本文選題：SOX4 + 妊娠期糖尿病��； 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：第一部分:SOX4基因作為妊娠期糖尿病分子診斷標(biāo)志物有效性的研究背景:全基因組關(guān)聯(lián)分析研究(GWAS)發(fā)現(xiàn)CDKAL1基因單核苷酸多態(tài)性(SNP)位點rs7756992與糖尿病患病風(fēng)險呈正相關(guān)。隨后研究發(fā)現(xiàn)SOX4基因與此SNP位點連鎖,攜帶rs7756992 SNP人群表現(xiàn)為SOX4基因表達(dá)升高,說明SOX4基因的高表達(dá)與糖尿病的患病風(fēng)險存在正相關(guān)關(guān)系。同時,SOX4是哺乳動物發(fā)育過程中高表達(dá)的轉(zhuǎn)錄因子,SOX4在胚胎發(fā)育期的表達(dá)很可能參與了孕婦生理性提高血糖濃度的過程,暗示SOX4與血糖濃度調(diào)節(jié)相關(guān)。因此,我們推測SOX4的表達(dá)參與了孕婦正常生理性血糖調(diào)節(jié),在妊娠期糖尿病孕婦中,SOX4的過度表達(dá)引起了過度調(diào)控,導(dǎo)致妊娠期糖尿病(GDM)的發(fā)生。目的:鑒定SOX4在GDM孕婦和正常孕婦的血清循環(huán)RNA、血清蛋白、血細(xì)胞RNA、血細(xì)胞蛋白中的濃度存在差異。方法:采集GDM孕婦(n=60)及正常對照孕婦(n=60)靜脈血后分離血細(xì)胞并制備血清。1)SOX4血清循環(huán)RNA濃度:應(yīng)用Qiagen循環(huán)RNA提取試劑盒制備孕婦循環(huán)RNA,通過q RT-PCR方法檢測SOX4循環(huán)RNA濃度。2)SOX4血清蛋白濃度:應(yīng)用冷凍干燥方法濃縮蛋白,將每組30個樣本合并,采用Western-blot方法檢測血清SOX4蛋白濃度。3)SOX4血細(xì)胞RNA濃度:采用Qiagen試劑盒提取血細(xì)胞總RNA,通過q RT-PCR方法檢測樣本中SOX4血細(xì)胞RNA濃度。4)SOX4血細(xì)胞蛋白濃度:應(yīng)用RIPA裂解液裂解細(xì)胞并提取總蛋白,將每組15個樣本合并,應(yīng)用Western-blot方法檢測血細(xì)胞中SOX4蛋白濃度。結(jié)果:在妊娠期糖尿病孕婦中,血清循環(huán)RNA、血清蛋白、血細(xì)胞RNA、血細(xì)胞蛋白中SOX4濃度較正常組均有升高,應(yīng)用SPSS18.0軟件分析,差異具有統(tǒng)計學(xué)意義(t0.001)。結(jié)論:在妊娠期糖尿病中,孕婦血清和血細(xì)胞中的SOX4 RNA和蛋白質(zhì)均有升高。但SOX4蛋白含量較低,需合并多個病人樣本以完成Western-blot實驗,在更高靈敏度的Elisa試劑盒研發(fā)成功前不可作為標(biāo)志物檢測。SOX4循環(huán)RNA具有作為妊娠期糖尿病診斷的意義,但本文受樣本量的限制,未能準(zhǔn)確計算SOX4循環(huán)RNA作為標(biāo)志物的靈敏度和準(zhǔn)確性。本研究仍需大規(guī)模樣本量研究以評價其作為妊娠期糖尿病標(biāo)志物的有效性。第二部分:SOX4高表達(dá)誘導(dǎo)胰島素抵抗機理的研究背景:II型糖尿病發(fā)病率高,占全世界糖尿病人的90%,其發(fā)病機制復(fù)雜,遺傳和環(huán)境因素共同作用可能導(dǎo)致II型糖尿病的發(fā)生。妊娠期糖尿病與II型糖尿病表現(xiàn)類似,均表現(xiàn)為血糖增高和胰島素抵抗,因此,妊娠期糖尿病與II型糖尿病應(yīng)存在相似的分子機制。近期全基因組關(guān)聯(lián)分析研究(GWAS)和基礎(chǔ)研究發(fā)現(xiàn)SOX4基因的高表達(dá)與rs7756992單核苷酸突變亞型II型糖尿病緊密相關(guān),同時文獻研究表明SOX4也是胚胎發(fā)育期高表達(dá)的轉(zhuǎn)錄調(diào)控因子,因此,我們推論孕期體內(nèi)環(huán)境改變可能促進SOX4的表達(dá),參與孕期血糖生理調(diào)控。當(dāng)SOX4表達(dá)量被異常刺激時,引起了妊娠期糖尿病。GDM孕婦體內(nèi)SOX4異常高表達(dá)已經(jīng)在第一部分研究,但SOX4誘導(dǎo)胰島素抵抗的分子機制仍不清楚,此分子機制的研究有助于理解妊娠期糖尿病的發(fā)病機制,為妊娠期糖尿病的精準(zhǔn)醫(yī)療提供理論基礎(chǔ)。目的:研究SOX4誘導(dǎo)胰島素抵抗的分子機制。方法:以小鼠脂肪細(xì)胞系3T3-L1為模型,慢病毒介導(dǎo)SOX4及TNF-α基因過表達(dá)和沉默;通過Western-blot研究目的蛋白的表達(dá);通過q RT-PCR方法研究目的基因m RNA的表達(dá);通過報告基因載體研究靶基因啟動子的活性;Elisa方法用于檢測TNF-α的濃度和糖吸收能力。綜合研究SOX4通過TNF-α和PI3K/AKT信號通路調(diào)控Glut4的膜定位,進而調(diào)控糖吸收的分子機制。結(jié)果:SOX4與TNF-α形成正反饋調(diào)節(jié)回路,進而抑制PI3K/AKT信號通路,并抑制了Glut4的膜定位,進而抑制了細(xì)胞的糖吸收能力。結(jié)論:SOX4通過TNF-α和PI3K/AKT信號通路抑制Glut4的膜定位和細(xì)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R714.256

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相關(guān)期刊論文 前1條

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