14-3-3及其调控RhoA/ROCK信号通路在涡虫中枢神经再生中的功能研究

The regeneration of central nervous system (CNS) is regulated by the multiple signaling pathways. The inhibition of Rho/ROCK pathway can promote axon regeneration and functional recovery in the injured CNS. 14-3-3 proteins can interact with RhoA-GEF as a integrator in the signaling pathway. However, whether 14-3-3 affects the regeneration of CNS through regulating RhoA/ROCK signaling pathway and its regulating mechanism is unclear. Our previous studies revealed that 14-3-3 ε, RhoA and ROCK expressed in CNS, and involved in the process of the regeneration of planarian CNS. To address the expression pattern and function of 14-3-3 in the CNS regeneration, Firstly，we will clone the sequence of 14-3-3 genes in the planarian which occupies an important position in the evolution. Next, the location, expression patterns and function of 14-3-3 during planarian regeneration will be confirmed by immunofluorescence, in situ hybridization and RNAi. Thirdly, the interaction proteins with 14-3-3 are screened using co-immunoprecipitation, and analyze its relationship with the regeneration of planarian CNS. Finally, in order to confirm the function and mechanism of 14-3-3 regulated RhoA/ROCK pathway during the process of regeneration, we will further examine the effects on the planarian CNS regeneration and RhoA/ROCK pathway by interference of 14-3-3 and its interaction proteins. In conclusion, the function and molecular mechanisms of 14-3-3 regulated RhoA/ROCK pathway on planarian CNS regeneration will be illuminated by the aid of the knowledge of neurosciences and developmental biology. The project will provide the evidence of further researching the CNS regeneration of animal, and even provide new clue for the research on the mechanism of regeneration in vertebrates.

中枢神经再生受多条信号通路调控，抑制Rho/ROCK信号通路能促进损伤中枢神经的轴突再生和功能恢复。14-3-3是调控信号通路的整合子，能与RhoA-GEF相互作用。 然而，14-3-3能否通过调控RhoA/ROCK信号通路影响中枢神经再生及其调节机制的问题，目前尚不清楚。我们前期研究发现14-3-3ε、RhoA和ROCK主要表达在涡虫中枢神经，并参与中枢神经再生。本课题拟从克隆在进化上占据重要地位的涡虫14-3-3基因入手，利用原位杂交、免疫组化和RNAi技术，研究14-3-3在涡虫中枢神经再生中的分布、表达及功能；通过免疫共沉淀筛选出14-3-3相互作用蛋白并分析其与涡虫中枢神经再生的关系；体内干预14-3-3及其相互作用蛋白表达，检测其对涡虫再生神经和RhoA/ROCK信号通路的影响，明确其功能和调控机制。该研究将为动物中枢神经再生提供理论依据，进而为脊椎动物再生机制研究提供新思路。

中枢神经系统再生受多条信号通路调控，抑制Rho/ROCK信号通路能促进损伤中枢神经的轴突再 生和功能恢复。14-3-3是调控信号通路的整合子，能与RhoA-GEF相互作用。 本课题研究了涡虫14-3-3 在中枢神经再生中的功能以及14-3-3 调控RhoA/ROCK 信号通路在中枢神经再生中的机制，取得了以下数据和结果：1.涡虫14-3-3及相关基因的克隆及在再生中的表达变化和图式：发现Dj14-3-3α、ζ在咽部表达，Dj14-3-3ε在干细胞和脑部表达，DjGef、DjSlk等基因在中枢神经表达。2. 14-3-3 蛋白在涡虫再生过程中的组织分布及功能分析：干扰Dj14-3-3ε和DjGef后，涡虫脑部出现残缺；PAMPs刺激涡虫后，Dj14-3-3α、ζ表达量上调，与咽部再生无关，参与了涡虫早期免疫，且两者可能存在着互补关系。3. 涡虫再生过程中14-3-3 与相关基因之间的调控关系：当干扰Dj14-3-3ε后，Jnk表达量下调， Jnk表达于脑神经，并与脑神经再生有关。DjPsa表达于中枢神经系统，并与脑神经再生有关。DjSlk在成体和再生涡虫中枢神经表达，DjSlk干扰后，涡虫出现了皱缩、膨胀、解体等表型，可能其调控了细胞凋亡和增殖之间的平衡，从而影响涡虫再生。4. 涡虫14-3-3 相互作用蛋白的筛选与鉴定：发现Dj14-3-3ε与JNK能够相互作用，JNK调节Dj14-3-3ε，影响了脑神经再生。5. 涡虫Gef调控的Rac1及其下游基因受到14-3-3ε控制而影响脑神经再生。DjGef是14-3-3的下游基因，也是小G蛋白酶的主要调节因子。小G蛋白DjRac1表达于再生的中枢神经。其调节基因Cdc42表达于多能干细胞，而Rac1表达于脑神经，Rac1与FARP表达一致也参与了脑神经再生，且两者存在相互作用；DjRhoA表达于肠，在再生1、3天的脑原基中也有表达，干扰后对光不敏感，出现脑部残缺，严重影响了肠再生，即DjRhoA参与涡虫脑神经、视神经和肠的形成，且对成体干细胞的形成或迁移有影响。本研究从分子水平上探索两侧对称动物中枢神经再生的规律和机理，为动物中枢神经系统再生提供理论依据。