S0X4作为妊娠期糖尿病分子诊断标志物的鉴定及其致病机制的研究

发布时间：2018-06-07 12:46

本文选题：SOX4 + 妊娠期糖尿病　；参考：《重庆医科大学》2017年硕士论文

【摘要】：第一部分:SOX4基因作为妊娠期糖尿病分子诊断标志物有效性的研究背景:全基因组关联分析研究(GWAS)发现CDKAL1基因单核苷酸多态性(SNP)位点rs7756992与糖尿病患病风险呈正相关。随后研究发现SOX4基因与此SNP位点连锁,携带rs7756992 SNP人群表现为SOX4基因表达升高,说明SOX4基因的高表达与糖尿病的患病风险存在正相关关系。同时,SOX4是哺乳动物发育过程中高表达的转录因子,SOX4在胚胎发育期的表达很可能参与了孕妇生理性提高血糖浓度的过程,暗示SOX4与血糖浓度调节相关。因此,我们推测SOX4的表达参与了孕妇正常生理性血糖调节,在妊娠期糖尿病孕妇中,SOX4的过度表达引起了过度调控,导致妊娠期糖尿病(GDM)的发生。目的:鉴定SOX4在GDM孕妇和正常孕妇的血清循环RNA、血清蛋白、血细胞RNA、血细胞蛋白中的浓度存在差异。方法:采集GDM孕妇(n=60)及正常对照孕妇(n=60)静脉血后分离血细胞并制备血清。1)SOX4血清循环RNA浓度:应用Qiagen循环RNA提取试剂盒制备孕妇循环RNA,通过q RT-PCR方法检测SOX4循环RNA浓度。2)SOX4血清蛋白浓度:应用冷冻干燥方法浓缩蛋白,将每组30个样本合并,采用Western-blot方法检测血清SOX4蛋白浓度。3)SOX4血细胞RNA浓度:采用Qiagen试剂盒提取血细胞总RNA,通过q RT-PCR方法检测样本中SOX4血细胞RNA浓度。4)SOX4血细胞蛋白浓度:应用RIPA裂解液裂解细胞并提取总蛋白,将每组15个样本合并,应用Western-blot方法检测血细胞中SOX4蛋白浓度。结果:在妊娠期糖尿病孕妇中,血清循环RNA、血清蛋白、血细胞RNA、血细胞蛋白中SOX4浓度较正常组均有升高,应用SPSS18.0软件分析,差异具有统计学意义(t0.001)。结论:在妊娠期糖尿病中,孕妇血清和血细胞中的SOX4 RNA和蛋白质均有升高。但SOX4蛋白含量较低,需合并多个病人样本以完成Western-blot实验,在更高灵敏度的Elisa试剂盒研发成功前不可作为标志物检测。SOX4循环RNA具有作为妊娠期糖尿病诊断的意义,但本文受样本量的限制,未能准确计算SOX4循环RNA作为标志物的灵敏度和准确性。本研究仍需大规模样本量研究以评价其作为妊娠期糖尿病标志物的有效性。第二部分:SOX4高表达诱导胰岛素抵抗机理的研究背景:II型糖尿病发病率高,占全世界糖尿病人的90%,其发病机制复杂,遗传和环境因素共同作用可能导致II型糖尿病的发生。妊娠期糖尿病与II型糖尿病表现类似,均表现为血糖增高和胰岛素抵抗,因此,妊娠期糖尿病与II型糖尿病应存在相似的分子机制。近期全基因组关联分析研究(GWAS)和基础研究发现SOX4基因的高表达与rs7756992单核苷酸突变亚型II型糖尿病紧密相关,同时文献研究表明SOX4也是胚胎发育期高表达的转录调控因子,因此,我们推论孕期体内环境改变可能促进SOX4的表达,参与孕期血糖生理调控。当SOX4表达量被异常刺激时,引起了妊娠期糖尿病。GDM孕妇体内SOX4异常高表达已经在第一部分研究,但SOX4诱导胰岛素抵抗的分子机制仍不清楚,此分子机制的研究有助于理解妊娠期糖尿病的发病机制,为妊娠期糖尿病的精准医疗提供理论基础。目的:研究SOX4诱导胰岛素抵抗的分子机制。方法:以小鼠脂肪细胞系3T3-L1为模型,慢病毒介导SOX4及TNF-α基因过表达和沉默;通过Western-blot研究目的蛋白的表达;通过q RT-PCR方法研究目的基因m RNA的表达;通过报告基因载体研究靶基因启动子的活性;Elisa方法用于检测TNF-α的浓度和糖吸收能力。综合研究SOX4通过TNF-α和PI3K/AKT信号通路调控Glut4的膜定位,进而调控糖吸收的分子机制。结果:SOX4与TNF-α形成正反馈调节回路,进而抑制PI3K/AKT信号通路,并抑制了Glut4的膜定位,进而抑制了细胞的糖吸收能力。结论:SOX4通过TNF-α和PI3K/AKT信号通路抑制Glut4的膜定位和细胞的糖吸收能力。
[Abstract]:The first part: the research background of the SOX4 gene as a molecular diagnostic marker for gestational diabetes: the whole genome association analysis (GWAS) found that the CDKAL1 gene single nucleotide polymorphism (SNP) locus rs7756992 is positively correlated with the risk of diabetes. Subsequently, the study found that the SOX4 gene is linked to the SNP locus and carries the rs7756992 SNP person. The expression of group expression is that the expression of SOX4 gene is elevated, indicating that the high expression of SOX4 gene is positively related to the risk of diabetes. At the same time, SOX4 is a highly expressed transcription factor in mammalian development, and the expression of SOX4 in the embryonic development period may be involved in the process of pregnant women's physiological increase in blood glucose concentration, suggesting that SOX4 and blood glucose concentration are regulated. Therefore, we speculate that the expression of SOX4 participates in normal physiological glucose regulation in pregnant women. Over expression of SOX4 causes excessive regulation in pregnant women with gestational diabetes, leading to the occurrence of gestational diabetes (GDM). Objective: to identify the serum circulating RNA, serum protein, blood cell RNA, and blood cell eggs of SOX4 in GDM pregnant women and normal pregnant women. Methods: GDM pregnant women (n=60) and normal control pregnant women (n=60) and normal control pregnant women (n=60) were used to separate blood cells and prepare serum.1) SOX4 serum circulating RNA concentration: Qiagen circulating RNA extraction kit was used to prepare pregnant women circulating RNA, and Q RT-PCR method was used to detect the concentration of serum protein. The concentration of protein was concentrated in 30 samples of each group. The concentration of serum SOX4 protein concentration.3 was detected by Western-blot method and RNA concentration in SOX4 blood cells: the total RNA of blood cells was extracted by Qiagen kit and the RNA concentration of SOX4 blood cells in the samples was detected by Q RT-PCR method. Protein, 15 samples of each group were combined, and the concentration of SOX4 protein in blood cells was detected by Western-blot method. Results: in pregnant women with gestational diabetes, serum circulating RNA, serum protein, blood cell RNA, and SOX4 concentration in blood cell protein were higher than those of the normal group. The difference was statistically significant (t0.001) with SPSS18.0 software analysis (t0.001). Conclusion: the difference is statistically significant (t0.001). In gestational diabetes, the SOX4 RNA and protein in the serum and blood cells of pregnant women are elevated. But the SOX4 protein content is low, and multiple patient samples need to be combined to complete the Western-blot experiment. The.SOX4 cycle RNA can not be used as a marker before the more sensitive Elisa kit is developed, which has the meaning of the diagnosis of gestational diabetes. However, the sensitivity and accuracy of SOX4 cycle RNA as a marker is not accurately calculated by the limitation of sample size. This study still needs a large sample size study to evaluate its effectiveness as a marker for gestational diabetes. The second part: the background of the mechanism of insulin resistance induced by high expression of SOX4: the high incidence of II diabetes, 90% of the world's diabetic patients have complicated pathogenesis. The combination of genetic and environmental factors may lead to the occurrence of type II diabetes. Gestational diabetes is similar to type II diabetes and is characterized by high blood sugar and insulin resistance. Therefore, there should be a similar molecular mechanism in gestational diabetes and type II diabetes. The group association analysis (GWAS) and basic research found that the high expression of SOX4 gene is closely related to the rs7756992 single nucleotide mutation type II diabetes. Meanwhile, the literature study shows that SOX4 is also a transcriptional regulator of high expression in the embryonic development period. Therefore, we deduce that the changes in the body environment during pregnancy may promote the expression of SOX4 and participate in pregnancy. Physiological regulation of blood glucose. When the expression of SOX4 is abnormal, the abnormal high expression of SOX4 in pregnant women with gestational diabetes.GDM has been studied in the first part, but the molecular mechanism of SOX4 induced insulin resistance is still unclear. This molecular mechanism is helpful to understand the pathogenesis of gestational diabetes and for gestational diabetes. Objective: to provide a theoretical basis for precision medical treatment. Objective: To study the molecular mechanism of SOX4 induced insulin resistance. Methods: the mouse adipocyte line 3T3-L1 was used as a model, the lentivirus mediated SOX4 and TNF- alpha gene overexpression and silence; the expression of the target protein was studied by Western-blot; the expression of m RNA in the target gene was studied by the Q RT-PCR method; The Elisa method was used to detect the activity of the target gene promoter, and the Elisa method was used to detect the concentration of TNF- alpha and the absorptive capacity of sugar. The molecular mechanism of Glut4 was regulated by TNF- alpha and PI3K/AKT signaling pathway, and then the molecular mechanism of sugar absorption was regulated. Results: SOX4 and TNF- alpha formed a positive feedback regulation loop, and then inhibited the PI3K/AKT signaling pathway, and suppressed the PI3K/AKT signal pathway. The membrane localization of Glut4 was made and the sugar absorption capacity of the cells was inhibited. Conclusion: SOX4 inhibits the membrane location of Glut4 and the sugar absorption capacity of Glut4 through the TNF- alpha and PI3K/AKT signaling pathways.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.256

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