【作者】 刘永萍；

【导师】 王保捷；

【作者基本信息】 中国医科大学 ， 法医学， 2021， 博士

【摘要】 目的:精神分裂症是一种以情绪损害、认知缺陷和社交功能障碍为特征的慢性、致残性精神疾病。精神分裂在全世界影响已经超过2000万人,给家庭和社会带来了沉重的负担。此外,精神分裂症也是法医精神病领域鉴定的重要内容,但目前其鉴定缺乏客观的指标。因此精神分裂症病因及发病机制的研究受到越来越多的关注。精神分裂症受到遗传因素和环境因素的共同影响,其病因复杂,研究显示遗传因素在精神分裂症的易感性以及病程发展过程中扮演十分重要的角色。精神分裂症的病因及发病机制涉及多种神经递质系统的假说,涉及的神经递质包括谷氨酸、多巴胺、五羟色胺及γ-氨基丁酸。其中,谷氨酸是人类中枢神经系统重要的兴奋性神经递质,通过与受体结合发挥生物学作用。谷氨酸受体分为4大类18种亚型,其中NR1受体由于在大脑中被普遍转录而受到了最为广泛的关注。NR1受体由GRIN1基因编码,被报道与抑郁症、多动症、阿尔茨海默症、帕金森等多种神经精神疾病相关。然而,目前对于GRIN1基因与精神分裂症的关联性研究结果并没有统一,中国北方汉族人群的数据较为缺乏,并且对人类GRIN1基因的表达具有调控作用的基因区域研究也相对不足。因此,本研究将对中国北方汉族群体GRIN1基因部分SNP的分布进行调查,探讨其与精神分裂症是否具有关联以及对法医物证学的应用价值。同时,将验证在GRIN1基因5’和3’端对基因表达有的影响的调控序列,为进一步的功能研究打下基础。研究方法:以316名中国北方汉族无关健康志愿者和309名中国北方汉族精神分裂症患者作为调查对象。对扩增的GRIN1基因5’端-2334bp～+86bp的片段进行Sanger DNA测序分析,应用Haploview 4.2软件对SNP的分布进行Hardy-Weinberg平衡检验,应用Powerstats v1.2软件对法医学参数进行计算。通过病例-对照研究探讨SNP的分布及其组成的单倍型与精神分裂症是否存在关联。应用基因克隆与基因重组技术,以5’端不同单倍型及5’和3’端不同长度DNA序列截断体为重组子,构建表达载体。转染不同细胞系后,利用双荧光素酶报告基因技术检测其相对荧光强度,以研究对GRIN1基因表达具有调控作用的功能序列。结果:本研究共检测到rs112421622、rs138961287、rs117783907、rs181682830、rs7032504、rs144123109和rs11146020等7个SNP位点。全部SNP位点的基因型分布符合Hardy-Weinberg平衡。Rs138961287、rs117783907和rs11146020等3个SNP的等位基因频率分布相对较好,DP值在0.5左右。连锁分析结果显示rs138961287和rs117783907处于连锁不平衡状态,且rs11146020分别于rs138961287和rs117783907具有连锁不平衡关系。病例-对照研究结果显示rs138961287的Ins T等位基因、rs117783907的T等位基因和rs11146020的C等位基因在精神分裂症组(15.2%、15.2%和14.7%)和对照组(20.9%、21.7%和20.1%)之间的频率分布均存在显著性差异。7个SNP所形成的6种单倍型中,单倍型H1和H2在病例组(77.8%和12.6%)与对照组(70.9%和16.8%)之间的频率分布均存在显著性差异(p分别为0.005和0.046)。研究显示,重组单倍型载体H1和H2在HEK-293、SK-N-SH和U87细胞中的相对荧光强度不具有统计学差异(p分别为0.178、0.956和0.501)。截断体重组载体F10和F11在3种细胞系间的相对荧光强度均具有显著性差异(p分别为0.036、4.091E-06和1.606E-06)。此外,在SK-N-SH和U87细胞中,重组表达载体F10和F12的相对荧光强度分别相较于F9和F11呈显著上升趋势(p分别为0.003和3.086E-06)。在GRIN1基因3’UTR区,完整的重组表达载体F1在HEK-293、SK-N-SH和U87细胞系中的相对荧光强度与pmir GLO-Basic载体相比均显着降低(p分别为0.002、6.321E-26和3.390E-14),重组表达载体F6的相对荧光强度相较于F5显着升高(p分别为0.004、3.294E-06和3.294E-06)。此外,在SK-N-SH细胞中,F4和F7的相对荧光强度分别相较于F3和F6显著性下降(p分别为4.711E-05和8.413E-05)。生物信息学分析提示has-mi R-212-5p、has-mi R-324-3和has-mi R-326可能识别F6-F7之间的功能序列,而has-mir-491-5p可能识别F3-F4之间的功能序列。F1分别与has-mi R-212-5p mimic、has-mi R-324-3 mimic、has-mi R-326 mimic、has-mir-491-5p mimic和对照mi RNA NC mimic共转染,F1再分别与has-mi R-212-5p inhibitor、has-mi R-324-3 inhibitor、has-mi R-326 inhibitor、has-mir-491-5p inhibitor和对照mi RNA NC inhibitor共转染的结果表明,在HEK-293、SK-N-SH细胞和U87中,F1+has-mi R-491-5p mimic的相对荧光强度较F1+mi RNA mimic NC显著下降(p分别为0.021、0.000267和0.029)。而F1+has-mi R-491-5p inhibitor的相对荧光强度与F1+mi RNA inhibitor NC的相比,在三个细胞系中相对荧光素酶表达的统计学差异均消失。结论:1.GRIN1基因rs117783907、rs138961287和rs11146020在中国北方汉族群体中的多态性分布较好,具有一定的法医学应用价值。2.在中国北方汉族群体中,GRIN1基因rs117783907的T、rs138961287的Ins T以及rs11146020的C等位基因在中国北方汉族群体中可能是精神分裂症发生的保护性因素。3.在中国北方汉族群体中,GRIN1基因5’端单倍型H1与H2可能与精神分裂症的易感性相关。4.GRIN1基因5’端单倍型H1与H2对基因的表达没有影响。5.在神经细胞中,GRIN1基因5’端-337bp～-159bp和-704bp～-556bp的片段中可能包含转录抑制序列,而-556bp～-337bp则可能包含转录增强序列,可为基因调控的机制研究提供参考。7.mi R-491-5p可以下调GRIN1基因在HEK-293、SK-N-SH和U87细胞系中的表达。更多还原

【Abstract】 Objective: Schizophrenia is a chronic and disabling mental illness characterized by emotional impairment,cognitive deficits and social dysfunction.Schizophrenia has affected more than 20 million people all over the world,and has brought a heavy burden to families and society.In addition,schizophrenia is also an important part of the identification of forensic psychiatry,but its identification currently lacks objective indicators.Therefore,the research on the etiology and pathogenesis of schizophrenia has received more and more attention.Schizophrenia is affected by both genetic and environmental factors,and its etiology is complex.Studies have shown that genetic factors play a very important role in the susceptibility and course of schizophrenia.The etiology and pathogenesis of schizophrenia involve the hypothesis that multiple neurotransmitter systems are involved,including glutamate,dopamine,serotonin and gamma-aminobutyric acid.Among them,glutamate is an important excitatory neurotransmitter in the human central nervous system,which plays a biological role by binding to receptors.Glutamate receptors are divided into 4 categories and 18 subtypes.Among them,NR1 receptor has received the most extensive attention.The NR1 receptor is encoded by the GRIN1 gene and has been reported to be associated with a variety of neuropsychiatric disorders,including depression,ADHD,Alzheimer’s disease and Parkinson’s disease.However,the current research results on the association between the GRIN1 gene and schizophrenia are not unified.There is a lack of data on the Han population in northern China,and the research on the ways in which GRIN1 gene expression is regulated is relatively insufficient.Therefore,this study will investigate the distribution of part of the SNP of the GRIN1 gene in the Han population in northern China,and explore whether it is associated with schizophrenia and its application value to forensic evidence.At the same time,the regulatory sequences that affect gene expression at the 5’and 3’ends of the GRIN1 gene will be verified to lay the foundation for further functional research..Methods: 316 unrelated healthy volunteers and 309 schizophrenic patients of the Han nationality in northern China were selected as the survey subjects.Sanger DNA sequencing analysis was performed on the amplified GRIN1 gene 5’end-2334 bp ~+86bp fragment.The Haploview 4.2 software was used to perform Hardy-Weinberg equilibrium test on the distribution of SNPs,and the Powerstats v1.2 software was used to calculate forensic parameters.A case-control study was conducted to explore whether the distribution of SNPs and its haplotypes are related to schizophrenia.Gene cloning and gene recombination techniques are used to construct expression vectors with different haplotypes at the 5’end and truncated DNA sequences with different lengths at the 5’and 3’ends as recombinants.After transfection of different cell lines,the relative fluorescence intensity of the dual luciferase reporter gene technology was used to study the functional sequences that have a regulatory effect on the expression of GRIN1 gene.Results: In this study,7 SNP loci were detected,including rs112421622,rs138961287,rs117783907,rs181682830,rs7032504,rs144123109 and rs11146020.The genotype distribution of all SNP loci accorded with Hardy-Weinberg equilibrium.The polymorphisms of the three SNPs rs138961287,rs117783907 and rs11146020 were relatively well distributed,and the DP value was all about 0.5.The results of linkage analysis showed that rs138961287 and rs117783907 were in a state of linkage disequilibrium.The results of the case-control study showed that the frequency distribution of the Ins T of rs138961287,the T allele of rs117783907 and the C allele of rs11146020 were significantly different between the schizophrenia group(15.2%,15.2%,and 14.7%)and the control group(70.9% and 16.8%).Among the 6 haplotypes formed by 7 SNPs,there are significant differences in the frequency distribution of haplotypes H1 and H2 between the case group and the control group(p values were 0.005 and 0.046,respectively).Studies have shown that the relative fluorescence intensities of recombinant haplotype vectors H1 and H2 in HEK-293,SK-N-SH and U87 cells are not statistically different(p values were 0.178,0.956,and 0.501,respectively).The relative fluorescence intensities of the truncated recombinant vectors F10 and F11 in the three cell lines are all significantly different(p values were 0.036,4.091E-06,and 1.606E-06,respectively).In addition,in SK-N-SH and U87 cells,the relative fluorescence intensities of recombinant expression vectors F10 and F12 were significantly higher than those of F9 and F11(p values were 0.003 and 3.086E-06,respectively).In the 3’UTR region of the GRIN1 gene,the relative fluorescence intensity of the complete recombinant expression vector F1 in the HEK-293,SK-N-SH and U87 cell lines was significantly lower than that of the pmir GLO-Basic vector(p values were 0.002,6.321E-26,and 3.390E-14,respectively),the relative fluorescence intensity of recombinant expression vector F6 was significantly higher than that of F5(p values were 0.004,3.294E-06,and 3.294E-06,respectively).In addition,in SK-N-SH cells,the relative fluorescence intensities of F4 and F7 were significantly lower than those of F3 and F6(p values were 4.711E-05 and 8.413E-05,respectively).Bioinformatics analysis suggests that has-mi R-212-5p,has-mi R-324-3 and has-mi R-326 may recognize the functional sequence between F6 and F7,and has-mir-491-5p may recognize functional sequence between between F3 and F4.F1 was co-transfected with has-mi R-212-5p mimic,has-mi R-324-3 mimic,has-mi R-326 mimic,has-mir-491-5p mimic,and the control mi RNA NC mimic,then F1 was co-transfected with has-mi R-212-5p inhibitor,has-mi R-324-3 inhibitor,has-mi R-326 inhibitor,has-mir-491-5p inhibitor,and control mi RNA NC inhibitor.The results showed that in HEK-293,SK-N-SH,and U87 cells,the relative fluorescence intensity of F1+has-mi R-491-5p mimic was significantly lower than that of F1+mi RNA mimic NC(p values were 0.021,0.000267,and 0.029,respectively).Compared with the relative fluorescence intensity between F1+mi RNA inhibitor NC and F1+has-mi R-491-5p inhibitor,the statistical difference of relative luciferase expression in the three cell lines were all disappeared.Conclusion: 1.Rs117783907,rs138961287 and rs11146020 of GRIN1 gene are well distributed in the Han population in northern China,and have forensic application value.2.In the Han population in northern China,the T allele of rs117783907,the Ins T of rs138961287 and the C genotype of rs11146020 are protective factors for the occurrence of schizophrenia in the Han population in northern China.3.In the Han population in northern China,the haplotype H1 and H2 of the 5’ end of GRIN1 gene might be related to the susceptibility to schizophrenia.4.The two haplotypes composed of 7 SNPs at the 5’end of GRIN1 gene have no significant effect on gene expression.5.The different length sequences of the 5’regulatory region of the GRIN1 gene have different effects on gene expression in the three cell lines of HEK-293,SK-N-SH and U87,indicating that the regulatory mechanism of gene expression is different depending on the cell type,which might provide reference and help for further functional research.6.In nerve cells,-337 bp ~-159 bp and-704 bp ~-556 bp at the 5’end of GRIN1 gene contain transcription repression regions,while-556 bp ~-337 bp contain enhanced transcription enhancement regions,which can provide a reference for the study of gene regulation mechanisms.7.mi R-491-5p can down-regulate the expression of GRIN1 gene in HEK-293,SK-N-SH and U87 cell lines.更多还原