【作者】 田文敏；

【导师】 沈岩； 许琪；

【作者基本信息】 北京协和医学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 重性抑郁障碍(major depressive disorder, MDD)是一种病因尚未阐明的多基因复杂性状疾病,因其具有高患病率(16.2%)和高自杀率(3.4%)的特点,给家庭和社会带来了沉重的负担。一直以来,研究者们普遍认为5-羟色胺和多巴胺等单胺类神经递质的异常是导致MDD发生的主要原因,但难以解释通过增加单胺类递质水平发挥抗抑郁效果的一些经典抗抑郁药起效时间延迟,以及部分患者经抗抑郁药治疗后无法获得理想疗效的现象。近年来,大量基础及临床研究发现MDD的发生可能在结构及分子水平上存在神经可塑性的改变,进而提出了MDD的神经营养假说。神经营养因子在海马神经发生和突触可塑性方面具有重要作用。因此,神经营养因子功能障碍可能参与了部分MDD的发生。在本课题组的前期工作中,在小样本中系统的筛查了以NMDA受体为基础的LTP通路上相关基因与MDD患者认知功能障碍的关系,发现表皮生长因子(epidermal growth factor, EGF)与MDD患者的认知功能损害相关。基于MDD的神经营养假说以及本课题组的前期工作,我们系统研究了EGF与MDD的关系。首先,在463例MDD患者和413例对照中,筛查了EGF基因的功能SNPs,采用病例-对照研究方法分析了EGF基因与MDD的相关性。其次,在120例患者中,进一步验证EGF与MDD患者认知功能障碍的关系。随后,采用ELISA的方法检测了210例患者和223例对照血浆中EGF的含量,并比较了二者的差异。最后,在体外细胞水平研究了功能SNP对EGF表达量的影响。最终发现,EGF rs11569017T/T基因型与MDD相关(X2=11.07,p=0.0039,corrected p=0.03),随后我们以rs11569017为条件SNP,分析了该位点与其它SNPs的顺式相互作用,发现rs11569017-rs11569126的相互作用与MDD显著相关(χ2=13.08, p=0.0003, corrected p=0.0027),其中T-A构成的单倍型是MDD的危险因素(χ2=6.17, OR=1.73, p=0.01, corrected p=0.04)。EGF基因多态性位点rs2250724、rs11568943和rs11569126的基因型与WAIS-RC的言语分和操作分强相关,进一步验证了EGF基因与MDD患者的认知功能障碍相关。在蛋白质水平,我们发现MDD患者血浆中EGF含量(66.75±4.86pg/ml)显著低于对照组(114.50±6.28pg/ml,p<0.0001)。并且在患者中rs11569017T/T基因型携带者血浆中EGF含量显著低于A/A和A/T基因型携带者(global p=0.001)。体外细胞学研究发现,EGFrs11569017T突变型质粒转染的HEK293T细胞上清EGF含量显著低于EGFWT质粒转染的细胞(p=0.001)。在本工作中,我们从基因、蛋白质和细胞三个层面上系统地研究了EGF与MDD的关系。综合三个层面的研究结果发现,EGF中rs11569017的变异可能通过影响EGF的表达量,从而增加MDD的易感性。并且这种改变可以在患者血浆中反映出来。在未来,血浆中EGF水平可能作为一个生物标志物用于重性精神疾病的辅助诊断。更多还原

【Abstract】 Major depressive disorder (MDD) is a common psychiatric disease, and its precise mechanism is poorly understood. Epidemiological studies have shown that the lifetime prevalence of the disease is～16.2%and the suicide rate in patients with MDD is～3.4%. Therefore, MDD has contributed to heavy worldwide social burdens.It has long been noted that abnormalities of monoamine transmitters, such as serotonin (5-HT) and dopamine (DA), are very likely to be involved in the development of MDD. However, these hypotheses failed to explain why the therapeutic responses to antidepressant drugs delay, even partially to be remitted. In recent years, a number of basic and clinical studies have found that the occurrence of MDD may result from the changes in neuronal plasticity at structural and molecular levels. Based on these findings, the neurotrophin hypothesis of MDD was proposed. Neurotrophic factors play an important role in neurogenesis and neuronal plasticity, suggesting that the aberrant function of neurotrophic factors may involve in the pathophysiology of MDD.In a previous study, we took the cognitive function as the endophenotype and carried out the study of the predisposing genes related to the NMDAR-LTP pathway. The results showed that EGF (epidermal growth factor) gene was associated with cognitive impairments in MDD patients.In combination of the neurotrophic mechanism of MDD and our preliminary work, we carried out the study on the relationship between EGF and MDD systematically.At the beginning, in case-control study, eight single nucleotide polymorphisms (SNPs) in the functional regions of EGF gene were genotyped in463patients with MDD and413control subjects, of which210patients and223controls underwent determination of plasma EGF levels using the enzyme-linked immunosorbent assay (ELISA). Further more, in order to verify the relationship between EGF and cognitive impairments in MDD patients, we used the association study in120MDD patients with fully clinical data. Finally, to test the effects of functional SNPs on EGF expression in vitro, EGF levels in the supernatants of cultured HEK293T cells were measured. In case-control study, none of the SNPs in EGF gene showed allelic association with MDD, but the TT genotype of rs11569017showed significant genotypic association with MDD (x2=11.07, p=0.0039, corrected p=0.03). Following this, we found that the cis-phase interaction between rs11569017and rs11569126was strongly associated with the illness (x2=13.08,p=0.0003, corrected p=0.0027), and the T-A haplotype was an risk factor to MDD (x2=6.17, OR=1.73, p=0.01, corrected p=0.04). The genotype of rs2250724, rs11568943and rs11569126were associated with the Verbal Scale Score and Performance Scale Score of WAIS-RC significantly, confirmed the relationship between EGF and cognitive impairments in MDD patients. The EGF levels in plasma were significantly lower in the patients group than the control group (p<0.0001). The EGF levels were also significantly lower in patients with the rs11569017-TT genotype than those with either the AA genotype or the AT genotype (global p=0.001). The rs11569017T allele affected the expression of EGF gene significantly (p=0.0004).In our works, we analyzed the relationship between EGF and MDD at genetic, proteinic and cellular levels. In combination of these studies, we found that rs11569017in EGF gene may influence the expression of EGF, then contribute to the etiology of MDD. Besides, the quantity changes could be reflected in the plasma of MDD patients. So the plasma EGF level may be a useful bio-marker for auxiliary diagnosis of major psychiatric disorders in future.更多还原