以RNA-Seq技术为主探究三法三穴对CCI模型鼠镇痛的启动机制

前期研究主要结论为：三法三穴干预后，①用RNA-Seq检测出DRG中有226个差异基因，有显著差异的代谢途径有Wnt、IL-17、MAPK等信号通路；②可以上调抑制性神经递质、下调炎性因子；初步回答了推拿治疗神经病理性疼痛的部分机制。推拿如何启动镇痛机制成为新的科学问题。为此本课题选取坐骨神经慢性压迫性损伤（CCI）模型鼠，采用推拿手法模拟仪定性定量模拟三法（拨法、点法、揉法）作用于三穴（殷门、阳陵泉、承山），以DRG、脊髓背角为主要观察点；通过行为学研究及形态学观察寻找启动的表型证据；通过RNA-Seq技术检测干预不同时间点的差异基因，结合GO功能和KEGG通路分析筛选与镇痛相关的差异基因及其通路；通过RT-PCR、Western Blot、免疫荧光进一步检测相关基因的表达及在通路中的作用，明确启动基因及其在表达时间、表达部位上的特点。综合分析，揭示“推拿如何启动镇痛机制”这一科学问题。

The main conclusions of previous studies are as follows: after the intervention of the three methods and three acupoints technique, ①226 differentially expressed genes were detected in DRG by RNA-Seq, which mainly enriched on Wnt, IL-17 and MAPK signaling pathways; ②it could up regulate inhibitory neurotransmitters and down regulate inflammatory factors; it preliminarily answered the partial mechanism of Tuina in the treatment of neuropathic pain. How Tuina initiates the analgesic mechanism has become a new scientific issue. This research will select chronic constriction injury of sciatic nerve (CCI) rats as the model, and utilize a Tuina-technique simulator to simulate three methods ( plucking, point-pressing, and kneading method) on three acupoints (Yinmen, Yanglingquan, Chengshan), and choose DRG and dorsal horn as the main observation sites. Then, behavioral research and morphological observation will be used to looking for the phenotypic evidences; RNA-Seq will be used to detect the differentially expressed genes at different times and screen the analgesic genes combined with GO function and KEGG pathway; RT-PCR, Western Blot and immunofluorescence will be used to further detect the expression of related genes and their roles in the pathway, so as to clarify the initiation genes and their expression times and sites. Finally, answer the question of “how does Tuina initiate the analgesic mechanism”.