骨髓间充质干细胞与牙髓细胞共培养体系在牙髓再生中的应用研究

发布时间：2018-06-01 04:28

本文选题：骨髓间充质干细胞 + 牙髓细胞　；参考：《广西医科大学》2016年硕士论文

【摘要】：目的：体外建立骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells, BMMSCs)与牙髓细胞(Dental Pulp Cells, DPCs)的共培养体系,观察和评价两种细胞在共培养后各自细胞特性的变化和相互影响,探索最佳的两种细胞在共培养体系中的细胞比例和培养时间,体内评估该共培养体系培养后的细胞应用于牙髓再生中的可行性,为牙髓再生种子细胞的选择提供新思路及实验基础。方法：1、体外分离、培养及鉴定BMMSCs和DPCs。将细胞在0.4 μm的Transwell小室中进行共培养,分组情况如下：①单一BMMSCs;②单一DPCs; ③BMMSCs和DPCs共培养组。共培养6天后,用MTT法绘制以上各组细胞的生长曲线并分别提取RNA及蛋白,分别进行聚合酶链反应(PCR)及蛋白免疫印迹(WB)检测牙本质涎磷蛋白(DSPP)、碱性磷酸酶(ALP)、基质细胞抗原-1(Stro-1)和CD145的表达情况。2、为探索最佳的共培养细胞比例和体外共培养时间,将细胞在0.4μm的Transwell小室中进行共培养,BMMSCs与DPCs按照10：I、I：I、1：5和1：10的比例分别共培养3、6、9、12天。提取以上各组细胞的mRNA,qPCR检测DSPP、ALP、Stro-1和CD146的表达并进行统计学分析。3、将人牙牙根段的根管进行机械及化学预备,将牙根段随机分为4组,每组4个样本,将不同的细胞成分在水凝胶支架中混匀,并注入根管内,分组情况如下：①BMMSCs组；②DPCs组；③共培养组；④水凝胶支架空白组。将以上制备好的牙根段,移植到裸鼠背部,6周后取出标本,制作HE染色切片,并于显微镜下观察组织生长情况。结果：1、分离培养的SD大鼠骨髓间充质细胞,24小时后可见细胞贴壁呈比较均一的圆球状,3天后呈长梭形、纺锤样,随着培养时间延长,细胞增殖分裂聚集呈簇似漩涡样。流式细胞仪检测间充质干细胞表面标记物CD29,CD44表达阳性；造血干细胞标记物CD45,CD34表达阴性。用成骨诱导培养基、成脂诱导培养基及成软骨诱导培养基中诱导21天、21天和14天后,分别进行茜素红、油红0及阿尔新蓝染色阳性。牙髓细胞传至第三代后形态均一,长梭形,呈成纤维样细胞形态。免疫组织化学染色提示,角蛋白染色阴性,而波形丝蛋白染色阳性,提示培养的牙髓细胞为间充质来源,可用于后续实验。2、通过共培养体系,可以提高BMMSCs牙髓细胞特异标志物DSPP和ALP的表达,降低干细胞标志物Stro-1和CD146的表达,显示有向牙髓细胞向分化的趋势；同时也可以提高DPCs干细胞标志物的表达,降低牙髓细胞标志物的表达,提示有去分化的趋势,激活分化为其它细胞的潜能。3、共培养细胞比例与时间改变,会对共培养细胞的分化情况有明显影响。在BMMSCs与DPCs按照1：1与1：5的比例共培养时,可以较高效的诱导BMMSCs分化及DPCs表达干细胞特异标记物；体外共培养条件下,BMMSCs牙髓细胞标志物表达增加、干细胞标志物表达下降在共培养9-12天增加更为明显,DPCs干细胞标记物在共培养6-9天增加明显。4、BMMSCs与DPCs按照1：1比例共培养9天,并装配至多肽水凝胶支架充盈至经机械化学预备后的根管内,牙根段移植至裸鼠背部6周后,发现共培养组能再生组织结构与正常牙髓相似的类牙髓组织,而单一细胞组再生组织量较少且组织结构与正常牙髓差异较大。结论：1、在共培养体系中,BMMSCs与DPCs相互影响,增殖速率及mRNA、蛋白的表达发生变化；BMMSCs受DPCs的影响出现牙髓细胞向分化的趋势,DPCs出现去分化表现,结果提示共培养条件在一定程度可调控共培养细胞的增殖与分化,用低比例的DPCs获得更多来源的适合牙髓组织再生的种子细胞。2、在BMMSCs与DPCs共培养体系中,细胞共培养比例为1：1、1：5,体外共培养时间为9天左右时,细胞增殖与分化互相影响最为明显。3、体内皮下异位实验的结果显示,该共培养体系培养的细胞可以作为牙髓组织工程再生中的种子细胞使用。
[Abstract]:Objective: to establish a co culture system of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and dental pulp cells (Dental Pulp Cells, DPCs) in vitro, to observe and evaluate the changes and interaction of the two cells in co culture, and to explore the proportion of the best two cells in the co culture system and the proportion of the cells in the co culture system. Culture time, in vivo evaluation of the viability of the cells used in dental pulp regeneration in vivo, and provide new ideas and experimental basis for the selection of dental pulp regeneration seed cells. Methods: 1, in vitro isolation, culture and identification of BMMSCs and DPCs. cells in the Transwell chamber of 0.4 micron m. The following groups are as follows: 1 Single BMMSCs; (2) single DPCs; (3) BMMSCs and DPCs co culture group. After 6 days of co culture, the growth curves of these cells were plotted by MTT and RNA and protein were extracted respectively. Polymerase chain reaction (PCR) and protein immunoblotting (WB) were used to detect dentin sialosphosin (DSPP), alkaline phosphatase (ALP), matrix cell antigen -1 (Stro-1) and 45.2, in order to explore the best co culture cell ratio and in vitro co culture time, the cells were co cultured in the 0.4 m Transwell chamber, and BMMSCs and DPCs were co cultured for 3,6,9,12 days in proportion to 10:I, I:I, 1:5 and 1:10 respectively. Statistical analysis.3, the root canal of the tooth root canal was mechanically and chemically prepared, and the root segments were randomly divided into 4 groups, each group had 4 samples, and the different cell components were mixed in the hydrogel scaffold and injected into the root canal. The groups were as follows: (1) group BMMSCs; (2) DPCs group; (3) co culture group; (4) the blank group of hydrogel scaffold. The prepared root segments were transplanted into the back of nude mice. After 6 weeks, the specimens were taken out and the HE staining sections were made. The tissue growth was observed under the microscope. Results: 1, the bone marrow mesenchymal cells were separated and cultured in SD rats. After 24 hours, the cells displayed a relatively uniform round ball. After 3 days, the spindle shaped, spindle like, and the culture time prolonged, The cell proliferation and division were clustered like swirling like vortices. Flow cytometry was used to detect the surface markers of mesenchymal stem cells (CD29, CD44), hematopoietic stem cell markers, CD45, CD34 negative. It was induced by osteogenic induction medium, fat induced medium and cartilage induced medium for 21 days, and alizarin red and oil, respectively, after 21 days and 14 days. Red 0 and alnew blue staining were positive. The dental pulp cells were spread to third generations, with long spindle shape and fibroblast like cells. Immunohistochemical staining suggests that keratin is negative, and the coloration of the plasma protein is positive, suggesting that the cultured dental pulp cells are the source of mesenchymal cells, which can be used for subsequent experimental.2 and can be extracted through co culture system. The expression of DSPP and ALP, a specific marker of high BMMSCs dental pulp cells, reduced the expression of stem cell markers Stro-1 and CD146, showing a tendency to differentiate into dental pulp cells, and could also improve the expression of the markers of DPCs stem cells and reduce the expression of the markers of dental pulp cells, suggesting the trend of dedifferentiation and activation to differentiate into other cells. The potential.3, co culture cell proportion and time change, will have a significant influence on the differentiation of co cultured cells. When BMMSCs and DPCs co culture 1:1 and 1:5, it can induce BMMSCs differentiation and DPCs expression of stem cell specific markers more efficiently. Under co culture conditions, the expression of BMMSCs dental pulp cell markers is increased. The decrease of expression of cell markers was more obvious at 9-12 days of co culture. DPCs stem cell markers increased.4 in co culture for 6-9 days, BMMSCs and DPCs were co cultured for 9 days according to 1:1 ratio, and assembled into the root canal of the polypeptide hydrogel scaffold after mechanochemical preparation, and the root segments were transplanted to the back of nude mice for 6 weeks, and the co culture group was found. The regenerated tissue structure is similar to the normal dental pulp, while the single cell group has less tissue and the difference between the tissue structure and the normal pulp. Conclusion: 1, in the co culture system, BMMSCs and DPCs interact with each other, the proliferation rate and the expression of mRNA, and the expression of protein are changed; BMMSCs is affected by DPCs. The trend of the dedifferentiation of DPCs shows that co culture conditions can regulate the proliferation and differentiation of co cultured cells to a certain extent, and use a low proportion of DPCs to obtain more seed cells that are suitable for the regeneration of dental pulp tissue,.2. In the co culture system of BMMSCs and DPCs, the proportion of cell co culture is 1:1,1:5, and in vitro co culture time. At about 9 days, the cell proliferation and differentiation affect the most obvious.3. The result of subcutaneous ectopic experiment in the body shows that the cells cultured in this co culture system can be used as seed cells in the regeneration of dental pulp tissue engineering.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R781

【相似文献】