突变型UNC5D在近视性高度屈光参差发病中的机制研究

发布时间：2018-04-18 15:59

本文选题：屈光参差 + 致病基因　；参考：《浙江大学》2015年博士论文

【摘要】：研究背景近视性高度屈光参差指双眼近视度高度不等,可诱发斜视、弱视及立体视功能障碍影响患者生活。该疾病发病隐匿,易造成较高度数眼视功能不可逆丧失。近视性高度屈光参差以双眼轴过度及不对称发育为特征,有一定遗传倾向性,目前尚无相关致病基因报道。本课题组在临床工作中密切关注该疾病患者,并收集了2个近视性高度屈光参差家系,对其中一个家系所有参与研究成员进行全面的眼科检查后发现所有患者右眼近视度数、眼轴长度及前房深度均高于左眼。该研究结果已于2012年在Optometry Vision Science上发表。课题组通过对致病基因和突变位点的筛选及定位,发现基因UNC5D是近视性高度屈光参差发生的易感基因,家系中所有5名患者UNC5D基因上第8个外显子中存在一处错义突变,导致蛋白质第433位精氨酸(Arg, R)被半胱氨酸(Cys, C)所替代。本研究拟通过突变型UNC5D基因体外克隆,探索该突变引起的相关基因表达差异及人成纤维细胞外基质成分变化的机制。研究目的本研究通过对突变型UNC5D引起相关基因表达差异及人成纤维细胞外基质成分变化机制的研究,推测UNC5D基因突变可能引起巩膜外细胞基质成分变化从而导致巩膜变薄,眼轴过度增长的发生机制,为深入研究突变型UNC5D基因导致近视性屈光参差眼轴不对称及过度发育的发病机理奠定基础。研究方法1应用生物信息学方法对突变蛋白结构及功能进行计算机分析与预测,初步判断突变对蛋白结构及功能的影响。(1)采用蛋白质疏水性分析(Kyte Doolittle Hydropathy Plots)软件对突变造成的蛋白质疏水性质的改变进行初步分析。(2)分别采用表型多态性分析(Polymorphism phenotypin v2, PolyPhen-2),格兰瑟姆得分差异(Granthem score difference, Align-GVGD),氨基酸替换损害性排序程序(Sorting Intolerant From Tolerant amino acid substitutions, SIFT)软件预测突变对蛋白功能的影响。2全基因合成野生型人UNC5D基因,利用DNA体外重组技术和PCR介导的定点诱变技术,构建野生型和突变型UNC5D基因Tet-on四环素调控慢病毒真核表达载体系统,分别与Tet-on调控元件共同感染HEK293T细胞,强力霉素(Doxycycline)诱导野生型和突变型UNC5D基因表达。提取诱导表达后野生组、突变组和未诱导组HEK293T细胞总RNA于Affymetrix 3' IVT芯片平台进行mRNA表达谱芯片检测,采用实时荧光定量PCR技术验证野生组、突变组及未诱导组HSPA6基因的表达差异。3构建含HA标签的野生型和突变型UNC5D基因pLVX-IRES-ZsGreenl'慢病毒真核表达载体,感染人真皮成纤维细胞(HDFs)。(1)采用实时荧光定量PCR技术验证野生组、突变组及对照组(空载体组)HSPA6基因的表达差异；(2)采用倒置荧光显微镜观察野生组和突变组UNC5D基因亚细胞定位差异；(3)采用免疫共沉淀技术检测HSP70B’蛋白与Smad2/3蛋白间相互作用；(4)TGF-β1处理野生组、突变组及对照组细胞,采用qPCR检测各组细胞COL1A1、MMP-1和MMP-2的mRNA水平表达差异,并以Western Blot验证COL1A1蛋白水平表达差异；(5)TGF-β1处理野生组、突变组及对照组细胞,采用Western Blot检测野生组和突变组Smad2/3蛋白磷酸化水平。研究结果1生物信息学方法对突变蛋白结构及功能分析与预测结果：(1)蛋白质疏水性分析提示：UNC5D蛋白第433位氨基酸由精氨酸突变为半胱氨酸,氨基酸由强亲水性突变为疏水性,造成突变型UNC5D蛋白该区域疏水性较野生型明显升高(2)分别采用表型多态性分析、格兰瑟姆得分差异及氨基酸替换损害性排序程序软件预测提示：突变极有可能损害蛋白功能。2成功构建野生型和突变型UNC5D基因Tet-on四环素调控慢病毒真核表达载体系统感染HEK293T细胞,野生型和突变型UNC5D基因Dox诱导表达成功。Affymetrix 3'IVT芯片平台mRNA表达谱芯片检测结果提示在mRNA水平突变组较野生组有15个基因表达上调,2个基因表达下调,其中HSPA6基因较野生组上调22.08倍,突变组HSPA6基因较未诱导组上调27.58倍,野生组较未诱导组HSPA6基因表达无差异。qPCR对HSPA6基因在各组表达差异验证结果与芯片检测结果趋势基本一致：突变组HSPA6基因相对表达量高于野生组7.82倍,高于未诱导组25.16倍,野生组HSPA6相对表达量高于未诱导组3.22倍,。3成功构建含HA标签的野生型和突变型UNC5D基因慢病毒真核表达载体pLVX-IRES-ZsGreenl感染人成纤维细胞。(1)qPCR验证野生组、突变组及对照组入成纤维细胞HSPA6基因表达差异提示：突变组HSPA6基因高于野生组6.02倍,突变组HSPA6基因高于对照组15.59倍,野生组HSPA6高于对照组2.59倍。(2)倒置荧光显微镜观察野生组和突变组人成纤维细胞UNC5D基因亚细胞定位未见明显差异。(3)免疫共沉淀结果提示突变组高表达的HSP70B'蛋白与Smad2/3蛋白间存在相互作用。(4) TGF-β1处理野生组、突变组及对照组细胞,qPCR检测发现突变组COL1A1较其他两组表达下调,MMP-2表达上调,MMP-1表达无显著差异,Western Blot亦提示突变组COL1A1蛋白表达低于较其他两组；(5)TGF-β1处理野生组、突变组及对照组细胞,Western Blot提示突变组Smad2/3蛋白磷酸化水平低于野生组。研究结论本研究发现突变型UNC5D基因可显著上调HSPA6基因表达,降低Smad2/3蛋白磷酸化水平,干扰TGF-β信号转导,导致人成纤维细胞外基质成分变化,推测该突变可引起巩膜成纤维细胞外基质成分变化,导致巩膜重建进而影响眼轴的发育,首次为近视性屈光参差相关致病基因的机制研究提供依据。
[Abstract]:On the background of high myopic anisometropia in the eyes of myopia with different height, can be induced by strabismus, amblyopia and stereopsis dysfunction in patients with the disease. Life affects the incidence of occult, caused by the high degree of visual function in eyes. The irreversible loss of high myopic anisometropia on binocular shaft and asymmetric characteristics of development, have a certain genetic predisposition at present, there is no related genes reported. The research group in clinical work, pay close attention to the disease, and collected 2 high myopic anisometropia family, one of the family members were all involved in the study of comprehensive eye examination found that all patients with right eye myopia, axial length and anterior chamber depth above the left eye. The results of the research have been published in the Optometry Vision Science in 2012. By the research group of pathogenic genes and mutation screening and localization, gene UNC 5D is susceptible to high myopic anisometropia occurred in the gene family of all 5 patients on the eighth exons of UNC5D gene a missense mutation exists in 433rd lead to protein arginine (Arg, R) (Cys, C) is a cysteine substitution. This study by mutant UNC5D in vitro gene cloning, explore the mutation gene expression caused by the differences and changes in fiber composition of the extracellular matrix. The mechanism of induced gene expression profiles of human fibroblast and extracellular matrix components change mechanism research on mutant UNC5D research purpose through this study, we speculated that UNC5D gene mutation can cause changes of scleral matrix composition cells resulting in scleral thinning, mechanism of axial excessive growth, for the further study of mutant UNC5D gene causes myopic anisometropia axial asymmetry and the excessive development of the pathogenesis of Dian Dingji research foundation. Methods 1 by bioinformatics analysis and prediction of protein structure and function of the computer, to determine the initial impact of the mutation on protein structure and function. (1) the analysis of protein hydrophobicity (Kyte Doolittle Hydropathy Plots) software to change the protein hydrophobicity caused by mutations were analyzed (2) were used. Analysis of phenotypic polymorphism (Polymorphism phenotypin V2, PolyPhen-2), Grantham (Granthem score difference, scores of Align-GVGD), amino acid substitution damage (Sorting Intolerant From Tolerant program amino acid substitutions SIFT) software to predict the mutation in the.2 gene synthesis of wild type UNC5D gene affect protein function, using site directed mutagenesis of DNA in vitro recombinant technology and PCR mediated, construction of wild type and mutant UNC5D gene Tet-on four cyclophilin control lentiviral eukaryotic expression As the carrier system, and regulatory elements of Tet-on co infected HEK293T cells, doxycycline (Doxycycline) induced by wild type and mutant UNC5D gene expression. The extraction of induced mutation group and wild group, non induced group HEK293T cell total RNA in Affymetrix 3'IVT chip platform mRNA microarray detection, using real-time fluorescence quantitative PCR technology verify the wild group, mutation and expression of group differences in the non induced group HSPA6 gene.3 was constructed with HA tag wild type and mutation type UNC5D gene pLVX-IRES-ZsGreenl' lentiviral eukaryotic expression vector infection of human dermal fibroblasts (HDFs). (1) by using real-time quantitative PCR technology to verify the wild group, control group and mutation group (vector group) HSPA6 gene differential expression; (2) using the inverted fluorescence microscope and UNC5D gene mutation group and wild group differences in subcellular localization; (3) by immunoprecipitation. The interaction was detected HSP70B protein and Smad2/3 protein; (4) TGF- beta 1 treatment group and control group of wild and mutant cells were detected by qPCR, cell COL1A1, the differential expression of MMP-1 and MMP-2 levels of mRNA, Western and Blot to verify the COL1A1 protein expression differences; (5) TGF- beta 1 treatment of wild group and control group, mutant cells group, using Western Blot to detect wild group and mutation group Smad2/3 protein phosphorylation level. The results of 1 methods of bioinformatics analysis and mutation of protein structure and function prediction results: (1) protein hydrophobicity analysis showed that UNC5D protein of 433rd amino acid mutation from arginine to cysteine. The amino acid mutation from hydrophilic to hydrophobic, causing mutant UNC5D protein the hydrophobic region compared to the wild type was significantly increased (2) were analyzed by phenotypic polymorphism, Grantham scores and amino acid substitution The damage of sorting program software to predict the likely damage tip: mutation.2 protein function construct of wild-type and mutant UNC5D gene Tet-on tetracycline regulated lentiviral eukaryotic expression vector system infected HEK293T cells, wild-type and mutant UNC5D gene expression induced by Dox.Affymetrix 3'IVT chip platform mRNA expression detection results suggest that at the level of mRNA mutation group a group of wild expression of 15 genes microarray, 2 genes down regulated the expression of the HSPA6 gene than the wild group increased 22.08 times, HSPA6 gene mutation group than non induced group increased 27.58 times, wild group than in the non induced group HSPA6 gene.QPCR expression was no significant difference of HSPA6 gene expression test results between the results and the chip basically the same trend in each group: the relative expression of HSPA6 gene mutation group was higher than the wild group 7.82 times, 25.16 times higher than that of non induced group, HSPA6 group of wild relative table As the amount of higher than non induced group 3.22 times,.3 successfully constructed HA tag containing wild type and mutant UNC5D gene lentiviral eukaryotic expression vector of pLVX-IRES-ZsGreenl infection in human fibroblasts. (1) qPCR verification wild group and mutation group and control group in fibroblast cells HSPA6 gene expression in HSPA6 gene mutation group was higher than that of Tip: the wild group 6.02 times, HSPA6 gene mutation group was higher than that of group 15.59 times that of the control group was higher than that of wild HSPA6 2.59 fold of control group. (2) under the inverted fluorescence microscope wild-type UNC5D gene in fibroblasts of subcellular localization showed no significant difference between the group and mutation (3). The results suggest the presence of interacting mutation group high expression of HSP70B'protein and Smad2/3 protein co immunoprecipitation. (4) TGF- beta 1 treatment group and control group of wild and mutant cells, detection of qPCR mutation was found in group COL1A1 than the other two groups of expression of MMP-2 expression, MMP-1 expression was not significantly The difference, Western Blot also indicated that the mutation group of COL1A1 protein was lower than that in the other two groups; (5) TGF- beta 1 treatment group and control group of wild and mutant cells group, Western Blot group suggested that the mutant phosphorylation of Smad2/3 protein than the wild group. Conclusions this study found that the mutant UNC5D gene can up regulate the expression of HSPA6 gene. Reduce the level of Smad2/3 protein phosphorylation, interference of TGF- beta signaling, leads to changes of fibroblast extracellular matrix components, speculated that the mutation can cause scleral changes of fibroblast extracellular matrix, thereby affecting the axial lead of scleral reconstruction development, provide the basis for the first time to study the mechanism of myopic anisometropia related genes.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R777.44

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