【作者】 黄悦；

【导师】 孙慧敏；

【作者基本信息】 天津医科大学 ， 眼科学， 2007， 博士

【摘要】 目的：中国山东、河南遗传性无虹膜两家系致病基因的定位及突变筛查；构建基因PAX6RNAi载体；探讨基因PAX6沉默对B3细胞的影响。方法：1、致病基因筛查定位。(1)、分别采集山东遗传性无虹膜合并先天性白内障家系、河南遗传性无虹膜家系的临床资料及血液标本,进行经典遗传学分析和细胞遗传学检查。(2)、提取家系成员的基因组DNA,采用定位克隆的策略,对山东家系进行全基因组微卫星标记的扫描,经多重PCR反应,Genescan分析基因型,数据文件在Linkage5.2软件上运算,连锁分析计算Lod Score,单倍体分析,精确定位。对河南家系则在已有研究结果上直接进行该区域定位排查,并进一步精细定位。(3)、从NCBI (National Center for biotechnology Information)等数据库中搜寻定位区域内的候选致病基因的基因序列,设计引物,PCR扩增测序,进行突变筛查。2、PAX6RNAi载体构建。(1)、选择4个PAX6基因RNA干扰靶序列,设计与之对应的shRNA序列,构建并验证shRNA重组质粒载体,构建过表达外源PAX6融合蛋白的293T细胞模型。用shRNA质粒载体进行外源靶序列筛选。(2)、构建shRNA慢病毒载体,用shRNA慢病毒载体进行内源目标序列筛选,与外源筛选结果共同确定有效的PAX6RNAi靶序列。3、应用有效的shRN/干扰慢病毒感染B3细胞,观察PAX6基因干扰后对B3细胞的影响。结果：1、致病基因筛查定位。(1)、根据经典遗传学分析,两家系为常染色体显性遗传,细胞遗传学检查未发现染色体异常。(2)、定位克隆把山东家系致病基因定位于微卫星Markers D11S915和D11S4101之间,河南家系致病基因定位于微卫星Markers D11S1755和D11S935之间。(3)、对候选基因PAX6进行PCR双向测序,发现山东家系中PAX6基因的第二个内含子的倒数第二个碱基腺嘌呤A发生缺失突变,导致可能的mRNA拼接错误,而河南家系中未发现PAX6基因突变。2、PAX6RNAi载体构建。(1)、针对候选的4个PAX6干扰靶点,成功构建了PAX6shRNA的重组质粒载体及表达FLAG tag、PAX6、红色荧光蛋白的融合蛋白表达系统,并在293T细胞上构建了PAX6过表达模型。外源筛靶结果验证了1#、2#和4#靶序列是有效的干扰靶位点。(2)、成功构建了PAX6shRNA重组慢病毒载体,在表达内源PAX6的晶状体上皮细胞系B3中进行靶点筛选,其结果与外源筛选结果一致,1#、2#和4#shRNA重组慢病毒对PAX6基因均有明显的干扰效果。3、PAX6沉默的B3细胞,凋亡细胞数目异常增加。结论：1、山东家系的PAX6基因在第二个内含子的倒数第二个碱基腺嘌呤A发生缺失突变(IVS2-2delA),导致该家系出现先天性无虹膜及先天性白内障表型。2、河南无虹膜家系的致病基因位点定位在Markers D11S1755和D11S935之间。3、找到了3个PAX6RNAi的有效靶序列。4、建立了PAX6RNAi质粒载体及慢病毒载体。5、PAX6基因沉默引起B3细胞过度凋亡。更多还原

【Abstract】 Objective:We tried to identify the genetic defect causing hereditary aniridia in two families from Shandong Prov. and Henan Prov. of China; To construct vectors of RNA interference (RNAi) of the pathogenics gene PAX6; To study the effect of RNAi-mediated PAX6gene silencing on B3cell.Methods:1. Pathogenic gene screening:(1). To investigate a family affected by aniridia and cataract in Shandong and another family affected by aniridia in Henan, genomic DNA was extracted from peripheral blood leucocytes using standard protocols with informed consent. The affected status was determined before genetic analysis Determine the pattern of inheritance, exclude the evidence of chromosome defects in karyotype analysis.(2). To Shandong family, a genomewide scan consisting of382microsatellite markers spaced at-10cM intervals was performed using ABI PRISM Linkage Mapping sets. To Henan family, only several markers was selected in a candidate region based on the result of Shandong family, Genotyping and haplotype construction were performed;(3). Identify the potential mutation by sequencing the candidate gene.2. Construct PAX6RNAi vector:(1). Choose four PAX6candidate target sequences for RNAi, design corresponding shRNA sequences, construct and verification the recombinant plasmids. Construct293T cell model of exogenous expression of the PAX6fusion protein. Use shRNA plasmid vector for exogenous target sequence screening.(2). Construct shRNA lentiviral vector, and use it for endogenous sequence screening, and identify effective RNAi target sequence compared with the results of the exogenous screening.3. Infected B3cell with effective shRNA entiviral vector., observation the influence of the B3cell.Results:1.(1). Two families show an autosomal dominant pattern of inheritance by the analysis. There was no evidence of defects in karyotype analysis.(2). Haplotype analysis indicated that disease gene lay between D11S915and D11S4101in Shandong family, lay between D11S1755and D11S935in Henan family.(3) The candidate gene is PAX6. Sequencing of PAX6gene showed a heterozygous deletion A→/in intron2in the Shandong family, that may lead to a mistake when mRNA splicing, but we can’t find any abnormal change in the Henan family.2.(1). PAX6shRNA recombinant plasmid vector was successfully constructed. pDsRed2-Nl-PAX6fusion protein expression system was successfully successfully constructed. Exogenous expression model of the PAX6in293T cells was constructed. Exogenous screen target results show that the1st. the2nd and No.4target sequence is effective interference target point.(2). PAX6shRNA lentiviral vector was constructed successfully. Endogenous screen target results are consistent with exogenous screening results.3. PAX6silencing in B3cells lead that the number of apoptotic cells abnormal increase.Conclusions:1. A deletion mutation occurred (IVS2-2delA) in the PAX6second intron in Shandong family, resulting in the family appeared congenital aniridia and congenital cataract phenotype.2. In Henan aniridia family, the disease gene lay between D11S935and D11S1755, but has not found the specific gene.3. Find three effective RNAi target sequence.4. Construct PAX6RNAi plasmid vector and lentiviral vector.5. PAX6silence caused excessive apoptosis of B3cells.更多还原