人眼轮匝肌来源骨骼肌干细胞治疗肌萎缩症的机制研究

Duchenne muscular dystrophy (DMD) is an X-linked recessive lethal inherited disease, and its incidence rate ranks the first in monogene genetic disease of nervous system. There is still no effective treatment of DMD presently, which is mainly caused by gene mutation of dystrophin, a kind of cytoskeleton protein on cell membrane in Xp21. Muscle cells are susceptible to contraction-induced damage due to lack of dystrophin protein. Progressive injury and repetitive regeneration of skeletal muscles exhaust the pool of satellite cells, which can repair muscle defect, and lead to eventually irreversible myonecrosis. Skeletal muscle-derived stem cell (SMSCs) transplantation is considered as a promising therapy of DMD. Human orbicularis oucli muscle (OOM) is spared from DMD due to the low expression of dystrophin. We have successfully isolated a kind of cell population(OOM-SMSCs) using a re-plating method, which possess stem cell-like characteristics. These cells demonstrate a high efficieny of myogenic differentiation and can participate in the in vivo muscle regeneration process. Therefore, we hypothesize that OOM-SMSCs transplantation is a new strategy for the treatment of DMD. In the present study, we aim to investigate the mechanism to improve muscle function by local intramuscular injection of myogenic differentiated human OOM-SMSCs into DMD nude mice model (mdx), which is constructed by CRISPR/Cas9 technology, and compare the results with those of the conventional cellular therapy via blood circulation pathway. The molecular mechanism of OOM-SMSCs in the treatment of mdx mice will be analyzed by means of cell tracing, single myofiber culture, muscle secondary injury and stem-cell secondary transplantation, cell immunology as well as other techniques. The feasibility of allogeneic cell transplantation will also be discussed to explore a new method for the clinical intervention of DMD.

杜氏肌萎缩症(DMD)是一种遗传性致死性肌肉疾病，因基因突变导致dystrophin蛋白缺乏，使肌细胞易受损伤，最终发生不可逆的肌坏死。肌肉干细胞移植是一种有前景的治疗方法。人眼轮匝肌因低表达dystrophin蛋白可免受DMD累及，申请人应用差速贴壁法从眼轮匝肌中分离出一类具有干细胞特性的细胞群体(OOM-SMSCs)。该细胞体外成肌诱导效率高，体内可再生修复肌肉损伤，因此猜测OOM-SMSCs移植是一种新的DMD治疗途径。本项目拟应用CRISPR/Cas9技术构建DMD模型(mdx裸小鼠)，局部肌肉注射成肌分化的人OOM-SMSCs，研究细胞移植对受损肌肉功能的改善，并与经血液循环途径的细胞治疗进行比较。通过干细胞示踪、单肌纤维培养、肌肉再次损伤、细胞二次移植、细胞免疫学等实验，分析OOM-SMSCs治疗mdx鼠的分子机制，探讨异基因细胞移植的可行性，为DMD的临床干预提供新思路。

杜氏肌萎缩症（DMD）是一种遗传性致死性肌肉疾病，肌肉干细胞移植是一种有前景的治疗方法。人眼轮匝肌因低表达dystrophin蛋白可免受DMD累及，因此猜测人眼轮匝肌来源的骨骼肌干细胞（OOM-SMSCs）移植是一种新的DMD治疗途径。我们应用I型胶原酶消化法和差速贴壁法从人眼轮匝肌组织中分离出一类具有干细胞特性的细胞群体。实验结果表明，人OOM-SMSCs能够贴壁生长，呈成纤维细胞样形态，表达骨骼肌来源干细胞相关表面抗原，并能体外多向诱导分化为脂肪细胞、成骨细胞和软骨细胞。OOM-SMSCs体外成肌肉分化时可融合形成肌管样结构，表达成肌分化特异性标记物Pax3、Pax7、Myf5和MyoD。细胞与基质胶（Matrigel）混合后植入裸鼠受损伤肌肉组织内，可以参与骨骼肌再生修复过程。因此认为，人眼轮匝肌组织中含有较强成肌分化潜能的骨骼肌干细胞，有望在肌肉组织再生和DMD疾病治疗方面获得进一步的应用。. 自体脂肪因来源广泛、获取方便等优势，已成为软组织塑形、缺损畸形修复的常用填充材料，但由于移植后脂细胞存活率差异大，导致术后远期效果难以准确预测。我们从移植后脂肪功能改变的角度探讨了自体脂移植高吸收率的原因。先后发现脂肪移植后周脂素蛋白2（Perilipin2）与周脂素蛋白1（Perilipin1）的协同作用，二者可用于观察脂肪细胞移植后的动态变化，Perilipin2是比Perilipin1更精确检测脂肪移植物中脂肪细胞再生的早期标志物。而微量脂肪注射是体内再生脂肪细胞的一种简单方法，并且在临床治疗面部皱纹的病人中获得了满意疗效。我们首次报道了东亚人的上眼睑存在皮下脂肪组织，这可能是导致亚洲人种上眼睑臃肿的重要因素之一。眼睑皮下脂肪细胞体积明显小于腹部的皮下脂肪细胞，但与眼眶隔内脂肪细胞的大小相似。而眼睑部位移植的腹部脂肪细胞在体内存活后，细胞体积仍然大于周围的眼眶脂肪细胞，再次证实了脂肪细胞移植后的再生理论。. 在间充质干细胞的条件培养基研究方面，我们先后制备了兔角膜化学烧伤和小鼠过敏性鼻炎模型，同样取得了较满意的实验结果，为干细胞提取物的产品研发与应用转化奠定了基础。

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