【作者】 陈强；

【导师】 周余来；

【作者基本信息】 吉林大学 ， 生物工程， 2007， 硕士

【摘要】 骨髓间充质干细胞(mesenchymal stem cells, MSCs)是骨髓内造血干细胞以外的非造血干细胞,具有多向分化潜能。骨髓间充质干细胞分化为多种功能细胞的研究进展,为治疗缺血性胰腺疾病提供了可能。目的:从细胞形态学对骨髓MSCs的生长特性进行观察,探讨骨髓MSCs分离纯化的方法;通过对动物模型注入标记的细胞,探讨MSCs对胰腺缺血损伤的治疗水平,并进一步探讨其治疗途径和机制。方法:抽取Wistar大鼠乳鼠骨髓,采用贴壁培养和组织块培养相结合的方法得到高纯度的骨髓MSCs,在含20% FBS的L-DMEM培养液中扩增培养。传3代后用EGFP重组腺病毒标记细胞,注入胰腺缺血性损伤动物模型胰腺局部,观察动物存活率,冰冻切片共聚焦显微镜观察标记细胞位置。结果:1、分离获得了高纯度贴壁生长的MSCs。MSCs原代培养呈均匀分布的集落样生长,呈梭形,传代培养中细胞均质性明显提高,细胞传代稳定;2、注入细胞的动物模型存活率明显高于对照组;3、共聚焦显微镜显示表明,注入细胞24h后,细胞可到达受损部位,并聚集在微循环系统中,以后逐渐大量进入受损部位,在循环系统及导管系统中有较高表达。结论:1、本实验通过对大鼠骨髓MSCs进行分离、体外培养,同时从细胞形态和生物学特性进行观察,认为采用贴壁培养和组织块培养法相结合,可以获得纯度较高和活性较强的骨髓MSCs,且方法简便易行;2、骨髓MSCs易分离培养,体外增殖和传代能力强,通过离体培养可使体内环境下低丰度的MSCs实现数量扩增;3、经培养纯化的MSCs无需体外诱导即可在体内应用,且效果良好;4、根据共聚焦显微镜对冰冻切片的观察,可证实MSCs修复缺血性胰腺损伤的主要途径是首先修复微循环系统,这可能就是MSCs修复缺血性胰腺损伤的机制。更多还原

【Abstract】 There are two results, that can be developed by tissues and organs repair regeneration, pancreas injury, acute and chronic pancreas inflammations. One is damaged cell replaced by the new same kind of cells, and function and construction can be recovered completely. The other is damaged cell cannot be regenerated, the tissue injury is replaced by fiber scar and lead to fibrosis, function and construction cannot be recovered completely as the situation before the damage. Division and proliferation of adult pancreas cells are difficult. For getting the ideal repair of function and construction after pancreas damage, it should have adult multifunctional stem cells or the other similar cells. And that is the ultimate way to solve the regeneration problem. Recently, it has not have break.Stem cell can be classified as embryo stem cell and adult stem cell. The main function of adult stem cells which founded in tissues at the present is the preparation for repairing damaged tissues. In bone marrow, not only have haemopoietic stem cells had the generation function and differentiation ability but mesenchymal stem cells (MSCs) have. In some situation, MSCs can beinduced to special tissue cells for replacing damaged tissue. MSCs have multi-directional differentiation potentiality and plasticity obviously. MSCs can migrate to some haematopoiesis tissues and differentiate to corresponding tissue cells in vivo, and have the ability to differentiate mesoderm and neuroectoderm tissue cells (such as osteoblast, chondroblast, adipocyte, fibroblast, endotheliocyte, myocyte, neurocyte and stroma-cell which can support hematopoiesis) in different induction condition (dexamethasone, TGF-β, FGF, insulin, for instance) in vitro.For knowing the immunity after MSCs being transplanted, some scholar used fluorescent labeling to label histoleucocyte which was non-passage and isolated directly from bone marrow and MSCs which was purified, and transplanted the labeling cells to rats’bodies. The result was no detection of non-passage histoleucocyte which labeled. It is considered that histoleucocyte could be rejected by immune system of host rat. Purified MSCs could survive, ecesis, and had no rejection in host rats; that proves that MSCs have unique immunogenicity and antigenicity is low even not have. It is possible to transplanting stem cells and ecesising successfully in vivo by the feature.The research is based on the feature mentioned. It discussed the repair function and mechanism by using pancreas injury animal models which used rats’MSCs cell cultured in vitro and injected after purifying.1. The cell culture of rats’MSCs in vitroCell adherence culture and tissue culture, which the research used, can get a large number of MSCs with high cell purity. G0-G1 cells are account 85.87% on the total cellular score determined by flow cytometer. It shows MSCs have infinite generation ability.2. The induction and regeneration of MSCs in vitroThe purified MSCs were added in osteoblast induction liquid and lipoblast induction liquid. After two weeks inducted, the cells were tested the production of cell induction by alkaline phosphatase and oil red staining. The result showed that MSCs have been inducted to osteoblast and lipoblast after inducted cell culture, and the specificity staining was positive. It proves the ability of MSCs’multi-differentiation.3. The survival rate of animal which injected MSCsTwo groups of animal model were set up, one was injected the MSCs (MSCs group), the other was injected the isotonic sodium chloride (group). The result showed the MSCs group was better than the NaCl2 group in aspects of mental status, activity, eating and drinking. To be observed 6 weeks continuously, the mortal rate of MSCs group is 10%, by contrast, that of the NaCl2 group is 80%. The differences between two groups were significant by statistics analysis.4. The observation of the position where MSCs settled down in vivoThe MSCs with Ad-EGFP labeled were transplanted into animal models. The animals were executed by luxation after transplantation 1 day, 3 day and 7 day. The pancreas tissues would be taken out, and be made into frozen section which would be observed with confocal microscopy. To observe the frozen section in the first 24 hours after injection of MSCs, the back ground was clean; there has only little non-specificity fluorescence; only one circular fluorescent image was showed in tissue frozen section and no fluorescent labeling was found. The circle is micro-circulatory system lumina tissue, and it was strong considered as venule by pathology specialist. Therefore, afterMSCs entered the damaged tissue, circulatory system was the first place where MSCs have been, and angei-endothelial tissue was the first repaired tissue. Duo to MSCs entered the angei-endothelial of damaged tissue’s circulatory system, small vessels got repaired, and it improved blood supply and ventilation of tissue. Damaged cell’s nutrition state improved, and got repaired. That leaded to tissue repaired. It might be one of the mechanisms of repairing damaged cells by MSCs.Some experiments showed that bone mesenchymal stem cells (BMSCs) can differentiate to pancreas cell by using inducing factor in vitro. It can find out in the research that BMSCs can differentiate to pancreas cells and substituted damaged or necrosis cells even though BMSCs was not induced to pancreas cells in vitro, so that the damaged tissue could recover quickly. The procedure happened at the proper circumstance with various kinds of biological factor in proper proportion if it was the emergency request of body.更多还原