基于基因芯片技术探讨HHIP对慢性不可预见性应激小鼠海马神经元的干预作用

Based on Gene Chip Technology Exploration of the Intervention Effect of HHIP on Hippocampal Neurons in Mice with Chronic Unpredictability Stress

目的观察刺猬相互作用蛋白(HHIP)基因对抑郁模型小鼠海马组织神经元的干预作用。方法采用慢性不可预见性温和刺激(CUMS)方法复制抑郁症模型。根据体质量通过分层法随机将小鼠分为模型组(30只)和对照组(20只)。慢性温和性应激刺激35 d后,糖水实验、走格实验、悬尾实验等评价其抑郁程度,然后取海马组织。应用基因芯片技术查找差异表达基因,通过聚合酶链式反应(PCR)和蛋白免疫印迹法(Western Blot)确认HHIP蛋白的表达下调。制备HHIP基因慢病毒,沉默HHIP基因进一步体外实验探索HHIP蛋白对海马组织神经细胞的干预作用。结果抑郁症模型小鼠呈现抑郁伴焦虑状态,基因芯片结果显示,焦虑、抑郁情绪与海马组织神经元,胶质细胞生育、分化及细胞外基因及其连通性有关。海马组织差异表达的mRNA有447个,其中mRNA下调281个,上调166个。下调基因包括Hedgehog信号通路中的3个基因HHIP、SHH、Claudin11。PCR及Western Blot显示HHIP mRNA及蛋白表达均降低。Western Blot法显示HHIP慢病毒感染后PC12细胞促凋亡因子Bcl-2表达下调,Bax表达上调,细胞凋亡百分数增加。结论HHIP参与了小鼠抑郁焦虑的发生,其发生机制可能与Hedgehog信号通路诱导了海马神经细胞凋亡有关。

Objective To observe the intervention effect of Hedgehog-interacting Proteain(HHIP)gene on hippocampal neurons of depression and anxiety model mice.Methods The depression model was established by chronic unpredictable mild stimulation(CUMS).According to body weight,the mice were randomly divided into model group(30 mice)and normal group(20 mice)by stratification method.After 35 days of chronic mild stress stimulation,the degree of depression was evaluated by sugar water experiment,walking test and tail hanging experiment,and then the hippocampus tissue was taken.The differentially expressed genes were identified by microarray.The down-regulated expression of HHIP protein was confirmed by polymerase chain reaction(PCR)and Western Blot.HHIP gene lentivirus was prepared,and HHIP gene was silenced to further explore the intervention effect of HHIP protein on hippocampal nerve cells in vitro.Results The depression model mice showed depression and anxiety.The results of gene chip technology showed that it was related to hippocampal neurons,glial cell fertility,differentiation,extracellular genes,and their connectivity.There were 447 mRNA differentially expressed in hippocampus,among which 281 mRNA were down-regulated and 166 mRNA were up-regulated.The down-regulated genes included three genes in the Hedgehog signaling pathway:HHIP,SHH,and Claudin11.PCR and Western Blot showed that HHIP mRNA and protein expression were decreased.After HHIP lentivirus packaging,Western Blot showed that the expression of Bcl-2 was down regulated,Bax expression was up-regulated and the percentage of apoptosis was increased in PC12 cells infected with HHIP lentivirus.Conclusion HHIP is involved in the occurrence of depression and anxiety in mice,and its mechanism may be related to the apoptosis of hippocampal nerve cells induced by Hedgehog signaling pathway.