牙髓干细胞增强上气道扩张肌抗疲劳性的生物学研究

Obstructive Sleep Apnea Hypopnea Syndrnome (OSAHS) is a common and life-threatening disorder characterized by repetitive collapse of the upper airway during sleep. The function of the upper airway dilator muscles are closely related with the stability of upper airway. Genioglossus is considered as "safe muscle of the upper airway". In our former research, the fracture of genioglossus fibers was observed under chronic intermittent hypoxia. Hypoxia inhibited myogenic differentiation of myoblasts and reduced the fatigue resistance of genioglossus. Dental pulp stem cells(DPSCs) are characterized by proliferation abilities and multi-lineage differentiation. Several studies have demonstrated that DPSCs have the potential of myogenic differentiation. In the present study, different clones of DPSCs will be isolated from different patients. The proliferation rates and differentiate potential (including osteogenic, adipogenic and chondrogenic differentiation) of these clones will be tested to identify the best clone as the candidate for the further study. The genioglossus myoblasts will be co-cultured with DPSCs in myogenic medium under hypoxia in vitro. The mRNA levels of myogenic differetiation related genes will be determined by qRT-PCR and the protein content will be detected by Western blot analysis. The expression of HIF-1α, Btg2 and Nur77 will be measured to find out their role in the myogenic differentiation of DPSCs. Based on our previous study, HIF-1a knockdown transgene mice model with chronic intermittent hypoxia will be raised and established. An appropriate amount of DPSCs will be injected into the local area of genioglossus via stereotactic injection method. The objective of in vivo study is to investigate the effects of DPSCs on recovering the function of genioglossus under chronic intermittent hypoxia condition. Both of the efficiency of myogenic regeneration and the recovery of the primary genioglossus will be investigated via morphological and histochemical analysis. The isometric contractile properties of the genioglossus will be investigated through EMG biological signal collecting and processing system. The mRNA and protein levels of myogenic differetiation related genes will be measured by qRT-PCR and Western blot analysis, respectively. The role and underlying biological mechanism of HIF-1α pathway in the protection of DPSCs for hypoxia-induced injury of genioglossus will be investigated.

阻塞性睡眠呼吸暂停低通气综合征是以睡眠时上气道反复阻塞为特征且具有致死危险的常见病。睡眠时上气道扩张肌功能与上气道稳定性密切相关，其中颏舌肌被称为“上气道安全肌”。研究表明，慢性间歇性低氧状态下颏舌肌肌纤维存在明显损伤，低氧抑制颏舌肌细胞成肌分化，降低颏舌肌抗疲劳性。牙髓干细胞（DPSCs）是一类具有高度增殖和多向分化潜能的间充质干细胞，可分化为成肌细胞。本课题拟筛选具有高度增殖和分化潜能的DPSCs克隆，通过分子生物学方法检测DPSCs在体外低氧条件下成肌分化能力，研究低氧诱导因子（HIF-1α）对DPSCs成肌分化的调控机制。而后，构建骨骼肌条件性敲除HIF-1α小鼠慢性间歇性低氧模型，通过立体定位注射法在颏舌肌局部注射DPSCs，通过分子生物学和组织化学方法评估DPSCs原位成肌分化效果及原有肌纤维损伤修复，评价肌功能状态，探索DPSCs增强颏舌肌抗疲劳性的分子生物学机制。

阻塞性睡眠呼吸暂停低通气综合征（OSAHS）是指睡眠过程中因上气道反复塌陷引起的呼吸暂停、通气不足，严重者常可引起心脑血管并发症，甚至猝死。上气道扩张肌的功能对于上气道开放和气道的大小起到了决定性作用，颏舌肌是最重要的上气道肌肉之一，也是研究OSAHS发病机制与治疗途径的关键。慢性间歇性低氧（CIH）抑制颏舌肌细胞成肌分化，降低颏舌肌抗疲劳性。牙髓干细胞（DPSCs）是一类具有高度增殖能力和多向分化潜能的间充质干细胞，已成为口腔组织再生工程中极具应用价值的种子细胞。本课题利用DPSCs成肌分化的特性，将DPSCs用于治疗CIH小鼠，期望通过形成新的肌组织，修复受损的原有肌纤维，恢复颏舌肌肌功能，增强其抗疲劳性。.课题组按照原实验方案，完成如下研究：首先研究了人牙髓干细胞（hDPSCs）改善低氧状态下成肌细胞分化抑制的体外研究，发现在氯化钴模拟的低氧条件下，C2C12成肌分化受到抑制，与DPSCs共培养条件能够能改善成肌细胞低氧损伤；其次应用人牙髓干细胞条件培养基（hDPSCs-CM），能显著减轻氯化钴诱导的颏舌肌成肌细胞低氧损伤，其机制可能与AMPK/PGC-1α通路的调节有关；再次，通过牙髓干细胞的优化成肌诱导分化形成类肌卫星干细胞，体内研究表明将该成肌诱导的牙髓干细胞（类肌卫星干细胞）局部注射在小鼠颏舌肌，能改善颏舌肌CIH损伤。.此外，课题组研究了低氧条件诱导成肌细胞焦亡的机制研究，发现CIH能激活GSDMD，诱导成肌细胞焦亡，可能与NF-κB/HIF-1α通路有关；同时我们比较了不同年龄段小鼠颏舌肌及其肌源性干细胞的差异，发现低氧条件下，年老型小鼠颏舌肌成肌细胞增殖与分化能力降低更明显，对低氧的耐受力更差。.本项目通过一系列生物学研究手段，研究了低氧状态下颏舌肌及其肌源性干细胞的病理生理学改变；通过将牙髓干细胞优化成肌诱导分化形成类肌卫星干细胞，并将其局部注射入CIH小鼠颏舌肌，增强CIH小鼠颏舌肌抗疲劳性，本项目为临床上有效利用DPSCs治疗OSAHS提供研究依据和理论基础。