大鼠骨髓干细胞的培养、诱导分化为骨骼肌治疗骨骼肌缺损的动物模型的实验研究

发布时间：2018-09-03 18:27

【摘要】：[目的] 验证大鼠骨髓干细胞(MSCs)体外培养的方法及其生物学特性，研究MSCs的多项分化潜能以及其在体外定向诱导分化为骨髂肌细胞，设计一组动物模型，分别将诱导后、未诱导的MSCs、阳性对照细胞注射到动物模型体内，研究MSCs在体内的诱导分化情况及其最终的形态变化；从而，找到肌肉缺损最佳的修复方法，为临床治疗开拓广阔的前景。 [方法] 首先培养大鼠骨髓干细胞(MSCs)、大鼠骨骼肌细胞(Mb，，主要用于阳性对照)，将体外培养的MSCs第三代用5—氮杂胞苷诱联合MyoD、TGF-β1、IGF-1导分化，用免疫组织化学方法鉴定诱导后的细胞。制做动物模型：取16只10周龄的Wistar大鼠，在其左后小腿胫前肌中部注射无水乙醇0.2ml，使其胫前肌中部变性坏死。右后小腿胫前肌中部注射无菌生理盐水0.2ml。将16只大鼠模型随机分成四组，第一组阴性对照，只注射培养基；第二组实验组，注射MSCs；第三组实验组，注射诱导后的MSCs；第四组阳性对照组，注射Mb。分别将第三代细胞、诱导后的细胞用BrdU标记，然后收集等待注射。动物模型建立4h后，就可以注射。注射部位双小腿胫前肌中部。在注射细胞后的第3d、6d、9d和12d分别取材进行形态观察和免疫学检查； [结果] 大鼠MSCs体外培养生长良好，形态不规则，流式细胞仪检测绝大部分细胞处于G1期(79.4％)，免疫组化检测CD44阳性，而CD34阴性。大鼠Mb体外培养生长良好，形态规则，流式细胞仪检测绝大部分细胞处于G1期(79.8％)，免疫组化检测Desmine，Myosin均阳性。经诱导后MSCs，形态多
[Abstract]:[objective] to verify the method and biological characteristics of rat bone marrow stem cell (MSCs) cultured in vitro, to study the differentiation potential of MSCs and its directional differentiation into osteoiliac muscle cells in vitro, and to design a group of animal models. The uninduced MSCs, positive control cells were injected into the animal model to study the differentiation of MSCs in vivo and its final morphological changes, so as to find the best method of repairing muscle defect and open up a broad prospect for clinical treatment. [methods] cultured rat bone marrow stem cell (MSCs),) rat skeletal muscle cells (Mb, was mainly used as positive control). The third generation of MSCs was induced by 5-azacytidine combined with MyoD,TGF- 尾 1IGF-1. The induced cells were identified by immunohistochemical method. Animal model: sixteen 10-week-old Wistar rats were injected with anhydrous ethanol 0.2 ml in the middle of the anterior tibial muscle of the left posterior leg to make the degeneration and necrosis of the middle part of the anterior tibial muscle. The right posterior leg anterior tibial muscle was injected with sterile saline 0.2 ml. Sixteen rats were randomly divided into four groups, the first group was negative control group, the other group was injected with culture medium, the second group was injected with MSCs;, the third group was injected with MSCs;, the fourth group was injected with MSCs; positive control group, and the second group was injected with Mb.. The third generation cells were labeled with BrdU and then collected for injection. The animal model can be injected 4 hours later. The injection site is the middle of the anterior tibial muscle of both legs. Morphological observation and immunological examination were performed on the 3rd day, 6th day and 12th day after injection. [results] Rat MSCs cultured in vitro grew well and had irregular morphology. Flow cytometry showed that most of the cells were in G1 phase (79.4%). Immunohistochemical staining showed that CD44 was positive, but CD34 was negative. Rat Mb grew well in vitro and its morphology was regular. Flow cytometry showed that most of the cells were in G1 phase (79.8%), and Desmine,Myosin was positive by immunohistochemistry. After induction, the morphology of MSCs, was numerous.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2;R687.2

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