先天性甲状腺功能减退伴中枢神经系统异常致病基因NKX2-1功能的研究

发布时间：2018-05-16 12:16

本文选题：NKX2-1相关疾病 + 先天性甲状腺功能减退　；参考：《北京协和医学院》2016年博士论文

【摘要】：目的先天性甲状腺功能减退症(Congenital hypothyroidism, CH)常由基因异常导致,NK2同源盒1 (NK2 homeobox 1, NKX2-1)基因突变是造成CH的较常见原因。其表达的组织特异性转录因子NKX2-1蛋白在甲状腺、肺和中枢神经系统中发挥重要作用。本课题组前期研究发现一对因矮小就诊并诊断为CH的双胞胎男童,因同时存在中枢神经系统症状,行全基因组检测发现NKX2-1基因存在杂合错义突变,突变位点为c.799G＞T (NM_001079668.2),氨基酸改变为p.Val235Phe (NP_001073136.1).因NKX2-1基因突变较为多样,临床表现也有很大的异质性,不同的位置突变可能会带来不同的临床表现。但NKX2-1突变的遗传型和表型的关联目前仍不明确,NKX2-1基因c.799GT杂合突变患者为什么存在相应表现,目前文献报道仍较少。本实验旨在从蛋白表达和转录活性层面对NKX2-1基因c.799GT杂合突变致病的机制进行探索,以加深对NKX2-1相关疾病发病机制的认识。方法首先获得野生型NKX2-1 cDNA克隆表达载体,通过定点诱变PCR的方式,获得包含目的突变(c.799G＞T)的突变型表达载体。通过阳离子脂质体瞬时转染技术,将野生型和突变型质粒表达载体瞬时转染进HEK293细胞,48小时后裂解细胞,行蛋白免疫印迹(Western blot)实验,观察野生型和突变型NKX2-1蛋白在HEK293细胞中的表达差异。在此基础上,进一步将野生型和突变型NKX2-1表达载体分别与含有甲状腺球蛋白(thyroglobulin, TG)、甲状腺过氧化物酶(thyroperoxidase, TPO)、表面活性物质蛋白-B(surfactant protein-B, SP-B)基因启动子序列的萤光素酶报告载体共转染,通过检测双萤光素酶的活性,研究野生型和突变型NKX2-1蛋白对TG, TPO和SP-B启动子的转录活性的影响,试图从转录层面探究NKX2-1基因c.799G＞T杂合突变对NKX2-1基因功能的影响。此外,本实验还使用生物信息学软件对突变型NKX2-1 p.V235F蛋白进行局部结构分析,从结构变化角度分析该突变对蛋白结构的影响。结果1.获得野生型和突变型NKX2-1表达载体以购买获得的NKX2-1表达载体pENTER-wtNKX2-1为模板,通过定点诱变PCR获得pENTER-mutNKX2-1表达载体(c.799GT),质粒测序证实定点诱变成功。2.野生型和突变型NKX2-1 p.V235F蛋白表达鉴定Western Blot实验发现,相较于野生型NKX2-1蛋白,突变型NKX2-1 p. V235F蛋白在HEK293细胞中的表达量明显减低,其表达量仅为野生型NKX2-1蛋白表达量的0.3倍(p0.05)。3.野生型和突变型NKX2-1蛋白转录活性鉴定表达载体与TG、TP0口SP-B启动子报告载体共转染,全量(TG、TPO、SP-B启动子组分别为800ng、900ng.850ng)转染pENTER-wtNKX2-1组萤光素酶活性分别是仅转染pENTER空载转染组的24.47、35.19和44.63倍,三组均具有显著统计学差异(p0.05),提示野生型NKX2-1蛋白对TG、 TPO和SP-B启动子有明显的激活作用。逐步减少PENTER-wtNKX2-1转染量时,TG启动子报告载体共转染组的萤光素酶活性呈现逐步减低的趋势,TPO与SP-B启动子共转染组无此趋势。全量转染pENTER-mutNKX2-1 时, TG, TPO和SP-B启动子报告载体共转染组的萤光素酶活性分别是全量转染PENTER-wtNKX2-1组萤光素酶活性的0.022、0.027和0.11倍、分别是全量转染pENTER空载组萤光素酶活性的0.12、0.19和6.05倍,三组均具有显著统计学差异(p0.05),提示突变型NKX2-1 p.V235F蛋白对TG, TPO启动子无激活作用,对SP-B启动子有弱激活作用。4.突变型NKX2-1 p.V235F蛋白对野生型NKX2-1蛋白转录活性的影响当恒定pENTER-wtNKX2-1半量转染、逐渐增加pENTER-mutNKX2-1的转染量(1/8、1/4、3/8、1/2量)时,TG、TPO和SP-B启动子报告载体共转染组各组的萤光素酶活性与pENTER-wtNKX2-l空载各为半量转染组相比均无统pENTER计学差异。进一步增加转染量时,也不能造成更加显著的总PENTER-mutNKX2-1萤光素酶活性减低。上述结果提示突变型蛋白并不影响野生型NKX2-1 p.V235FNKX2-1蛋白对TG、TPO和SP-B启动子的激活能力5.突变型蛋白突变位点局部结构变化分析NKX2-1 p.V235F突变型蛋白235位苯丙氨酸同位于螺旋3的临近氨基酸和位于螺旋NKX2-1Ⅱ的221位精氨酸均发生异常联系,其提供的表面电荷分布区域与野生型NKX2-1蛋白235位缬氨酸相比存在差异。结论相较野生型NKX2-1蛋白,突变型NKX2-1 p.V235F蛋白在HEK293细胞中的表达量明显减少。野生型NKX2-1蛋白可以激活TG、TPD口SP-B基因转录,其激活TG启动子呈现剂量依赖模式,激活TPO、SP-B启动子呈现非剂量依赖模式。突变型NKX2-1 p.V235F蛋白不能激活TG.TPO基因的转录,也不影响野生型NKX2-1蛋白激活TG、TPO基因转录的能力。突变型NKX2-1 p. V235F蛋白可微弱地激活SP-B基因的转录,不影响野生型NKX2-1蛋白激活SP-B基因转录的能力。结合NKX2-1基因c.799G＞T杂合突变激活TG转录时存在单倍剂量不足,考虑TG转录的减少可能是该基因突变患者存在CH的原因。突变型NKX2-1 p.V235F蛋白235位苯丙氨酸与周围氨基酸的异常联系和表面电荷分布的变化可能是导致该突变蛋白无法有效识别DNA序列并丧失转录因子作用的原因。
[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.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R722.1

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