肌成纤维细胞及其抗凋亡能力在颌骨放射性骨坏死发病机制中作用的实验研究

Osteoradionecrosis of jaws (ORNJ) is the most devastating long-term complication of radiotherapy in the head and neck area with slow progress and does not tend to heal spontaneously， which is very harmful for the patients quality of life. However, the precise pathogenesis of ORNJ has not been fully clarified yet. Many theories about how ORNJ occurs have been formulated and each has led sequential treatment protocol in past 90 years, but all of them have been proved to be obviously defective. The etiopathogenesis of ORNJ are still not consensual, which also makes obstacles to the prevention and management of ORNJ. And relative research has been the hotspot for the clinicians and pathologists. In recent years, a new theory for the pathogenesis of ORNJ has arisen and indicated ORNJ to be the radiation induced fibroatrophic disease, and some anti-fibrosis medicine, Pentoxifylline and Tocopherol, for example, have been applied to treat ORNJ and proved dramatical outcome. Despite its clinical effectiveness, this theory needs deeper study and experimental support. Therefore, we focus on the myofibroblast(MFB), the key effector cell in all fibrosis diseases, and assumed that ORNJ, like fibrosis in all other organs, is the result of a process of fibrogenesis. We are going to establish an animal model for ORNJ, then test the existence of MFB and observe its origination, differentiation,regulation and destination in by cell cultivation,immunohistochemical analysis，Tunel testand Flow cytometer. We will investigate the effect of TGF-β1 on the differentiation and regulation of MFB and the role of Fas/FasL pathway in the process of MFB acquiring the capacity of resistance to apoptosis and allowing their continuous matrix synthesis. we are also going to apply specific neutralizing antibody for TGF-β1 and FasL to intervene the differentiation and survival of MFB, and anti-fibrosis peptide cytokine(hepatocyte growth factor, HGF) will also be used to observe whether the ORNJ in experimental animals could be ameliorated and even reversed by them. In summary, our new hypothesis and experiments will partially unveil the pathophysiology mechanism of ORNJ, and will pave the way for a new and more effective therapy in the future.

放射性颌骨坏死（ORNJ）是头颈部恶性肿瘤患者放疗后常见且难治的并发症，严重影响生存质量。ORNJ的发病机制尚不明瞭，至今仍缺乏针对ORNJ非常有效的预防和治疗措施，此领域也一直是国内外研究热点。最新的放射性纤维萎缩假说认为ORNJ本质是一种放射性纤维化，而临床应用抗纤维化药物治疗也取得了明显的疗效，但缺乏实验依据和深入研究。我们以纤维化疾病中的主要效应细胞―肌成纤维细胞(myofibroblast，MFB)为切入点，通过建立ORNJ动物模型，培养MFB细胞并观察其细胞来源、分化和转归；研究TGF-β1对MFB分化的调控作用及Fas/FasL途径在MFB获得抗凋亡能力中的作用；最后应用TGF-β1和FasL的中和抗体及肝细胞生长因子进行体外体内干预实验，观察抑制MFB转化和促其凋亡是否可以改善和逆转动物的ORNJ，以期部分阐明ORNJ的发病机制，为探索预防和治疗的新方法提供理论依据。

放射性颌骨骨坏死（osteoradionecrosis of jaws, ORNJ）是破坏性最大的头颈部放疗晚期并发症，临床危害性大，但由于发病机制不清，缺乏有效的预防和治疗方法。近些年来提出的放射性纤维萎缩学说认为ORNJ是一种放射诱导的纤维化疾病，但此学说缺乏实验依据，ORNJ的具体纤维化过程并未完全阐明。肌成纤维细胞 (myofibroblast, MFB）是纤维化类疾病的主要效应细胞，也是纤维化治疗的重要靶点，但尚无实验研究MFB是否也在ORNJ的发生中起到了重要作用。本研究通过研究ORNJ中MFB的分布特点、分化调控、细胞来源及凋亡情况，并研究对ORNJ预防及治疗的措施，以期进一步阐明ORNJ的发病机制，为其预防和治疗提供新的思路和策略。研究内容及结论如下：.1.ORNJ动物模型的建立及MFB在其中的分布、凋亡特点.（1）成功建立了与临床实际接近、重复性好的ORNJ动物模型，可作为ORNJ纤维化机制研究平台。.（2）MFB在ORNJ组织中大量聚集，与MFB纤维化形成相关的上下游分子也呈高表达。MFB的表达具有时间和剂量相关性，与临床组织破坏的严重性呈正相关性。这些特点与其他纤维化疾病相同，说明MFB与ORNJ的发生和持续具有重要的相关性。.（3）ORNJ组织中MFB凋亡较正常组织中明显降低。.2.MFB的体外培养及特点研究.（1）首次探索出从ORNJ组织中培养MFB的方法，搭建了体外研究平台，对后续机制研究和治疗方法的评判有重要意义。.（2）培养的MFB具有分泌功能，TGF-β1可促进其分化增殖和发挥成纤维作用。进一步证实在ORNJ的发生中MFB起到了关键作用。.（3）体外培养的MFB凋亡降低，Fas/FasL表达增高，说明MFB存在凋亡抑制，通过Fas/FasL途径实现。.3.MFB细胞来源探究.（1）FBs和BMSCs可在TGF-β1诱导下向MFB分化并具有分泌功能，诱导后细胞凋亡下降，Fas/FasL表达增高。表明ORNJ局部存在MFB的细胞来源。.（2）体外使用富氢培养液培养MFB及诱导细胞，纤维化相关分子表达降低，提示富氢液是治疗ORNJ潜在的有效方法。.4.富氢液治疗作用的动物体内实验.使用富氢液干预放射前、后动物，对ONRJ具有一定的预防和治疗作用。进一步证实了ORNJ的本质和MFB的效应细胞作用，为ORNJ的预防和治疗提供新思路。.