發(fā)布時間：2018-04-18 15:59

  本文選題：屈光參差 + 致病基因�。� 參考：《浙江大學》2015年博士論文

【摘要】：研究背景近視性高度屈光參差指雙眼近視度高度不等,可誘發(fā)斜視、弱視及立體視功能障礙影響患者生活。該疾病發(fā)病隱匿,易造成較高度數(shù)眼視功能不可逆喪失。近視性高度屈光參差以雙眼軸過度及不對稱發(fā)育為特征,有一定遺傳傾向性,目前尚無相關致病基因報道。本課題組在臨床工作中密切關注該疾病患者,并收集了2個近視性高度屈光參差家系,對其中一個家系所有參與研究成員進行全面的眼科檢查后發(fā)現(xiàn)所有患者右眼近視度數(shù)、眼軸長度及前房深度均高于左眼。該研究結果已于2012年在Optometry Vision Science上發(fā)表。課題組通過對致病基因和突變位點的篩選及定位,發(fā)現(xiàn)基因UNC5D是近視性高度屈光參差發(fā)生的易感基因,家系中所有5名患者UNC5D基因上第8個外顯子中存在一處錯義突變,導致蛋白質(zhì)第433位精氨酸(Arg, R)被半胱氨酸(Cys, C)所替代。本研究擬通過突變型UNC5D基因體外克隆,探索該突變引起的相關基因表達差異及人成纖維細胞外基質(zhì)成分變化的機制。研究目的本研究通過對突變型UNC5D引起相關基因表達差異及人成纖維細胞外基質(zhì)成分變化機制的研究,推測UNC5D基因突變可能引起鞏膜外細胞基質(zhì)成分變化從而導致鞏膜變薄,眼軸過度增長的發(fā)生機制,為深入研究突變型UNC5D基因?qū)е陆曅郧鈪⒉钛圯S不對稱及過度發(fā)育的發(fā)病機理奠定基礎。研究方法1應用生物信息學方法對突變蛋白結構及功能進行計算機分析與預測,初步判斷突變對蛋白結構及功能的影響。(1)采用蛋白質(zhì)疏水性分析(Kyte Doolittle Hydropathy Plots)軟件對突變造成的蛋白質(zhì)疏水性質(zhì)的改變進行初步分析。(2)分別采用表型多態(tài)性分析(Polymorphism phenotypin v2, PolyPhen-2),格蘭瑟姆得分差異(Granthem score difference, Align-GVGD),氨基酸替換損害性排序程序(Sorting Intolerant From Tolerant amino acid substitutions, SIFT)軟件預測突變對蛋白功能的影響。2全基因合成野生型人UNC5D基因,利用DNA體外重組技術和PCR介導的定點誘變技術,構建野生型和突變型UNC5D基因Tet-on四環(huán)素調(diào)控慢病毒真核表達載體系統(tǒng),分別與Tet-on調(diào)控元件共同感染HEK293T細胞,強力霉素(Doxycycline)誘導野生型和突變型UNC5D基因表達。提取誘導表達后野生組、突變組和未誘導組HEK293T細胞總RNA于Affymetrix 3' IVT芯片平臺進行mRNA表達譜芯片檢測,采用實時熒光定量PCR技術驗證野生組、突變組及未誘導組HSPA6基因的表達差異。3構建含HA標簽的野生型和突變型UNC5D基因pLVX-IRES-ZsGreenl'慢病毒真核表達載體,感染人真皮成纖維細胞(HDFs)。(1)采用實時熒光定量PCR技術驗證野生組、突變組及對照組(空載體組)HSPA6基因的表達差異；(2)采用倒置熒光顯微鏡觀察野生組和突變組UNC5D基因亞細胞定位差異；(3)采用免疫共沉淀技術檢測HSP70B’蛋白與Smad2/3蛋白間相互作用；(4)TGF-β1處理野生組、突變組及對照組細胞,采用qPCR檢測各組細胞COL1A1、MMP-1和MMP-2的mRNA水平表達差異,并以Western Blot驗證COL1A1蛋白水平表達差異；(5)TGF-β1處理野生組、突變組及對照組細胞,采用Western Blot檢測野生組和突變組Smad2/3蛋白磷酸化水平。研究結果1生物信息學方法對突變蛋白結構及功能分析與預測結果：(1)蛋白質(zhì)疏水性分析提示：UNC5D蛋白第433位氨基酸由精氨酸突變?yōu)榘腚装彼?氨基酸由強親水性突變?yōu)槭杷?造成突變型UNC5D蛋白該區(qū)域疏水性較野生型明顯升高(2)分別采用表型多態(tài)性分析、格蘭瑟姆得分差異及氨基酸替換損害性排序程序軟件預測提示：突變極有可能損害蛋白功能。2成功構建野生型和突變型UNC5D基因Tet-on四環(huán)素調(diào)控慢病毒真核表達載體系統(tǒng)感染HEK293T細胞,野生型和突變型UNC5D基因Dox誘導表達成功。Affymetrix 3'IVT芯片平臺mRNA表達譜芯片檢測結果提示在mRNA水平突變組較野生組有15個基因表達上調(diào),2個基因表達下調(diào),其中HSPA6基因較野生組上調(diào)22.08倍,突變組HSPA6基因較未誘導組上調(diào)27.58倍,野生組較未誘導組HSPA6基因表達無差異。qPCR對HSPA6基因在各組表達差異驗證結果與芯片檢測結果趨勢基本一致：突變組HSPA6基因相對表達量高于野生組7.82倍,高于未誘導組25.16倍,野生組HSPA6相對表達量高于未誘導組3.22倍,。3成功構建含HA標簽的野生型和突變型UNC5D基因慢病毒真核表達載體pLVX-IRES-ZsGreenl感染人成纖維細胞。(1)qPCR驗證野生組、突變組及對照組入成纖維細胞HSPA6基因表達差異提示：突變組HSPA6基因高于野生組6.02倍,突變組HSPA6基因高于對照組15.59倍,野生組HSPA6高于對照組2.59倍。(2)倒置熒光顯微鏡觀察野生組和突變組人成纖維細胞UNC5D基因亞細胞定位未見明顯差異。(3)免疫共沉淀結果提示突變組高表達的HSP70B'蛋白與Smad2/3蛋白間存在相互作用。(4) TGF-β1處理野生組、突變組及對照組細胞,qPCR檢測發(fā)現(xiàn)突變組COL1A1較其他兩組表達下調(diào),MMP-2表達上調(diào),MMP-1表達無顯著差異,Western Blot亦提示突變組COL1A1蛋白表達低于較其他兩組；(5)TGF-β1處理野生組、突變組及對照組細胞,Western Blot提示突變組Smad2/3蛋白磷酸化水平低于野生組。研究結論本研究發(fā)現(xiàn)突變型UNC5D基因可顯著上調(diào)HSPA6基因表達,降低Smad2/3蛋白磷酸化水平,干擾TGF-β信號轉(zhuǎn)導,導致人成纖維細胞外基質(zhì)成分變化,推測該突變可引起鞏膜成纖維細胞外基質(zhì)成分變化,導致鞏膜重建進而影響眼軸的發(fā)育,首次為近視性屈光參差相關致病基因的機制研究提供依據(jù)。
[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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R777.44

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