【作者】 张凯；

【导师】 赵敏；

【作者基本信息】 上海交通大学 ， 精神病与精神卫生学， 2016， 博士

【摘要】 背景谷氨酸是中枢神经系统最重要的兴奋性神经递质,其AMPA受体介导兴奋性信号快速传递。g-氨基丁酸(GABA),是脑内最重要的抑制性神经递质,其中GABRA1在整个GABA受体中起到最重要的中枢抑制作用。有很多研究表明谷氨酸、GABA受体在阿片类、酒精等成瘾物质长期滥用导致的脑功能损伤中起到重要的作用,最终导致使用者产生成瘾物质使用障碍,但苯丙胺类中枢兴奋剂使用对谷氨酸、GABA受体表达的影响及可能的机制尚未见报道。目的探讨苯丙胺类兴奋剂使用对谷氨酸受体基因GRIA2和GABA受体基因GABRA1表达的影响,并寻找异常表达的可能调控机制,为苯丙胺类兴奋剂使用障碍预后判断和治疗靶点奠定理论基础。材料和方法1.通过ELISA法对苯丙胺类兴奋剂使用障碍者外周血谷氨酸受体GRIA2基因及GABA受体GABRA1基因编码蛋白进行检测,明确苯丙胺类兴奋剂长期慢性使用导致相关受体功能障碍。2.构建甲基苯丙胺暴露神经细胞模型,使用逆转录和Western blot等方法,检测甲基苯丙胺暴露后对神经细胞谷氨酸受体基因GRIA2和GABA受体基因GABRA1表达的影响。3.通过双荧光素酶报告基因实验及Western blot实验进行miR-181a与GRIA2、GABRA1基因靶向调控关系验证。4.Sequenom MassARRY质谱基因分析法检测GABRA1基因12个单核苷酸突变位点,寻找GABRA1基因表达异常的原因。结果1.苯丙胺类兴奋剂使用障碍组GRIA2基因编码蛋白表达(32.58±10.46)高于健康对照组(23.56±9.68)(p<0.01)。苯丙胺类兴奋剂使用障碍组GABRA1基因编码蛋白表达低于健康对照组(p<0.05)。2.细胞模型结果显示,甲基苯丙胺升高GRIA2 mRNA和蛋白表达,甲基苯丙胺同样升高GABRA1 mRNA和蛋白表达。3.荧光素酶报告基因实验和Western blot实验结果表明,miR-181a负向调控GRIA2,GABRA1与miR-181a无负向调控关系。4.正常对照组与苯丙胺类兴奋剂使用障碍组在GABRA1基因12个SNP位点等位基因与基因型频率比较结果显示,两组中各位点等位基因与基因型频率比较未发现统计学差异(p>0.05)。结论1.苯丙胺类兴奋剂使用损害谷氨酸受体GRIA2和GABA受体GABRA1表达。2.苯丙胺类兴奋剂使用影响miR-181a表达,进而负向调控谷氨酸受体GRIA2。3.单核苷酸突变并非GABRA1表达改变的原因。更多还原

【Abstract】 Background Glutamate is the most important excitatory neurotransmitter of the central nervous system,and neurotransmitter in the brain,plays the most important central inhibitory effect throughout the GABA receptor.There are a lot of research suggests that glutamate and GABA receptor play an important role in addictive substance use disorders.However,there are lack the reporte on the GRIA2 and GABRA1 expression of amphetamines type stimulant use disorder patients.Objective The study explore the expression of GRIA2 and GABRA1 of amphetamines type stimulant patients.Then look for the possible regulatory mechanism of abnormal expression.Provide theoretical basis for amphetamines type stimulant use disorder patients.Method 1.One hundred twenty-four ATS use disorder patients and Fifty-seven gender and age matched healthy controls were recruited.The GRIA2 and GABRA1 serum levels were assessed using the ELISA kits.2.Chronic methamphetamine treatments were performed to SH-SY5 Y cells,and then total RNA or protein of cells will be isolated to detect the expression of GRIA2 and GABRA1 after methamphetamine exposure.3.The psiCheck-2-GRIA2 and psiCheck-2-GRIK2 vector was used in the Dual Luciferase reporter gene assays in HEK 293 T cells and western blot in SH-SY5 Y cells,to examine whether miR-181 a negative regulate glutamate receptor genes GRIA2 and GABRA1.4.Twelve previously studied common SNPs of GABRA1 were selected to be genotyped using Sequenom MassARRY genotype assays.To investigate the associations between common genetic polymorphisms of GABRA1 and ATS use disorder,and detectd the associations between polymorphisms and aberrant expression of GABRA1.Result 1.The GRIA2 gene encoding protein expression of ATS patients(32.58±10.46)higher than that of healthy controls(23.56±9.68).GABRA1 gene encoding protein expression was downregulated among ATS use disorder patients.2.Reverse transcriptase PCR analysis showed that 2mM methamphetamine exposure increased GRIA2 and GABRA1 mRNA expression,respectively.GRIA2 protein expression is also increased in SH-SY5 Y cells following treatment with amphetamine.3.Based on the Dual Luciferase reporter gene assays result,GRIA2 is a direct target of miR-181 a.The relative luciferase activity of the reporter containing GRIA2 3’-UTR was significantly suppressed when miR-181 a was cotransfected.Western blot was performed to examine the effect of overexpression or knockdown of miR-181 a on the protein levels of GRIA2 in SH-SY5 Y cells.Based on the Dual Luciferase reporter gene assays result,GABRA1 maybe not direct targets of miR-181 a.4.No association between the SNPs of GABRA1 and ATS use disorders risk.Conclution 1.The GRIA2 gene encoding protein expression of ATS use disorder patients higher than that of healthy controls.2.2mM and 4mM MA exposure for 48 h increased GRIA2 mRNA expression.3.GRIA2 is a direct target of miR-181 a.miR-181 a may act as a suppressor through downregulating the expression of GRIA2.GABRA1 maybe not direct targets of miR-181 a.4.No association between the SNPs of GABRA1 and ATS use disorders risk.更多还原