本文選題：NKX2-1相關(guān)疾病 + 先天性甲狀腺功能減退　； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：目的先天性甲狀腺功能減退癥(Congenital hypothyroidism, CH)常由基因異常導(dǎo)致,NK2同源盒1 (NK2 homeobox 1, NKX2-1)基因突變是造成CH的較常見(jiàn)原因。其表達(dá)的組織特異性轉(zhuǎn)錄因子NKX2-1蛋白在甲狀腺、肺和中樞神經(jīng)系統(tǒng)中發(fā)揮重要作用。本課題組前期研究發(fā)現(xiàn)一對(duì)因矮小就診并診斷為CH的雙胞胎男童,因同時(shí)存在中樞神經(jīng)系統(tǒng)癥狀,行全基因組檢測(cè)發(fā)現(xiàn)NKX2-1基因存在雜合錯(cuò)義突變,突變位點(diǎn)為c.799G＞T (NM_001079668.2),氨基酸改變?yōu)閜.Val235Phe (NP_001073136.1).因NKX2-1基因突變較為多樣,臨床表現(xiàn)也有很大的異質(zhì)性,不同的位置突變可能會(huì)帶來(lái)不同的臨床表現(xiàn)。但NKX2-1突變的遺傳型和表型的關(guān)聯(lián)目前仍不明確,NKX2-1基因c.799GT雜合突變患者為什么存在相應(yīng)表現(xiàn),目前文獻(xiàn)報(bào)道仍較少。本實(shí)驗(yàn)旨在從蛋白表達(dá)和轉(zhuǎn)錄活性層面對(duì)NKX2-1基因c.799GT雜合突變致病的機(jī)制進(jìn)行探索,以加深對(duì)NKX2-1相關(guān)疾病發(fā)病機(jī)制的認(rèn)識(shí)。方法首先獲得野生型NKX2-1 cDNA克隆表達(dá)載體,通過(guò)定點(diǎn)誘變PCR的方式,獲得包含目的突變(c.799G＞T)的突變型表達(dá)載體。通過(guò)陽(yáng)離子脂質(zhì)體瞬時(shí)轉(zhuǎn)染技術(shù),將野生型和突變型質(zhì)粒表達(dá)載體瞬時(shí)轉(zhuǎn)染進(jìn)HEK293細(xì)胞,48小時(shí)后裂解細(xì)胞,行蛋白免疫印跡(Western blot)實(shí)驗(yàn),觀察野生型和突變型NKX2-1蛋白在HEK293細(xì)胞中的表達(dá)差異。在此基礎(chǔ)上,進(jìn)一步將野生型和突變型NKX2-1表達(dá)載體分別與含有甲狀腺球蛋白(thyroglobulin, TG)、甲狀腺過(guò)氧化物酶(thyroperoxidase, TPO)、表面活性物質(zhì)蛋白-B(surfactant protein-B, SP-B)基因啟動(dòng)子序列的螢光素酶報(bào)告載體共轉(zhuǎn)染,通過(guò)檢測(cè)雙螢光素酶的活性,研究野生型和突變型NKX2-1蛋白對(duì)TG, TPO和SP-B啟動(dòng)子的轉(zhuǎn)錄活性的影響,試圖從轉(zhuǎn)錄層面探究NKX2-1基因c.799G＞T雜合突變對(duì)NKX2-1基因功能的影響。此外,本實(shí)驗(yàn)還使用生物信息學(xué)軟件對(duì)突變型NKX2-1 p.V235F蛋白進(jìn)行局部結(jié)構(gòu)分析,從結(jié)構(gòu)變化角度分析該突變對(duì)蛋白結(jié)構(gòu)的影響。結(jié)果1.獲得野生型和突變型NKX2-1表達(dá)載體以購(gòu)買獲得的NKX2-1表達(dá)載體pENTER-wtNKX2-1為模板,通過(guò)定點(diǎn)誘變PCR獲得pENTER-mutNKX2-1表達(dá)載體(c.799GT),質(zhì)粒測(cè)序證實(shí)定點(diǎn)誘變成功。2.野生型和突變型NKX2-1 p.V235F蛋白表達(dá)鑒定Western Blot實(shí)驗(yàn)發(fā)現(xiàn),相較于野生型NKX2-1蛋白,突變型NKX2-1 p. V235F蛋白在HEK293細(xì)胞中的表達(dá)量明顯減低,其表達(dá)量?jī)H為野生型NKX2-1蛋白表達(dá)量的0.3倍(p0.05)。3.野生型和突變型NKX2-1蛋白轉(zhuǎn)錄活性鑒定表達(dá)載體與TG、TP0口SP-B啟動(dòng)子報(bào)告載體共轉(zhuǎn)染,全量(TG、TPO、SP-B啟動(dòng)子組分別為800ng、900ng.850ng)轉(zhuǎn)染pENTER-wtNKX2-1組螢光素酶活性分別是僅轉(zhuǎn)染pENTER空載轉(zhuǎn)染組的24.47、35.19和44.63倍,三組均具有顯著統(tǒng)計(jì)學(xué)差異(p0.05),提示野生型NKX2-1蛋白對(duì)TG、 TPO和SP-B啟動(dòng)子有明顯的激活作用。逐步減少PENTER-wtNKX2-1轉(zhuǎn)染量時(shí),TG啟動(dòng)子報(bào)告載體共轉(zhuǎn)染組的螢光素酶活性呈現(xiàn)逐步減低的趨勢(shì),TPO與SP-B啟動(dòng)子共轉(zhuǎn)染組無(wú)此趨勢(shì)。全量轉(zhuǎn)染pENTER-mutNKX2-1 時(shí), TG, TPO和SP-B啟動(dòng)子報(bào)告載體共轉(zhuǎn)染組的螢光素酶活性分別是全量轉(zhuǎn)染PENTER-wtNKX2-1組螢光素酶活性的0.022、0.027和0.11倍、分別是全量轉(zhuǎn)染pENTER空載組螢光素酶活性的0.12、0.19和6.05倍,三組均具有顯著統(tǒng)計(jì)學(xué)差異(p0.05),提示突變型NKX2-1 p.V235F蛋白對(duì)TG, TPO啟動(dòng)子無(wú)激活作用,對(duì)SP-B啟動(dòng)子有弱激活作用。4.突變型NKX2-1 p.V235F蛋白對(duì)野生型NKX2-1蛋白轉(zhuǎn)錄活性的影響當(dāng)恒定pENTER-wtNKX2-1半量轉(zhuǎn)染、逐漸增加pENTER-mutNKX2-1的轉(zhuǎn)染量(1/8、1/4、3/8、1/2量)時(shí),TG、TPO和SP-B啟動(dòng)子報(bào)告載體共轉(zhuǎn)染組各組的螢光素酶活性與pENTER-wtNKX2-l空載各為半量轉(zhuǎn)染組相比均無(wú)統(tǒng)pENTER計(jì)學(xué)差異。進(jìn)一步增加轉(zhuǎn)染量時(shí),也不能造成更加顯著的總PENTER-mutNKX2-1螢光素酶活性減低。上述結(jié)果提示突變型蛋白并不影響野生型NKX2-1 p.V235FNKX2-1蛋白對(duì)TG、TPO和SP-B啟動(dòng)子的激活能力5.突變型蛋白突變位點(diǎn)局部結(jié)構(gòu)變化分析NKX2-1 p.V235F突變型蛋白235位苯丙氨酸同位于螺旋3的臨近氨基酸和位于螺旋NKX2-1Ⅱ的221位精氨酸均發(fā)生異常聯(lián)系,其提供的表面電荷分布區(qū)域與野生型NKX2-1蛋白235位纈氨酸相比存在差異。結(jié)論相較野生型NKX2-1蛋白,突變型NKX2-1 p.V235F蛋白在HEK293細(xì)胞中的表達(dá)量明顯減少。野生型NKX2-1蛋白可以激活TG、TPD口SP-B基因轉(zhuǎn)錄,其激活TG啟動(dòng)子呈現(xiàn)劑量依賴模式,激活TPO、SP-B啟動(dòng)子呈現(xiàn)非劑量依賴模式。突變型NKX2-1 p.V235F蛋白不能激活TG.TPO基因的轉(zhuǎn)錄,也不影響野生型NKX2-1蛋白激活TG、TPO基因轉(zhuǎn)錄的能力。突變型NKX2-1 p. V235F蛋白可微弱地激活SP-B基因的轉(zhuǎn)錄,不影響野生型NKX2-1蛋白激活SP-B基因轉(zhuǎn)錄的能力。結(jié)合NKX2-1基因c.799G＞T雜合突變激活TG轉(zhuǎn)錄時(shí)存在單倍劑量不足,考慮TG轉(zhuǎn)錄的減少可能是該基因突變患者存在CH的原因。突變型NKX2-1 p.V235F蛋白235位苯丙氨酸與周圍氨基酸的異常聯(lián)系和表面電荷分布的變化可能是導(dǎo)致該突變蛋白無(wú)法有效識(shí)別DNA序列并喪失轉(zhuǎn)錄因子作用的原因。
[Abstract]:Objective congenital hypothyroidism (Congenital hypothyroidism, CH) is often caused by genetic abnormalities. The mutation of NK2 homologous box 1 (NK2 homeobox 1, NKX2-1) is the most common cause of CH. The expression of the tissue specific transcription factor NKX2-1 protein plays an important role in the thyroid, lung and central nervous system. In the previous study, a pair of twin boys with short visit and CH diagnosis was found. Because of the simultaneous presence of central nervous system symptoms, the whole genome detection found that the NKX2-1 gene had a heterozygous missense mutation, the mutation site was c.799G > T (NM_001079668.2), and the amino acid change was p.Val235Phe (NP_001073136.1). The mutation of the NKX2-1 gene was more varied. The clinical manifestations also have great heterogeneity, and different position mutations may bring different clinical manifestations. However, the relationship between the genotype and phenotype of the NKX2-1 mutation is still unclear. Why the NKX2-1 gene c.799GT heterozygous mutation has the corresponding performance, the present literature is still less. This experiment aims at protein expression and transcriptional activity. The mechanism of the heterozygous mutation of the NKX2-1 gene c.799GT was explored to deepen the understanding of the pathogenesis of NKX2-1 related diseases. Method first obtained the wild type NKX2-1 cDNA cloning expression vector, and obtained the mutant expression vector containing the target mutation (c.799G > T) through the fixed-point mutagenesis of PCR. The transient expression vector containing the target mutation (c.799G > T) was obtained by the cationic liposome transient. Transfection technique, the wild type and mutant plasmid expression vector were transiently transfected into HEK293 cells. After 48 hours, the cells were lysed and the protein immunoblotting (Western blot) experiment was performed to observe the differences in the expression of wild type and mutant NKX2-1 protein in HEK293 cells. On the basis of this, the wild type and mutant NKX2-1 expression vectors were further studied. Thyroglobulin, TG, thyroperoxidase, TPO, -B (surfactant protein-B, SP-B) gene promoter sequences of the surfactant protein -B (surfactant protein-B, SP-B) gene promoter were co transfected. By detecting the activity of double fluorosin enzyme, the wild type and mutant NKX2-1 protein were studied for TG, TPO and SP-B. The effect of the transcriptional activity of the promoter was tried to explore the effect of the NKX2-1 gene c.799G > T heterozygous mutation on the NKX2-1 gene function from the transcriptional level. In addition, this experiment also used the bioinformatics software to analyze the local structure of the mutant NKX2-1 p.V235F protein, and analyzed the effect of the mutation on the structure of the protein from the structural change angle. Results 1. The wild type and mutant NKX2-1 expression vector was used to purchase the acquired NKX2-1 expression vector pENTER-wtNKX2-1 as the template, and the pENTER-mutNKX2-1 expression vector (c.799GT) was obtained by the fixed-point mutation of PCR. The plasmid sequencing confirmed that the.2. wild type and the mutant NKX2-1 p.V235F egg white expression of the NKX2-1 p.V235F egg white expression was confirmed by the plasmid sequencing, and the Western Blot experiment was found, compared with the wild one. The expression of mutant NKX2-1 P. V235F protein in HEK293 cells decreased obviously in HEK293 cells, the expression amount was only 0.3 times of the wild type NKX2-1 protein expression (P0.05).3. wild type and mutant NKX2-1 protein transcriptional activity identification expression vector and TG, TP0 mouth SP-B promoter report carrier co transfection. 800ng, 900ng.850ng) transfected pENTER-wtNKX2-1 group fluoro enzyme activity was 24.47,35.19 and 44.63 times of pENTER only transfection group, three groups had significant statistical difference (P0.05), suggesting that wild type NKX2-1 protein had obvious activation effect on TG, TPO and SP-B promoter. The activity of fluoro enzyme in the promoter co transfection group showed a tendency to decrease gradually, and there was no trend in the co transfection group of TPO and SP-B promoter. The fluoro enzyme activity of the TG, TPO and SP-B promoter co transfected group was the 0.022,0.027 of the full transfection of the PENTER-wtNKX2-1 group of the PENTER-wtNKX2-1 group. And 0.11 times, respectively, 0.12,0.19 and 6.05 times the activity of total transfection of pENTER, respectively. The three groups had significant statistical differences (P0.05), suggesting that the mutant NKX2-1 p.V235F protein had no activation effect on TG, TPO promoter, and the.4. mutant NKX2-1 p.V235F protein of SP-B promoter was used for the transcription of wild type NKX2-1 protein. When transfection of constant pENTER-wtNKX2-1 half quantity and increasing the transfection amount of pENTER-mutNKX2-1 (1/8,1/4,3/8,1/2), the activity of TG, TPO and SP-B promoter in the co transfection group had no pENTER difference compared with that of the half volume transfection group with pENTER-wtNKX2-l no-load, and the transfection amount was further increased. No more significant total PENTER-mutNKX2-1 fluorinase activity was reduced. The above results suggest that the mutant protein does not affect the local structural changes of the mutation loci of the 5. mutant protein of the wild type NKX2-1 p.V235FNKX2-1 protein to the TG, TPO and SP-B promoters, and the analysis of the NKX2-1 p.V235F mutant protein 235 phenylalanine same position The adjacent amino acids of spiral 3 and 221 - bit arginine located in spiral NKX2-1 II have abnormal relation, and the distribution of surface charge is different from that of wild type NKX2-1 protein 235 - valine. Conclusion compared with wild type NKX2-1 protein, the expression of mutant NKX2-1 p.V235F protein in HEK293 cells is significantly reduced. Type NKX2-1 protein activates TG, TPD port SP-B gene transcription, which activates the TG promoter to present a dose dependent mode, activates TPO, and the SP-B promoter presents a non dose dependent mode. The mutant NKX2-1 p.V235F protein does not activate the transcription of the TG.TPO gene, nor does it affect the activation of the NKX2-1 protein of the NKX2-1 protein, and the ability of the gene transcription. 235F protein weakly activates the transcription of SP-B gene, and does not affect the ability of the wild type NKX2-1 protein to activate the SP-B gene transcription. The NKX2-1 gene c.799G > T heterozygous mutation activates the TG transcript, and the decrease of the TG transcription may be the reason for the existence of CH in the mutation of the gene. The mutant NKX2-1 p.V235F protein is 235 bits. The abnormal relationship between phenylalanine and the surrounding amino acids and the change of surface charge distribution may be the reason that the mutant protein can not effectively identify the DNA sequence and lose the role of the transcription factor.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R722.1

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8 中南大學(xué)湘雅醫(yī)院急診科 教授 羅學(xué)宏;中年女性“發(fā)�！� 警惕甲減來(lái)襲[N];醫(yī)藥經(jīng)濟(jì)報(bào);2010年

9 吉林省中醫(yī)藥科學(xué)院 李貴滿;甲狀腺功能減退 中醫(yī)藥應(yīng)對(duì)有方[N];中國(guó)中醫(yī)藥報(bào);2010年

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6 宋娜;甲狀腺功能減退與妊娠期高血壓疾病關(guān)系探討[D];吉林大學(xué);2014年

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9 曾憲忠;成年期甲狀腺功能減退對(duì)雄性小鼠空間學(xué)習(xí)記憶及海馬內(nèi)神經(jīng)顆粒素表達(dá)的影響[D];安徽醫(yī)科大學(xué);2007年

10 何秀波;高頻超聲對(duì)橋本氏甲狀腺炎甲狀腺功能減退的診斷研究[D];中南大學(xué);2014年

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