神经及相关功能性神经肽对牙周组织再生过程的影响

Innervation and nerve regeneration play important roles in the field of regenerative medicine. Periodontal tissue regeneration is an unsolved problem until now.There are many factors that affect the quantity and quality of regenerated periodontal tissues and the treatment results of periodontal disease are still questionable both in clinic and animal models. Currently, many measures have been taken to try to gaining more functional periodontal tissue regeneration, especially periodontal tissue engineering technique which is on the basis of seeding cells, biomaterial as a scaffold and a variety of growth factors. Even though,it is considered as a promising method in tissue regeneration,obviously,this progressive idea is too incomprehensive to attribute the countless elements participated in the regenerative process only to three main factors, and fall into neglect of the tissue regeneration is a systemic precise process. .As it is known that nervous system and calcitonin gene related peptide(CGRP) are crucial to bone development, repair and remodeling. But, the roles of nerve and its related neuropeptide in the process of periodontal tissue regeneration are still unknow. We hypothesize that to achieve ideal periodontal tissue regeneration, nerve and sufficient calcitonin gene related peptide in the pathological changed periodontal region are predominately important. So the exact function of nerve and neuropeptides need further investigation. .We found in previous studies that there were very severe periodontal tissues destruction in gross and tissue level after excision of inferior alveolar nerve compared with that in un-excised in rats and recombinant beta-nerve growth factor could accelerate the process of periodontal tissue regeneration in Beagles. .Based on our solid preliminary work, we try to illustrate further the function of nerve and related neuropeptides especially calcitonin gene related peptide in the process of periodontal tissue regeneration in this application. What we will do as follows: (1) Function of CGRP on periodontal ligament cells(PDLCs) in vitro and in vivo; (2)Changes of neuropeptides after transection of the inferior alveolar nerve in gene and protein levels using Realtime PCR and Western blot methods; (3)Effect of exogenous CGRP and CGRP gene modified rPDLCs on periodontal tissue regeneration in tissue and molecular level in denervated rats. The significance of this project is to discover the basic function of nerve and neuropeptides in periodontal tissue regeneration and to find a better way for igniting and promoting the process of periodontal tissue regeneration.

再生组织的神经支配与重建是再生医学领域的研究热点和新动向。病变牙周组织再生困难且再生组织质量存在问题，神经及相关功能性神经肽在此过程中的作用有待阐明。本项目组发现，离断大鼠下牙槽神经后，实验性牙周炎组织破坏严重，而神经生长因子可以促进牙周组织再生。我们提出"神经及相关因子与牙周组织的精确再生密切相关，降钙素基因相关肽是牙周组织再生的重要调节因子之一"。本项目拟在前期工作基础上重点解决：①神经肽（CGRP）对牙周膜细胞增殖与成骨方向分化的影响②离断下牙槽神经后对牙周组织中神经肽特别是对骨再生具有重要作用的CGRP分泌及牙周组织再生过程的影响③外源性CGRP对牙周组织再生潜能的激发作用。从分子、细胞及组织多层面探讨神经与牙周组织再生能力的关系。项目的意义在于揭示神经因素对促进牙周组织再生并提高再生组织的质量方面的作用，为深入研究基于神经因素的牙周组织再生性治疗的新策略及靶点分子提供理论依据。

牙槽骨是全身骨组织中代谢最活跃的骨质，神经血管在牙槽骨改建过程中的作用不容忽视，其主要是通过肽能神经分泌的神经肽对骨组织起作用的。降钙素基因相关肽( calcitonin gene related peptide, CGRP)是骨组织中分布最为广泛的一种神经肽, 主要分布于骨代谢活跃的部位, CGRP直接或间接地通过调节成骨细胞和破骨细胞的分化及活性来影响骨折愈合。.本项目在体外实验中，常规条件下全骨髓法培养Wistar大鼠BMSC，采用 Realtime-PCR 及 Western Blot 方法检测了细胞的CGRP 受体CRLR 和 Ramp1，CCK-8法检测了不同浓度CGRP促进细胞增殖的作用，并检测出适宜浓度CGRP蛋白可促进rBMSC的成骨分化；进而采用CGRP基因慢病毒转染rBMSC/rBMSC的CGRP基因沉默后检测出CGRP有促进细胞增殖及成骨分化的作用，其作用机制可能是通过调节OPG/RANKL的比例来促进成骨。体内实验选用Wistar大鼠作为受试对象，建立离断下牙槽神经和牙周缺损双模型，采用Realtime-PCR、Western Blot、免疫组织化学染色方法检测大鼠牙周组织中CGRP、SP及骨改建相关因子OPG、RANKL的表达变化，失神经1w、2w后CGRP、SP、OPG的表达降低，随后开始上升至正常水平，RANKL的表达与OPG相反，失神经后下颌骨密度降低，骨小梁稀疏，HE、Masson染色观察到大鼠失神经后延缓了牙周组织缺损的愈合；后续实验分别将转染CGRP基因的rBMSC复合胶原膜及CGRP基因沉默的rBMSC复合胶原膜植入牙周缺损区，发现CGRP明显促进失神经后的大鼠牙周组织缺损的愈合。.本课题组研究结果表明CGRP在缺损牙周组织愈合过程中有重要作用，在牙周组织再生研究领域中具有重要的理论意义。与其它组织的再生不同，缺损牙周组织再生的难点在于其持续存在的炎症微环境，拓展实验研究表明炎性微环境影响干细胞神经肽的分泌及成骨分化能力；在牙周炎伴牙周骨缺损的大鼠实验中发现炎性环境可加剧缺损早期的炎症反应，增加破骨细胞的分化，从而影响牙周骨缺损的修复。如何实现抗炎和促再生的有效结合，如何在促进成骨细胞分化的同时抑制破骨细胞分化，是我们进一步需要研究的问题，以期达到更理想的牙周再生效果。