本文選題：NKX2-1相關疾病 + 先天性甲狀腺功能減退�。� 參考：《北京協和醫(yī)學院》2016年博士論文

【摘要】：目的先天性甲狀腺功能減退癥(Congenital hypothyroidism, CH)常由基因異常導致,NK2同源盒1 (NK2 homeobox 1, NKX2-1)基因突變是造成CH的較常見原因。其表達的組織特異性轉錄因子NKX2-1蛋白在甲狀腺、肺和中樞神經系統(tǒng)中發(fā)揮重要作用。本課題組前期研究發(fā)現一對因矮小就診并診斷為CH的雙胞胎男童,因同時存在中樞神經系統(tǒng)癥狀,行全基因組檢測發(fā)現NKX2-1基因存在雜合錯義突變,突變位點為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,臨床表現也有很大的異質性,不同的位置突變可能會帶來不同的臨床表現。但NKX2-1突變的遺傳型和表型的關聯目前仍不明確,NKX2-1基因c.799GT雜合突變患者為什么存在相應表現,目前文獻報道仍較少。本實驗旨在從蛋白表達和轉錄活性層面對NKX2-1基因c.799GT雜合突變致病的機制進行探索,以加深對NKX2-1相關疾病發(fā)病機制的認識。方法首先獲得野生型NKX2-1 cDNA克隆表達載體,通過定點誘變PCR的方式,獲得包含目的突變(c.799G＞T)的突變型表達載體。通過陽離子脂質體瞬時轉染技術,將野生型和突變型質粒表達載體瞬時轉染進HEK293細胞,48小時后裂解細胞,行蛋白免疫印跡(Western blot)實驗,觀察野生型和突變型NKX2-1蛋白在HEK293細胞中的表達差異。在此基礎上,進一步將野生型和突變型NKX2-1表達載體分別與含有甲狀腺球蛋白(thyroglobulin, TG)、甲狀腺過氧化物酶(thyroperoxidase, TPO)、表面活性物質蛋白-B(surfactant protein-B, SP-B)基因啟動子序列的螢光素酶報告載體共轉染,通過檢測雙螢光素酶的活性,研究野生型和突變型NKX2-1蛋白對TG, TPO和SP-B啟動子的轉錄活性的影響,試圖從轉錄層面探究NKX2-1基因c.799G＞T雜合突變對NKX2-1基因功能的影響。此外,本實驗還使用生物信息學軟件對突變型NKX2-1 p.V235F蛋白進行局部結構分析,從結構變化角度分析該突變對蛋白結構的影響。結果1.獲得野生型和突變型NKX2-1表達載體以購買獲得的NKX2-1表達載體pENTER-wtNKX2-1為模板,通過定點誘變PCR獲得pENTER-mutNKX2-1表達載體(c.799GT),質粒測序證實定點誘變成功。2.野生型和突變型NKX2-1 p.V235F蛋白表達鑒定Western Blot實驗發(fā)現,相較于野生型NKX2-1蛋白,突變型NKX2-1 p. V235F蛋白在HEK293細胞中的表達量明顯減低,其表達量僅為野生型NKX2-1蛋白表達量的0.3倍(p0.05)。3.野生型和突變型NKX2-1蛋白轉錄活性鑒定表達載體與TG、TP0口SP-B啟動子報告載體共轉染,全量(TG、TPO、SP-B啟動子組分別為800ng、900ng.850ng)轉染pENTER-wtNKX2-1組螢光素酶活性分別是僅轉染pENTER空載轉染組的24.47、35.19和44.63倍,三組均具有顯著統(tǒng)計學差異(p0.05),提示野生型NKX2-1蛋白對TG、 TPO和SP-B啟動子有明顯的激活作用。逐步減少PENTER-wtNKX2-1轉染量時,TG啟動子報告載體共轉染組的螢光素酶活性呈現逐步減低的趨勢,TPO與SP-B啟動子共轉染組無此趨勢。全量轉染pENTER-mutNKX2-1 時, TG, TPO和SP-B啟動子報告載體共轉染組的螢光素酶活性分別是全量轉染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)計學差異(p0.05),提示突變型NKX2-1 p.V235F蛋白對TG, TPO啟動子無激活作用,對SP-B啟動子有弱激活作用。4.突變型NKX2-1 p.V235F蛋白對野生型NKX2-1蛋白轉錄活性的影響當恒定pENTER-wtNKX2-1半量轉染、逐漸增加pENTER-mutNKX2-1的轉染量(1/8、1/4、3/8、1/2量)時,TG、TPO和SP-B啟動子報告載體共轉染組各組的螢光素酶活性與pENTER-wtNKX2-l空載各為半量轉染組相比均無統(tǒng)pENTER計學差異。進一步增加轉染量時,也不能造成更加顯著的總PENTER-mutNKX2-1螢光素酶活性減低。上述結果提示突變型蛋白并不影響野生型NKX2-1 p.V235FNKX2-1蛋白對TG、TPO和SP-B啟動子的激活能力5.突變型蛋白突變位點局部結構變化分析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。結論相較野生型NKX2-1蛋白,突變型NKX2-1 p.V235F蛋白在HEK293細胞中的表達量明顯減少。野生型NKX2-1蛋白可以激活TG、TPD口SP-B基因轉錄,其激活TG啟動子呈現劑量依賴模式,激活TPO、SP-B啟動子呈現非劑量依賴模式。突變型NKX2-1 p.V235F蛋白不能激活TG.TPO基因的轉錄,也不影響野生型NKX2-1蛋白激活TG、TPO基因轉錄的能力。突變型NKX2-1 p. V235F蛋白可微弱地激活SP-B基因的轉錄,不影響野生型NKX2-1蛋白激活SP-B基因轉錄的能力。結合NKX2-1基因c.799G＞T雜合突變激活TG轉錄時存在單倍劑量不足,考慮TG轉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系和表面電荷分布的變化可能是導致該突變蛋白無法有效識別DNA序列并喪失轉錄因子作用的原因。
[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.

【學位授予單位】：北京協和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R722.1

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