【作者】 谭婕；

【导师】 吴建华；

【作者基本信息】 湖北中医药大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 2型糖尿病(Type 2 Diabetes Mellitus, T2DM)是一种多因素致病的疾病,它是由复杂的遗传因素和环境因素共同作用的结果,目前对它的病因及发病机制尚无明确的阐述。其中胰岛素信号转导通路一直是人们研究的重点,而组成胰岛素信号转导通路的相关分子更成为研究热点,在过去20多年的广泛研究中许多分子的功能作用逐步得到证实。而糖原合成激酶-3(Glycogen Synthase Kinase 3, GSK-3)作为一种抑制糖原合成的限速酶为人们所熟知,参与多种信号的调控,其中包括胰岛素细胞信号传导,以糖原合成激酶-3β(Glycogen Synthase Kinase 3β, GSK-3β)亚型的作用为主,因此GSK-3β与2型糖尿病发生的相关性研究受到越来越多的重视。近年国内外均有相关研究,提出GSK-3β的高表达与胰岛素敏感性降低呈相关性,探讨研究GSK-3β在患者体内功能活性的异常增高等问题,同时开发GSK-3β抑制剂也已经成为新药开发热点。比如,近年来有研究已经发现GSK-3β在2型糖尿病患者的骨骼肌及肝脏中蛋白表达量较健康人有明显的增高,一些GSK-3的抑制剂在2型糖尿病以及阿尔茨海默病患者的离体细胞中及动物模型中也能起到一定的治疗作用。基于以上资料,我们了解到当2型糖尿病发生时,GSK-3β在2型糖尿病患者体内的蛋白表达量发生了异常改变,GSK-3β与2型糖尿病的发生存在关联性。但这种关联性尚不足以阐明GSK-3β本身对于2型糖尿病患者的主要作用机制,我们希望通过追溯到其基因表达的层面上来了解GSK-3β在人体中的表达状态。那么现在我们想知道GSK-3β作为一种丝／苏氨酸蛋白激酶,当激活各个相关途径的时候,在患者体内基因表达是否发生改变,最终可否影响与人体胰岛素正常表达相关的各种信号转导途径。为了验证GSK-3β是否是主动参与2型糖尿病致病的重要因素,我们选取糖原合成激酶-3β为研究对象,采用SYBR实时荧光PCR技术研究2型糖尿病患者外周血有核细胞中的GSK-3β基因表达,探讨其与2型糖尿病患者致病机制的相关性。目的运用实时相对定量PCR技术检测GSK-3β基因在2型糖尿病患者和健康人外周血有核细胞中表达量,探讨GSK-3β在2型糖尿病患者体内基因表达的临床意义。方法于早晨抽取2型糖尿病患者和健康人空腹静脉血3ml,肝素抗凝,行常规生化检测空腹血糖,总胆固醇,甘油三酯。设计GSK-3β基因引物,运用实时荧光RT-PCR相对定量法检测40例糖尿病患者和40例健康人PBMC内基因表达水平量,应用Delta-Delta Ct法处理原始数据,比较并分析GSK-3β基因在人体表达的临床意义及应用价值。结果1.2型糖尿病患者组空腹血糖、血浆总胆固醇、甘油三酯均高于健康对照组(P<0.05),年龄无差别(P>0.05)。2.2型糖尿病患者组外周血有核细胞中GSK-3β的mRNA相对表达量为0.94±0.34,健康对照组为0.92±0.19,比较两者无明显差异(P>0.05)。结论在2型糖尿病患者外周血有核细胞中GSK-3β的基因相对表达量与健康人对照组相比无明显差异。在2型糖尿病的发生时GSK-3β在人体的基因表达未发生明显的异常改变,而抑制GSK-3β在人体中的基因表达可以通过阻断部分信号传导而起到降低血糖的作用,但外周血有核细胞中的GSK-3β并不能成为治疗2型糖尿病的靶位基因。更多还原

【Abstract】 Type 2 diabetes is a disease with multifactorial pathogenesis. This result is leaded by many complex genetic factors and environmental factors together, so there is no clearly articulated about its etiology and pathogenesis nowadays. The insulin cell signal transduction is constantly the focus of the study, and the molecules related with the Insulin signal transduction has also become the research hotspot. Through extensive researches the function of these molecules have gradually been confirmed in the past 20 years. Glycogen synthase kinase-3 is well-known as a rate-limiting enzyme to inhibit glycogen synthesis, and participate in the regulation of a variety of signals, including insulin-secreting cells signal transduction, GSK-3βisoforms chiefly. Hence GSK-3βand Type 2 diabetes the correlation study was payed more and more attention. In recent years, high expression of GSK-3βand decreased insulin sensitivity was related to study the function of GSK-3βactivity at home and abroad. The development of GSK-3βinhibitors have become the hotpot of new drug research. For example, the protein expression of GSK-3βin skeletal muscle and liver significantly increased in type 2 diabetes more than in healthy subjects. And GSK-3 inhibitors have effective therapeutic effect in animal models of type 2 diabetes and Alzheimer’s disease. However, the mechanism of GSK-3βis not clear. Based on these data, we have understand that the protein expression of GSK-3βin type 2 diabetes patient may affect its function. As a serine/threonine protein kinase,the gene expression of GSK-3βin patients which is whether or not changed leads to its impact in their original normal signal transduction pathways. while activating each relative pathway. In order to verify whether the GSK-3βactively participates in an important pathogenic factor in T2DM, we select the glycogen synthase kinase-3βas the research object, using SYBR real-time fluorescent PCR technology research in the nucleated cells of type 2 diabetes patients, GSK-3βgene expression to explore in patients with type 2 diabetes pathogenesis of relevance.Object:To evaluate the relative quantitative real-time PCR detection of GSK-3βgene in type 2 diabetes patients and healthy expression of peripheral blood nucleated cells, the amount of GSK-3βin type 2 diabetes patients the clinical significance of gene expression in vivo.Methods:Collected in the morning in type 2 diabetic patients and healthy fasting venous blood, EDTA anticoagulant, then tested fasting plasma glucose, total cholesterol and triglyceride. Designed GSK-3βgene primers and detected 40 cases of diabetic patients and 40 healthy human PBMC gene expression with real-time fluorescence relative quantitative RT-PCR. Apply Delta-Delta Ct method to deal with raw data, compare and analyze the clinical significance of expression in the human body and application value of GSK-3βgene.Result:1. The fasting blood glucose, serum total cholesterol and triglycerides of the type 2 diabetic patients group were higher than the healthy control group (P<0.05), but the age is no difference (P>0.05).2. The type 2 diabetic patient group’s GSK-3βin the relative mRNA expression levels is 0.94±0.34, and the healthy group is 0.92±0.19, which compared no significant difference between the two groups (P> 0.05).Conclusion:In peripheral blood monucleated cells of type 2 diabetic patients, GSK-3βgene relative expression has no significant difference compared with the healthy control group. GSK-3βin the pathogenesis of type 2 diabetes does not have a significant impact, while restraining the GSK-3βgene relative expression can block part of the signal transduction and play a role in lowering blood glucose, but GSK-3βcan not be the target gene for the treatment of type 2 diabetes.更多还原