分析CUMS大鼠脑5-HT介导信号转导及基因多态性变异挖掘“女子以肝为先天”理论基础

A large clinic and experimental evidences have suggested that the female patient with depression has been higher more than the male by 2-3 times,which means that prevelence and morbidity of depression have been obviously differences among the sex.Based on the theory "Woman with liver as a congenital",the present research would aim to duplicate human depression using CUMS,and determine the changes of hippocanpus intracellular signal conduction pathway, AC-cAMP-PKA-CREB pathway and related gene polymorphism.Using the RTFQ-PCR,RFLP-PCR and so on ,it would has been determined that gene expression level of ACmRNA/PKAmRNA/CREBmRNA ,and the variations of single nucleotide polymorphism(SNP) in uptargets 5-HT1A & 2A Receptor gene,and downtargets BDNF & Trk Receptor gene.Therefore,the software ETDT would been used to analyze the transmission disequelibrium test(TDT) in SNPs of them.It would been found that the vulnerable gene of the female and pathophysiological mechanism and the variations in gene polumorphism of gender differences in prelevence and morbidity of the depression, meanwhile compleined the cellular substances of the theory "Woman with liver as a congenital" and discovered the gender defferences of clinical usage by the antidepressant Xiaoyaosan.

抑郁症发病率和药物疗效存在明显性别差异，女性发病高于男性2-3倍，其机制研究国内外报道尚少。基于“女子以肝为先天”理论，研究大鼠脑5-HT介导“AC-cAMP-PKA-CREB”信号转导差异与上下游蛋白酶活性变化、基因多态性变异与性别发病关系。采用RTFQ-PCR和RFLP-PCR等技术，检测AC、PKA、CREB蛋白表达以及ACmRNA、PKAmRNA、CREBmRNA基因表达变化，再进行上游靶点5-HT1A受体、5-HT2A受体与下游靶蛋白BDNF与受体TrkB基因上单核苷酸多态性基因分型分析，最后借助ETDT系统对每个位点进行传递/不平衡分析检验。揭示5-HT介导“AC-cAMP-PKA-CREB”信号转导差异与性别差异的关系，发现抑郁症雌性易感基因位点与发病性别差异机制，阐明“女子以肝为先天”理论的物质载体，为补充抑郁症发病理论和性别差异化治疗提供新理论新思路。

目的：基于“女子以肝为先天” 的理论，观察CUMS雌雄大鼠海马和皮层5-HT介导AC-cAMP-PKA-CREB信号转导通路相关蛋白、基因表达差异以及上游5-HT1AR、5-HT2AR及下游基因BDNFmRNA和TrkBmRNA表达和基因多态性变化，探讨抑郁症发病的性别差异机制，挖掘“女子以肝为先天”理论的物质基础。方法：（1）复制CUMS模型，运用IHC方法检测雌雄SD大鼠海马与皮层5-HT介导蛋白AC、PKA、CREB表达情况；（2）运用ELISA方法检测海马、皮层和血清中cAMP含量变化。（3）用Real-time qPCR法检测雌雄大鼠海马和皮层中ACmRNA、PKAmRNA、CREBmRNA表达变化，（4）运用Real-time PCR技术，检测雌雄大鼠皮层5-HT1ARmRNA、5-HT2ARmRNA表达变化；(5）运用基因测序技术检测大鼠皮层5-HT2AR、BDNF、TrkB基因的单核苷酸多态性。结果：在海马和皮层中，ACmAC/PKAmPKA/CREBmCREB/5-HT1ARmRNA/5-HT2ARmRNA均出现性别差异性表达；在TrkB基因多态性位点rs107475362A/G上，在各组中存在3种基因型：AA型、AG型、GG型，分布频率分别为64.3%、28.6%、7.1%。等位基因A、G出现的频率分别为78%、21.4%。氟西汀组内雌雄大鼠等位基因A出现的频率分别为68.75%和87.55%，雄性高于雌性；G出现的频率分别为31.25%和12.5%，雌性高于雄性，差异有统计学意义(P<0.05)。结论：本研究表明，5-HT介导AC-cAMP-PKA-CREB信号转导通路存在性别差异性调节，说明在抑郁症发病机制、中西药作用过程中存在不同的靶点，是抑郁症性别差异的机制之一，也是“女子以肝为先天”理论的相关物质基础。5-HT介导AC-cAMP-PKA-CREB信号转导通路可以作为进一步深入研究抑郁症发病理论和“女子以肝为先天”的物质基础和思路方法。