【作者】 聂铭博；

【导师】 洪光祥； 陈振兵；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 硕士

【摘要】 转化生长因子-β(TGF-β)是一种多功能细胞因子。近年发现,TGF-β在致组织和器官纤维化中起着重要作用,其致纤维化作用和调控机制已成为当前研究热点。肌源性干细胞(Muscle-derived Stem Cells, MDSC)是从骨骼肌组织中分离出的一类新型干细胞,表达造血干细胞的表型Sca-1和CD34,能向多种组织分化,自我更新能力强,在细胞治疗和基因工程研究中其表现明显优于肌卫星细胞。本文旨在探讨转化生长因子β1(TGF-β1)对失神经骨骼肌肌源性干细胞内生成结缔组织生长因子(CTGF)的影响,为今后进一步探讨TGFβ1- CTGF通路在致失神经骨骼肌纤维化的作用及机制打下基础。本实验分为两部分1.失神经小鼠肌源性干细胞培养与鉴定:采用双酶(胶原酶XI和胰蛋白酶)消化法消化分离小鼠失神经骨骼肌组织,用差速贴壁法纯化,成功地获得原代肌源性干细胞,进行免疫组化染色证实为Sca-1+/CD34+细胞,然后在高血清条件下进行传代增殖,了解其细胞生物学特性。2.将获得的肌源性干细胞,传代以便获得数量足够的细胞,然后在细胞培养基中加入TGF-β1,观察不同浓度培养相同时间和相同浓度培养不同时间下TGF-β1作用于体外培养小鼠失神经骨骼肌肌源性干细胞后,细胞内CTGF生成的变化。实验结果表明:失神经小鼠MDSC能够在TGF-β1刺激下分泌CTGF,在10μg/L浓度范围内存在明显的剂量依赖关系,在3～12h时间范围内呈时间依赖关系。说明TGF-β1-CTGF通路可能通过对肌源性干细胞,也就是干细胞水平影响、参与了失神经骨骼肌组织纤维化进程。更多还原

【Abstract】 The TGF-βis a factor of multiple functions. It is founded that TGF-βhad act key roles in the course of the fibrosis in the organs and tissue. So, the mechanism of the fibrosis caused by TGF-βis a popular issue in our research.Muscle-derived stem cells are a new stem cell found in skeletal muscles, and express similar surface antigens, as observed in hematopoietic stem cells, Sca-1 and CD34. MDSC can differentiate to variant tissues and posses the strong power in self-renewal. In the study of cell therapy and gene engineering MDSC is better than skeletal muscle satellite cell.The aim is to research the The influences of TGF-β1 on the denervated mouse MDSCs’producting the CTGF in vitro, and make the found for our following research in the mechanism of TGFβ1- CTGF pathway in the denervated muscular fibrosis. The experiment is composed of two parts1. the identification of the cultured MDSC: we digested skeletal muscle tissues from new born mice using the dual-enzyme (collagenase XI and trypsin) digestion method, purified the cells by preplate technique and successfully cultivated muscle-derived stem cells in vitro. Immunohisochemistry staining showed that the muscle-derived stem cells expressed Sca-1 and CD34. Then the muscle-derived stem cells were cultivated and passaged in DMEM including 20% FBS. We observed their biological characteristic.2.We obtain and proliferate the MDSC so that we have enough cells for research. The TGF-β1 was added into the DMEM including 20% FBS. We observed the variation of the CTGF’s production in MDSC treated with DMEM TGF-β1.The results showed that: the MDSC from denervated mice can be helpful to the production of the CTGF in MDSC. The dose-dependent effect exist between the groups from 1ng/ml to 10ng/ml ,the time dependency exist between the groups from 3h to 12h.It is showed that the TGF-β1-CTGF pathway could act as an key role in the denervated muscular fibrosis by the influence to the MDSC.更多还原