针刀干预对PI3K/Akt/mTOR信号通路介导颈椎病颈肌细胞自噬与凋亡研究

The occurrence and development of Cervical Spondylosis(CS) is closely related to the injuries of cervical muscles. The key pathogensis of CS is spasm of cervical muscles after chronic injuries leading to relative position change of cervical vertebras or cervical vertebras and other adjacent tissues, resulting in the syndrome. Aiming at the chronic injuried cervical muscles, acupotomy therapy has authenic effect. Our previous study has shown that the key effective phase of acupotomy therapy to CS is to repair chronically injuried cervical muscles. However, the mechanism of repairing injuried cervical muscles is not clear. Our hypothesis is that acupotomy therapy may regulate autophagy and apoptosis of cervical muscles mediated by the PI3K/Akt/mTOR singaling pathway in the repairment of injuried cervical muscles. The CS rabbits model will be made in the study and treated by acupotomy therapy. Transmission electron microscope will be used to observe the autophagy body and apoptosis body, and ultrastructure of cervical muscles as well. Experimental methods of immunohistochemistry, RT-PCR and Western Blot will be applied to detect mRNA and protein of PI3K/Akt/mTOR signaling pathway and its downstream relevant factors of autophagy and apoptosis. It is expected to explore the scientific basis of acupotomy therapy in the treatment of CS with the theory of Jingjin that is choosing the tender points and injuried muscle points as therapeutic points, and the theory of acupotomy therapy of "treatig bone diseases from muscles ". And it is expected to provide scientific proofs to the acupotomy therapy in the treatment of CS.

颈椎病（CS）的发生发展与颈椎相关肌肉系统病变密切相关，其关键机制是颈椎相关肌肉发生损伤后痉孪导致颈椎间或颈椎与其相邻组织相对位置发生改变后所引起的综合征。针刀治疗CS主要针对劳损颈肌松解，其疗效确切，前期研究表明针刀治疗CS的主要起效环节是修复劳损颈肌，但其如何修复劳损颈肌的机制不明。我们假说针刀治疗CS可能通过调节PI3K/Akt/mTOR信号通路介导颈肌细胞自噬与凋亡而修复劳损颈肌。本项目通过复制CS模型，针刀干预并与电针对照，干预后电镜观察颈肌细胞的自噬体、凋亡小体及颈肌超微结构；免疫组化、RT-PCR、Western Blot等检测PI3K/Akt/mTOR通路及其下游自噬和凋亡相关因子的mRNA和蛋白表达，以揭示针刀治疗颈椎病“以痛为输”、“以筋结为腧”的治则及针刀疗法“骨病调筋”理论的科学内涵，为临床针刀治疗CS提供科学依据。

颈椎病（CS）的发生发展与颈椎相关肌肉系统病变密切相关，其关键机制是颈椎相关肌肉发生损伤后痉孪导致颈椎间或颈椎与其相邻组织相对位置发生改变后所引起的综合征。临床针刀“调筋治骨”颈椎病疗效可靠，研究表明针刀治疗CS的可修复劳损颈肌，但其如何修复劳损颈肌的机制不明。本研究通过假说针刀治疗CS可能通过调节PI3K/Akt/mTOR信号通路介导颈肌细胞自噬与凋亡而修复劳损颈肌，从PI3K/Akt/mTOR信号通路对针刀干预修复劳损颈肌的机制进行了深入研究。.颈肌病理实验证实颈椎病模型兔颈肌病变明显，而针刀干预可明修复劳损颈肌。电镜观察颈肌超微结构显示针刀干预对颈肌有良好的修复作用，减少颈肌细胞的自噬和凋亡。. 进一步研究表明，上述针刀疗效机制可能是通过调节PI3K/Akt/mTOR信号通路及其下游因子，针刀干预可上调PI3K、P-Akt、mTOR蛋白和mRNA表达，下调PTEN、Beclin1蛋白和mRNA表达，下调LC3II和LC3II/LC3I蛋白和mRNA比值，下调Caspase-9、Caspase-3蛋白和mRNA表达，上调Bcl-xL的蛋白和mRNA表达。表明针刀通过调控PI3K/Akt/mTOR信号通路及其下游因子，可上调颈肌抑制自噬和凋亡因子的蛋白和mRNA表达水平，下调促自噬和凋亡因子的蛋白和mRNA表达水平，从而减少颈肌细胞的自噬和凋亡。. 通过上述研究内容的积极开展，本课题观察了颈椎病颈肌PI3K/Akt/mTOR信号通路在颈椎病发病中的作用以及针刀干预对劳损颈肌修复作用，揭示其机制可能通过调控PI3K/Akt/mTOR信号通路及其下游自噬和凋亡相关因子减少劳损颈肌自噬和凋亡，从而促进劳损颈肌细胞再生修复。本研究初步证实了针刀“调筋治骨”理论的科学性，为下一步深入研究针刀“调筋治骨”理论的科学内涵奠定了工作基础，为研究针刀治疗颈椎病提供了科研思路，也为临床上推广针刀治疗颈椎病奠定理论基础和实验依据。