本文選題：先天性甲狀腺功能減退癥 + 促甲狀腺素受體�。� 參考：《青島大學》2014年碩士論文

【摘要】：先天性甲狀腺功能減退癥(CH)是嬰幼兒最常見的內(nèi)分泌疾病之一,主要表現(xiàn)為甲狀腺功能部分或完全喪失導致的生長發(fā)育落后和智力發(fā)育遲緩。80%-85%的CH患者繼發(fā)于甲狀腺發(fā)育異常,與促甲狀腺激素受體(TSHR)等基因突變有關(guān)；其余15%-20%的患者則是由甲狀腺激素合成缺陷導致,其中,雙氧化酶2(DUOX2)基因突變后因不能氧化NADPH提供H202,致使碘的有機化障礙,最終導致CH。 本實驗第一部分以89例山東地區(qū)甲狀腺異位導致CH的患兒為研究對象,采集外周血提取DNA, PCR擴增第10外顯子,采用直接測序的方法對TSHR基因進行突變篩查。在89例研究對象第10外顯子測序結(jié)果中,發(fā)現(xiàn)1個錯義突變c.1269GA(p.V424I),1個SNP位點(rs1991517,c.2181GC,變異頻率18.5%)。對小鼠、大鼠、貓、豬、牛、斑馬魚和人的TSHR蛋白進行同源性比較,發(fā)現(xiàn)第424密碼子編碼的氨基酸位于TSHR的保守區(qū),該位點的纈氨酸被異亮氨酸取代(p.V4241),表明山東地區(qū)甲狀腺異位導致CH的患者中,TSHR基因第10外顯子突變率較低。 在前期的研究中,本課題組對10例甲狀腺功能減退伴甲狀腺腫大的患兒進行DUOX2基因突變篩查,共發(fā)現(xiàn)2個DUOX2基因新的錯義雜合突變：c.106OCT (p. R354W)和c.G3616A(p.A1206T)。本實驗第二部分對新發(fā)現(xiàn)的2個突變進行功能學研究,以闡明DUOX2基因突變導致甲減的致病機理。構(gòu)建DUOX2野生型或突變型以及DUOXA2野生型真核表達載體,轉(zhuǎn)染293T細胞,轉(zhuǎn)染后48h加入50uM Amplex Red reagent和0.2U/ml辣根過氧化物酶,37℃避光孵育2h,利用多功能酶標儀監(jiān)測595nm熒光強度,計算H202濃度；以DUOX2野生型或突變型與DUOXA2野生型真核表達載體共轉(zhuǎn)染Hela細胞,36h后加入終濃度為100μg/ml的放線菌酮(CHX)阻斷細胞內(nèi)蛋白質(zhì)合成,之后0-24h內(nèi)取5個時間點分別裂解細胞,提取總蛋白,用Western blot技術(shù)對DUOX2進行定量檢測,研究DUOX2突變后蛋白半衰期的改變。結(jié)果表明：無論野生型還是突變型DUOX2質(zhì)粒單獨轉(zhuǎn)染293T細胞后都不能生成H202,而與DUOXA2共轉(zhuǎn)染293T細胞后,野生型產(chǎn)生H202能力顯著提高,而突變型生成H202能力則表現(xiàn)為部分缺陷型；DUOX2基因突變后表達的蛋白半衰期縮短,可能與突變蛋白更易降解有關(guān)。
[Abstract]:Congenital hypothyroidism (CHS) is one of the most common endocrine diseases in infants and young children. It is mainly characterized by growth retardation caused by partial or complete loss of thyroid function and mental retardation. 80-85% of Ch patients are secondary to thyroid dysplasia. The other 15% to 20% of the patients were caused by the deficiency of thyroid hormone synthesis. After the mutation of the dioxygenase 2DUOX2 gene, the NADPH could not be oxidized to provide H2022, which resulted in the organic disorder of iodine, which resulted in CH2. In the first part of this experiment, 89 children with Ch caused by thyroid heterotopia in Shandong province were studied. The peripheral blood samples were extracted, the exon 10 was amplified by PCR, and the mutation of TSHR gene was screened by direct sequencing. One missense mutation c.1269 GAp.V424Ihe and one SNP locus, rs1991517, c.2181GC, were found in 89 subjects. The mutation frequency was 18.5g. The homology of TSHR protein in mice, rats, cats, pigs, cattle, zebrafish and human was compared. It was found that the amino acid encoded at codon 424 was located in the conserved region of TSHR. The valine of this locus was replaced by isoleucine, indicating that the mutation rate of exon 10 of TSHR gene was lower in patients with Ch caused by thyroid ectopic in Shandong area. In the previous study, 10 children with hypothyroidism and goiter were screened for DUOX2 gene mutation. Two new missense heterozygosity mutations of DUOX2 gene were found in 10 children with hypothyroidism and goiter. R354W) and c. G3616A, p. A1206T _ _ _ In the second part of this experiment, the two new mutations were studied in order to elucidate the pathogenesis of hypothyroidism caused by DUOX2 gene mutation. DUOX2 wild-type, mutant and DUOXA2 wild-type eukaryotic expression vectors were constructed and transfected into 293T cells. After 48 h transfection, 50uM Amplex Red reagent and 0.2U/ml horseradish peroxidase were incubated at 37 鈩，

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