 h后出现多饮、多食、多尿、血糖增高、呆滞、皮毛松散、体重下降等表现,血糖大于16.7mmol/l,糖尿病模型造模成功。3.骨密度结果显示：在术后第1周,三组大鼠骨痂局部骨密度测量值没有明显统计性差异(P0.05)；但在术后第2,3,4,5周,我们发现糖尿病大鼠组骨痂局部骨密度值随着时间逐渐增加,但在相应时间点其密度值明显低于对照组,其差异具有统计学意义；hUC-MSCs组骨密度值在术后第2,3,4,5周高于糖尿病组,差异具备显著性(P0.05),但是与对照组相比较,差异无统计学意义(P0.05)。4.组织学及免疫组化显示：糖尿病鼠软骨细胞及成骨细胞成熟延迟,骨痂形成较慢。免疫组化显示：骨折后1-3周TGF-β1、 BMP-2在细胞内广泛表达,特别是在骨痂和骨膜内,而糖尿病组骨痂组织中表达明显低于正常组与hUC-MSCs治疗组。hUCMSCs组和糖尿病组无明显差别。5. BMP-2、TGF-β1实时荧光定量RT-PCR检查显示：正常组断端骨痴组织BMP-2在第2周达高峰,而糖尿病组在第3周达高峰；正常组断端骨痴组织TGF-β1在第3周达高峰,而糖尿病组在第4周达高峰；糖尿病组TGF-β1 BMP-2表达比正常对照组均推迟1周,BMP-2？TGF-β1在 hUC-MSCs组表达在第2、3周均较糖尿病组高,但和正常对照组相比差别不大。结论：1.在体外应用酶消化法能获得人脐带间充质干细胞,人脐带间充质干细胞具有多向分化能力,可分化为成骨细胞及脂肪细胞。2.糖尿病大鼠骨折后骨折愈合速度慢、愈合延迟。3.糖尿病降低骨折处骨痂骨密度,影响骨折部位骨痂形成。4.糖尿病明显降低TGVF-β1、BMP-2表达,进而影响大鼠骨折处骨痂形成,是引起骨折愈合差的原因之一。5.应用人脐带间充质干细胞治疗后,糖尿病大鼠骨痂处细胞因子BMP-2、TGF-β1表达增多,骨密度提高,这可能是干细胞治疗改善骨折愈合的分子机制之一,可为临床治疗糖尿病骨折提供理论支持。
[Abstract]:Background: Diabetes Mellitus (DM) is a systemic disease caused by hyperglycemia, which causes endocrine and metabolic disorders. It can cause multiple organ and multiple system injuries, including bone, nerve and kidney. Diabetes patients are relatively or absolutely deficient in insulin, and metabolic disorders of minerals and tissues. Both clinical and experimental studies show that sugar Urinary diseases can change the biomechanical properties of bone and affect the healing of fractures. Many studies have shown that the healing time of diabetic fractures is about two times that of non diabetic patients. Although millions of fractures occur each year, most of the healing is satisfactory, but 5% to 10% will continue to delay healing or nonunion and even lead to false joint formation. Human umbilical cord mesenchymal stem cells (hUCMSCs) and bone marrow mesenchymal stem cells (human bone marrow MSCs, hBMSCs) are the same kind of self as a kind of self. Adult stem cells are updated and pluripotent. But the extraction of bone marrow mesenchymal stem cells requires invasive operation and less extraction. In addition, as the age increases, the ability of hBMSCs to renew and proliferate is reduced. Recently, human umbilical cord mesenchymal stem cells are becoming more and more concerned. Human umbilical cord mesenchymal stem cells can be treated to bone. The cartilage, ligaments, tendons, muscles and fat are differentiated. They are rich in origin, low in cost, and without invasive operation. In addition, hUCMSCs has low immune rejection and no tumor teratogenicity. Therefore, it has a broad application prospect in the fields of tissue engineering, stem cell transplantation and gene therapy, which has become a hot spot in stem cell research. We have provided new ideas for the treatment of fracture healing of diabetes. The causes of diabetes affecting fracture healing are complex, and the change of cytokines during the healing process is one of the most important factors. TGF- beta 1 and BMP-2 have a very important role in the healing process. Research shows that TGF- beta 1 and BMP-2 are bone remodeling and bone remodeling. TGF- beta 1 promotes the growth of mesenchymal stem cells and osteoblasts in the process of fracture healing. BMP-2 is a very important factor in the osteogenesis and endochondral osseous bone in the membrane. It has been studied more during the process of fracture healing, but the change of TGF- beta 1L and BMP-2 during the healing process of diabetes mellitus The study is relatively less. The application of human umbilical cord mesenchymal stem cells in the treatment of diabetic fracture healing is less. We used rats to make a diabetic fracture animal model to study the changes of TGF- beta 1 and BMP-2 during the healing process of diabetes. To observe the changes of TGF-p1 and BMP-2 during the healing process of diabetic fracture. To provide a theoretical basis for the clinical treatment of diabetes fractures. Objective: the study of 1. human umbilical cord mesenchymal stem cells and the effect of osteogenic differentiation of.2. diabetes on fracture healing and the changes of TGF-pl and BMP-2 during the fracture healing process,.3. human umbilical cord mesenchymal stem cells were injected. The effect of the fracture site on the healing of diabetic fracture. Research methods: isolation, identification and differentiation of human umbilical cord mesenchymal stem cells from 1 human umbilical cord mesenchymal stem cells, isolation and culture of human umbilical cord mesenchymal stem cells cultured in.1.1 cells. Obtaining healthy, full month, Caesarean birth umbilical cord, stripping umbilical cord wharton'sjelly glue, full scissors and enzyme digestion to obtain umbilical cord mesenchymal Stem cells, external generation, purification, amplification, and inverted microscope observation of cell morphology.1.2 human umbilical cord mesenchymal stem cells surface molecular markers detection collected and cultured third generation of hUC-MSCs, adding CD14-PE.CD73.PE, CD105-PE.CD34-FITC..CD44-FITC.CD45-FITC.CD90-FITC.MHC II -FITC monoclonal antibody, 1gGl-PE. 1gG1-FITC as the same type control, 2 In 4 hours, CD molecules on the cell surface were identified by flow cytometry to identify the osteogenic differentiation and lipid differentiation of.1.3 human umbilical cord mesenchymal stem cells. 1.3.1 collected third generation of hUC-MSCs for osteogenic induction culture. After 7 days of induction, alkaline phosphatase staining was performed. Alizarin red staining was performed for 21 days after induction of differentiation, and calcified matrix was detected to precipitate.1.. 3.2 third generations of hUC-MSCs were collected with fat induced culture medium and 14 days after induction of differentiation and culture. The animal model of.2 diabetes animal fracture was made and grouped by oil red 0. 75 male Wister rats were randomly divided into three groups: diabetes group (n=25), HUCMSCs group (n=25), normal control group (n=25). Diabetes mellitus (n=25). The diabetic rat model was established by intraperitoneal injection of four pyrimidine (160mg/kg) in the disease group and the HUCMSCs group. When the blood glucose concentration was greater than 16.7mmol/l, the diabetic rat model was successfully induced. The midway death and the failure of the rat model were supplemented to ensure that the number of rats used in each group was 5. After success, the three groups of animals were operated on the 1/3 junction of the left tibia at the upper and middle tibia at the left tibia to cause the fracture of the left tibia in the upper and middle of the left tibia at the upper and middle tibia at the left tibia. Then the.HUCMSCs group was fixed at the fracture site and injected into the fracture end by injecting the syringe to the fracture end to induce the third generation of the third generation stem cells (1x107 /Kg), the normal control group and the diabetes. The disease group was injected with the same amount of physiological saline for the control of.2.2 bone density test for 1 weeks, 2 weeks, 3 weeks, 4 weeks, 5 weeks, each group randomly selected 5 rats in each group, and then used DEXA bone densitometer to scan the newborn callus BMD at the fracture site and record the data. The callus was located at the center of 1 cm from near to far. Each group was measured three times. Before each measure, the specimens were reset. The average value of.2.3 histology and immuno chemical examination was 1,2,3,4,5 weeks after the operation. 5 rats were randomly selected from each group. Under the anesthetic state, the bone fracture end was taken as the center, and half of the upper and lower 0.5cm specimens were cut. Half After being taken out, stored in liquid nitrogen (for fluorescence quantitative RT-PCR detection), half immediately placed in 10% formalin and fixed for 24 hours, 10%ED'A decalcified. After the embedding, the paraffin slice machine cut the bone tissue into 5 mu m slices. Routine pathological section was made and the immunohistochemical detection was used to analyze the.Leica-QwinV3 image analysis software according to the instructions. Changes in the gray value of TGF- beta 1 and BMP-2 were detected by.2.4 fluorescence quantitative RT-PCR. After extraction of 5 tibial callus samples in each group, the fluorescence quantitative RT-PCR detection.QRT-PCR was carried out by SYBR green PCR kit, without the template and the reverse transcription reactant as negative control. The PCR program was set with GAPDH as the internal parameter of RT-PCR. The fluorescent quantitative PC was set. R was used to detect the related data and analyze the results. 1. after hUCMSCs. inoculation was used to effectively isolate and purify 24 inoculation, the morphology of the adherent cells was long shuttle type, polygon or fibroblast like form, the size of the cells was uniform, and the whirl grew after 9 days. The flow cytometry was divided and the third generation cells highly expressed CD44, CD73, CD90 and CD105, low Expression of D14, CD34, CD45 and MHCI I. The alkaline phosphatase staining was strongly positive after osteogenesis, and alizarin red staining showed obvious calcium nodules. Oil red O staining showed that after injection of four oxalacil in the red.2. rats of the cytoplasm, most of them appeared polydipsia, polyuria, hyperglycemia, dull, loose skin and weight loss after 24-48 h. At present, the blood sugar was more than 16.7mmol/l, and the results of.3. bone density in the model of diabetes model showed that there was no significant statistical difference between the three groups of rat callus local bone mineral density (P0.05) at first weeks after the operation, but at week 2,3,4,5 after the operation, we found that the bone mineral density value of the callus in the diabetic rats was gradually increased with time, but at the corresponding time. The density value of the inter point was significantly lower than that of the control group. The difference of BMD in hUC-MSCs group was higher than that of the diabetic group at week 2,3,4,5 after the operation, the difference was significant (P0.05), but compared with the control group, the difference was not statistically significant (P0.05).4. histology and immunohistochemistry showed that the chondrocytes and osteogenesis of diabetic rats were fine. TGF- beta 1 and BMP-2 were widely expressed in the callus and periosteum at 1-3 weeks after the fracture, especially in the callus and periosteum, and the expression of the bone callus in the diabetic group was significantly lower than that in the.HUCMSCs group and the diabetic group of the normal group and the hUC-MSCs group,.5. BMP-2, TGF- beta 1 real-time fluorescent quantitative RT-PC. The R examination showed that the tissue BMP-2 in the normal group reached the peak at second weeks, while the diabetic group reached the peak in the third week, and the TGF- beta 1 at the broken end of the normal group reached the peak in third weeks, and the diabetes group reached the peak in the fourth week, and the TGF- beta 1 BMP-2 expression in the diabetic group was 1 weeks late than that in the normal control group, and BMP-2? TGF- beta 1 was expressed in the hUC-MSCs group in the hUC-MSCs group. 2,3 weeks were higher than those in the diabetic group, but there was little difference compared with those in the normal control group. Conclusion: 1. the human umbilical cord mesenchymal stem cells can be obtained by enzyme digestion in vitro. Human umbilical cord mesenchymal stem cells have multiple differentiation ability and can differentiate into osteoblasts and adipocytes.2. diabetic rats with slow healing speed and delayed healing of.3. Diabetes reduces the bone scab bone density at the fracture site, affects the formation of.4. diabetes in the fracture site and significantly reduces the expression of TGVF- beta 1, BMP-2, and then affects the formation of callus in the fracture of the rat, which is one of the reasons for the poor healing of the fracture. After the treatment of.5. application human umbilical cord mesenchymal stem cells, the expression of BMP-2 and TGF- beta 1 in the callus of the diabetic rats is increased. The increase of bone mineral density may be one of the molecular mechanisms of stem cell therapy to improve fracture healing. It can provide theoretical support for clinical treatment of diabetic fractures.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.2;R683
本文编号：2152468
[Abstract]:Background: Diabetes Mellitus (DM) is a systemic disease caused by hyperglycemia, which causes endocrine and metabolic disorders. It can cause multiple organ and multiple system injuries, including bone, nerve and kidney. Diabetes patients are relatively or absolutely deficient in insulin, and metabolic disorders of minerals and tissues. Both clinical and experimental studies show that sugar Urinary diseases can change the biomechanical properties of bone and affect the healing of fractures. Many studies have shown that the healing time of diabetic fractures is about two times that of non diabetic patients. Although millions of fractures occur each year, most of the healing is satisfactory, but 5% to 10% will continue to delay healing or nonunion and even lead to false joint formation. Human umbilical cord mesenchymal stem cells (hUCMSCs) and bone marrow mesenchymal stem cells (human bone marrow MSCs, hBMSCs) are the same kind of self as a kind of self. Adult stem cells are updated and pluripotent. But the extraction of bone marrow mesenchymal stem cells requires invasive operation and less extraction. In addition, as the age increases, the ability of hBMSCs to renew and proliferate is reduced. Recently, human umbilical cord mesenchymal stem cells are becoming more and more concerned. Human umbilical cord mesenchymal stem cells can be treated to bone. The cartilage, ligaments, tendons, muscles and fat are differentiated. They are rich in origin, low in cost, and without invasive operation. In addition, hUCMSCs has low immune rejection and no tumor teratogenicity. Therefore, it has a broad application prospect in the fields of tissue engineering, stem cell transplantation and gene therapy, which has become a hot spot in stem cell research. We have provided new ideas for the treatment of fracture healing of diabetes. The causes of diabetes affecting fracture healing are complex, and the change of cytokines during the healing process is one of the most important factors. TGF- beta 1 and BMP-2 have a very important role in the healing process. Research shows that TGF- beta 1 and BMP-2 are bone remodeling and bone remodeling. TGF- beta 1 promotes the growth of mesenchymal stem cells and osteoblasts in the process of fracture healing. BMP-2 is a very important factor in the osteogenesis and endochondral osseous bone in the membrane. It has been studied more during the process of fracture healing, but the change of TGF- beta 1L and BMP-2 during the healing process of diabetes mellitus The study is relatively less. The application of human umbilical cord mesenchymal stem cells in the treatment of diabetic fracture healing is less. We used rats to make a diabetic fracture animal model to study the changes of TGF- beta 1 and BMP-2 during the healing process of diabetes. To observe the changes of TGF-p1 and BMP-2 during the healing process of diabetic fracture. To provide a theoretical basis for the clinical treatment of diabetes fractures. Objective: the study of 1. human umbilical cord mesenchymal stem cells and the effect of osteogenic differentiation of.2. diabetes on fracture healing and the changes of TGF-pl and BMP-2 during the fracture healing process,.3. human umbilical cord mesenchymal stem cells were injected. The effect of the fracture site on the healing of diabetic fracture. Research methods: isolation, identification and differentiation of human umbilical cord mesenchymal stem cells from 1 human umbilical cord mesenchymal stem cells, isolation and culture of human umbilical cord mesenchymal stem cells cultured in.1.1 cells. Obtaining healthy, full month, Caesarean birth umbilical cord, stripping umbilical cord wharton'sjelly glue, full scissors and enzyme digestion to obtain umbilical cord mesenchymal Stem cells, external generation, purification, amplification, and inverted microscope observation of cell morphology.1.2 human umbilical cord mesenchymal stem cells surface molecular markers detection collected and cultured third generation of hUC-MSCs, adding CD14-PE.CD73.PE, CD105-PE.CD34-FITC..CD44-FITC.CD45-FITC.CD90-FITC.MHC II -FITC monoclonal antibody, 1gGl-PE. 1gG1-FITC as the same type control, 2 In 4 hours, CD molecules on the cell surface were identified by flow cytometry to identify the osteogenic differentiation and lipid differentiation of.1.3 human umbilical cord mesenchymal stem cells. 1.3.1 collected third generation of hUC-MSCs for osteogenic induction culture. After 7 days of induction, alkaline phosphatase staining was performed. Alizarin red staining was performed for 21 days after induction of differentiation, and calcified matrix was detected to precipitate.1.. 3.2 third generations of hUC-MSCs were collected with fat induced culture medium and 14 days after induction of differentiation and culture. The animal model of.2 diabetes animal fracture was made and grouped by oil red 0. 75 male Wister rats were randomly divided into three groups: diabetes group (n=25), HUCMSCs group (n=25), normal control group (n=25). Diabetes mellitus (n=25). The diabetic rat model was established by intraperitoneal injection of four pyrimidine (160mg/kg) in the disease group and the HUCMSCs group. When the blood glucose concentration was greater than 16.7mmol/l, the diabetic rat model was successfully induced. The midway death and the failure of the rat model were supplemented to ensure that the number of rats used in each group was 5. After success, the three groups of animals were operated on the 1/3 junction of the left tibia at the upper and middle tibia at the left tibia to cause the fracture of the left tibia in the upper and middle of the left tibia at the upper and middle tibia at the left tibia. Then the.HUCMSCs group was fixed at the fracture site and injected into the fracture end by injecting the syringe to the fracture end to induce the third generation of the third generation stem cells (1x107 /Kg), the normal control group and the diabetes. The disease group was injected with the same amount of physiological saline for the control of.2.2 bone density test for 1 weeks, 2 weeks, 3 weeks, 4 weeks, 5 weeks, each group randomly selected 5 rats in each group, and then used DEXA bone densitometer to scan the newborn callus BMD at the fracture site and record the data. The callus was located at the center of 1 cm from near to far. Each group was measured three times. Before each measure, the specimens were reset. The average value of.2.3 histology and immuno chemical examination was 1,2,3,4,5 weeks after the operation. 5 rats were randomly selected from each group. Under the anesthetic state, the bone fracture end was taken as the center, and half of the upper and lower 0.5cm specimens were cut. Half After being taken out, stored in liquid nitrogen (for fluorescence quantitative RT-PCR detection), half immediately placed in 10% formalin and fixed for 24 hours, 10%ED'A decalcified. After the embedding, the paraffin slice machine cut the bone tissue into 5 mu m slices. Routine pathological section was made and the immunohistochemical detection was used to analyze the.Leica-QwinV3 image analysis software according to the instructions. Changes in the gray value of TGF- beta 1 and BMP-2 were detected by.2.4 fluorescence quantitative RT-PCR. After extraction of 5 tibial callus samples in each group, the fluorescence quantitative RT-PCR detection.QRT-PCR was carried out by SYBR green PCR kit, without the template and the reverse transcription reactant as negative control. The PCR program was set with GAPDH as the internal parameter of RT-PCR. The fluorescent quantitative PC was set. R was used to detect the related data and analyze the results. 1. after hUCMSCs. inoculation was used to effectively isolate and purify 24 inoculation, the morphology of the adherent cells was long shuttle type, polygon or fibroblast like form, the size of the cells was uniform, and the whirl grew after 9 days. The flow cytometry was divided and the third generation cells highly expressed CD44, CD73, CD90 and CD105, low Expression of D14, CD34, CD45 and MHCI I. The alkaline phosphatase staining was strongly positive after osteogenesis, and alizarin red staining showed obvious calcium nodules. Oil red O staining showed that after injection of four oxalacil in the red.2. rats of the cytoplasm, most of them appeared polydipsia, polyuria, hyperglycemia, dull, loose skin and weight loss after 24-48 h. At present, the blood sugar was more than 16.7mmol/l, and the results of.3. bone density in the model of diabetes model showed that there was no significant statistical difference between the three groups of rat callus local bone mineral density (P0.05) at first weeks after the operation, but at week 2,3,4,5 after the operation, we found that the bone mineral density value of the callus in the diabetic rats was gradually increased with time, but at the corresponding time. The density value of the inter point was significantly lower than that of the control group. The difference of BMD in hUC-MSCs group was higher than that of the diabetic group at week 2,3,4,5 after the operation, the difference was significant (P0.05), but compared with the control group, the difference was not statistically significant (P0.05).4. histology and immunohistochemistry showed that the chondrocytes and osteogenesis of diabetic rats were fine. TGF- beta 1 and BMP-2 were widely expressed in the callus and periosteum at 1-3 weeks after the fracture, especially in the callus and periosteum, and the expression of the bone callus in the diabetic group was significantly lower than that in the.HUCMSCs group and the diabetic group of the normal group and the hUC-MSCs group,.5. BMP-2, TGF- beta 1 real-time fluorescent quantitative RT-PC. The R examination showed that the tissue BMP-2 in the normal group reached the peak at second weeks, while the diabetic group reached the peak in the third week, and the TGF- beta 1 at the broken end of the normal group reached the peak in third weeks, and the diabetes group reached the peak in the fourth week, and the TGF- beta 1 BMP-2 expression in the diabetic group was 1 weeks late than that in the normal control group, and BMP-2? TGF- beta 1 was expressed in the hUC-MSCs group in the hUC-MSCs group. 2,3 weeks were higher than those in the diabetic group, but there was little difference compared with those in the normal control group. Conclusion: 1. the human umbilical cord mesenchymal stem cells can be obtained by enzyme digestion in vitro. Human umbilical cord mesenchymal stem cells have multiple differentiation ability and can differentiate into osteoblasts and adipocytes.2. diabetic rats with slow healing speed and delayed healing of.3. Diabetes reduces the bone scab bone density at the fracture site, affects the formation of.4. diabetes in the fracture site and significantly reduces the expression of TGVF- beta 1, BMP-2, and then affects the formation of callus in the fracture of the rat, which is one of the reasons for the poor healing of the fracture. After the treatment of.5. application human umbilical cord mesenchymal stem cells, the expression of BMP-2 and TGF- beta 1 in the callus of the diabetic rats is increased. The increase of bone mineral density may be one of the molecular mechanisms of stem cell therapy to improve fracture healing. It can provide theoretical support for clinical treatment of diabetic fractures.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R587.2;R683
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