基于Rho/ROCK分子应答信号通路的美洲大蠊促组织修复“生肌”作用的分子机制研究

The group found that Periplaneta americana can lead fibroblast migration by Preliminary studies. Combining with the research methods of “regeneration medicine” and “molecular cytobiology”, we found a breakthrough and put forward the hypothesis: “The extracts of periplaneta americana regulate MLC molecular motor by Rho/ROCK, then lead repair cells move to wounds rapidly to promote tissue regeneration”..Focusing on Rho/ROCK regulating MLC molecular motor and starting from organic coupling between molecular signaling system inside the cell and mechanical system, We will establish fibroblasts model,siRNA Rho cell model,siRNA ROCK cell model, pretreating with C3 exoenzyme cell model，pretreating with Y27632 cell model; wound healing mice model ,pretreating with C3 exoenzyme mice model,pretreating whith Y27632 mice model and inspect the effect of Periplaneta americana on wound healing, cell migration, protein gene expression, MLC molecular motor activity and so on. The research will pay more attention to the relativity of the whole action-the cell behavior- mechanics- signaling system. Finally, we will analyse whether Rho/ROCK activate MLC molecular motor to induce cells migration and reveal molecular mechanism of the promoting tissue regeneration function of Periplaneta americana..After studying on relevance of “the overall effect- cell morphology- mechanics of machinery- signal path”, we not only may find molecular targets of promoting tissue regeneration function of Periplaneta americana, but also being good for enriching modern scientific connotation of traditional effect theory and providing a new method of researching animals of TCM..

前期研究发现动物类中药美洲大蠊具有诱导成纤维细胞迁移的特征，结合再生医学和分子细胞生物学的研究方法，以“诱导细胞迁移”为突破口，提出假说：美洲大蠊可能通过Rho/ROCK信号通路调控分子马达MLC活性，诱导修复细胞向创面迁移，达到生肌之目的。.以Rho/ROCK通路调控分子马达MLC活性为核心，从分子信号传导与机械力学的有机耦合性出发，分别建立正常、siRNA介导基因沉默、C3、Y27632抑制Rho/ROCK通路的体外培养成纤维细胞模型和体内皮肤溃疡小鼠模型，考察美洲大蠊对创面愈合、细胞迁移、Rho/ROCK通路相关蛋白基因表达、马达活性等的影响，重点关注整体效应-细胞行为-机械力学-信号通路的相关性，分析Rho/ROCK信号通路是否介导美洲大蠊诱导下的细胞迁移，揭示美洲大蠊“生肌”的分子机制。.本课题有利于探寻美洲大蠊生肌作用分子靶标，丰富生肌功效科学内涵，为动物类中药研究提供新思路。

摘要：本项目在前期研究中发现美洲大蠊具有诱导修复细胞迁移的特征，结合分子生物学的研究方法，以“诱导细胞迁移”为突破口，寻找美洲大蠊作用于成纤维细胞和角质形成细胞的分子信号应答通路，以及对下游靶基因转录和翻译的影响。课题组采用RhoA激酶活性检测试剂盒、Western Blot检测了核心蛋白MLC磷酸化激活蛋白的表达，发现无论在HSF人成纤维细胞系或在HaCaT人永生化上皮细胞中，RhoA激酶活性和总RhoA表达未见升高。p-MLC未检测到表达。说明美洲大蠊并未激活Rho/ROCK通路。通过对多条通路主要蛋白活性的筛选，发现美洲大蠊能上调HaCaT细胞TGF-β1/Smad3和JAK/STAT3通路中关键分子Smad3、JAK1、JAK2、STAT3的磷酸化水平，并从细胞、组织、动物层面分别验证了其对以上两条通路的激活作用，采用抑制剂做对比，证实美洲大蠊诱导下的HaCaT细胞的增殖和迁移与以上通路相关。同时，美洲大蠊能上调HSF细胞NF-κB和MAPK/Erk通路中P-IκBα、Rel A和P-Erk的表达，并从细胞、组织、动物分别验证了美洲大蠊对以上两条通路的激活作用，采用抑制剂做对比，证实美洲大蠊诱导下的HSF细胞的增殖和迁移与以上通路相关。课题组分别采用HSF和HaCaT为细胞模型，探索美洲大蠊诱导细胞增殖和迁移的分子机制。并从细胞、组织、动物层面分别证实了上述结论，为美洲大蠊生肌的分子机制研究奠定基础。