先天性核性粉尘样伴后极性白内障相关侯选基因定位及功能研究
发布时间：2019-06-09 11:39
【摘要】： 研究目的： 先天性白内障是儿童盲的首要原因。大约1／3的先天性白内障由遗传突变引起,其中最常见的是常染色体显性遗传。目前我国进行了大量先天性白内障疾病相关候选基因及其突变方式的研究,研究成果丰富了疾病相关基因库。而有关突变基因功能的研究尚有待开展。本研究拟对一先天性核性粉尘样伴后极性白内障大家系进行疾病相关候选基因的定位及功能研究。在进行遗传方式分析后,提取家系成员血DNA并克隆疾病相关侯选基因,通过DNA测序明确突变基因及位点。通过DNA体外重组技术构建携带突变基因的真核表达质粒,转染真核细胞后,在体外水平研究基因突变引起的生物学效应,阐明其引起先天性白内障的分子机制。 研究方法： 家系全体成员经眼部及全身检查后,根据系谱行遗传方式分析。白内障手术中将先证者晶状体用灌吸方式吸出,使用透射电镜观察晶状体纤维细胞结构。提取家系全体成员静脉血并抽提基因组DNA,使用聚合酶链反应(PCR)克隆核性白内障相关基因,经DNA测序明确突变基因及位点后,利用高效液相色谱分析(DHPLC)鉴定基因突变。以正常人血基因组DNA为模板,通过PCR反应获取野生型目的基因并通过DNA重组技术克隆至真核表达载体pEGFPN1。利用定点突变技术构建携带突变基因的质粒,分别转染至人晶状体上皮细胞(HLEB-3)及宫颈癌上皮细胞(HeLa),激光共聚焦显微镜下观察蛋白定位；免疫荧光技术检测晶状体上皮细胞中Connexin43 (Cx43)的表达分布及缝隙连接形成情况；使用G418抗生素筛选,建立稳定表达突变蛋白的细胞系；流式细胞分析术检测转染细胞的凋亡情况；Western-blot法检测凋亡相关蛋白caspase3的表达。 研究结果： 该家系为常染色体显性遗传,所有患者晶状体均表现为特殊的核性粉尘样伴后极性浑浊。透射电镜观察发现,先证者晶状体纤维细胞中存在小球样物质沉积。DNA测序示GJA3基因第2号外显子第5个碱基发生了G→A的置换(c.5GA),该突变使得GJA3编码的Connexin46(Cx46)蛋白N端第二个氨基酸发生甘氨酸到天冬酰胺的转变(p.G2N)。高效液相色谱分析(DHPLC)验证了该基因突变的存在。分别成功构建表达野生型Cx46蛋白的质粒pEGFPN1-wtCx46及表达突变蛋白的pEGFPN1-Cx46G2N,转染HeLa及HLEB-3细胞后发现突变蛋白呈团块样聚集,这种蛋白的聚集导致缝隙连接形成失败,并影响了Cx43形成缝隙连接的能力。G418筛选培养一周后,突变基因转染细胞全部死亡,提示该突变具有致死性。流式细胞分析证实了细胞凋亡比例增多,Western-blot发现总caspase3表达降低,而剪切的caspase3增多,提示了细胞凋亡通路的激活。结论： 本研究首次发现GJA3基因N端的一个新的先天性白内障疾病相关点突变G2N。该突变可以导致蛋白的聚集、缝隙连接形成失败,进而引起细胞凋亡。本研究拓展了先天性白内障的基因突变谱,揭示了缝隙连接蛋白突变导致先天性白内障发病的新机制。同时提示Cx46蛋白N端在维持晶状体正常生理状态中担当重要作用,为先天性白内障的预防和基因治疗提供了理论基础。
[Abstract]:The purpose of the study: Congenital cataract is the first of the children's blindness For the reason, about 1/3 of the congenital cataract is caused by a genetic mutation, the most common is the autosomal dominant Sex inheritance. At present, a large number of congenital cataract disease-related candidate genes and their mutation methods have been studied, and the research results are rich in the disease. The gene bank. The research on the function of the mutant gene is still To be carried out, a congenital nuclear dust sample with post-polar cataract is proposed to be used for the positioning and work of disease-related candidate genes. Can be studied. After the genetic analysis is carried out, the blood DNA of a family member is extracted and a disease-related candidate gene is cloned, and the mutant gene is clearly identified by DNA sequencing. And after transfection of the eukaryotic cells, the biological effect caused by the gene mutation in the in vitro level is detected, and the distribution of the congenital cataract is clarified. A sub-mechanism. Methods: All members of the family were examined by eye and eye. According to the inheritance pattern of the line spectrum line, the lens of the first witness in the cataract operation was aspirated by the suction method, and the transmission electron microscope was used. and observing the structure of the lens, extracting the venous blood of all the members of the family and extracting the genomic DNA, cloning the nuclear cataract-related genes by using a polymerase chain reaction (PCR), Identification of a gene mutation by HPLC. The wild-type target gene was obtained by PCR reaction using human blood genomic DNA as a template and cloned into a eukaryotic cell by a DNA recombination technique. the expression vector pEGFPN1 is constructed by using a fixed-point mutation technique to construct a plasmid carrying the mutant gene, and the plasmid is respectively transfected into the human lens epithelial cell (HLEB-3) and the cervical cancer epithelial cell (HeLa), and the laser is copolymerized The expression profile of connexin 43 (Cx43) in the lens epithelial cells and the formation of the gap junction were detected by immunofluorescent technique. The cell line of the stable expression of the mutant protein was established by using the G418 antibiotic. The flow cytometry was used to analyze the expression of connexin 43 (Cx43). The apoptosis of transfected cells was detected by Western-blot. caspa the expression of se3: the family is autosomal dominant, and all the patients are crystalline The body was characterized by a special nuclear dust sample with the following polarity turbidity. The transmission electron microscope (TEM) observed that first, DNA sequencing showed that the fifth base of exon 2 of the GJA3 gene had the substitution of G-A (c. 5GA), which resulted in the second amino acid in the N-terminal of the Connexin46 (Cx46) protein encoded by GJA3. Transformation of the raw glycine to the dearylamine (p. G2N). High performance liquid chromatography The gene mutation was verified by DHPLC. pEGFPN1-wtCx46 and pEGFPN1-Cx46G2N expressing the wild-type Cx46 protein and pEGFPN1-Cx46G2N expressing the mutant protein were successfully constructed. The loss and the ability to form a gap junction with Cx43. After one week of screening by G418, the mutant gene The cell apoptosis was confirmed by flow cytometry, and the expression of total caspase3 was reduced by Western-blot. (e) ase The results showed that GJA3 gene was found for the first time in this study. A new congenital cataract disease-related point mutation G2N at the N end. The mutation can result in The aggregation of the protein, the formation of the gap junction, and the apoptosis of the cells. This study expanded the gene mutation spectrum of the congenital cataract, and revealed that It is suggested that the N-terminal of Cx46 can play an important role in maintaining the normal physiological state of the lens.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R779.66
本文编号：2495520
[Abstract]:The purpose of the study: Congenital cataract is the first of the children's blindness For the reason, about 1/3 of the congenital cataract is caused by a genetic mutation, the most common is the autosomal dominant Sex inheritance. At present, a large number of congenital cataract disease-related candidate genes and their mutation methods have been studied, and the research results are rich in the disease. The gene bank. The research on the function of the mutant gene is still To be carried out, a congenital nuclear dust sample with post-polar cataract is proposed to be used for the positioning and work of disease-related candidate genes. Can be studied. After the genetic analysis is carried out, the blood DNA of a family member is extracted and a disease-related candidate gene is cloned, and the mutant gene is clearly identified by DNA sequencing. And after transfection of the eukaryotic cells, the biological effect caused by the gene mutation in the in vitro level is detected, and the distribution of the congenital cataract is clarified. A sub-mechanism. Methods: All members of the family were examined by eye and eye. According to the inheritance pattern of the line spectrum line, the lens of the first witness in the cataract operation was aspirated by the suction method, and the transmission electron microscope was used. and observing the structure of the lens, extracting the venous blood of all the members of the family and extracting the genomic DNA, cloning the nuclear cataract-related genes by using a polymerase chain reaction (PCR), Identification of a gene mutation by HPLC. The wild-type target gene was obtained by PCR reaction using human blood genomic DNA as a template and cloned into a eukaryotic cell by a DNA recombination technique. the expression vector pEGFPN1 is constructed by using a fixed-point mutation technique to construct a plasmid carrying the mutant gene, and the plasmid is respectively transfected into the human lens epithelial cell (HLEB-3) and the cervical cancer epithelial cell (HeLa), and the laser is copolymerized The expression profile of connexin 43 (Cx43) in the lens epithelial cells and the formation of the gap junction were detected by immunofluorescent technique. The cell line of the stable expression of the mutant protein was established by using the G418 antibiotic. The flow cytometry was used to analyze the expression of connexin 43 (Cx43). The apoptosis of transfected cells was detected by Western-blot. caspa the expression of se3: the family is autosomal dominant, and all the patients are crystalline The body was characterized by a special nuclear dust sample with the following polarity turbidity. The transmission electron microscope (TEM) observed that first, DNA sequencing showed that the fifth base of exon 2 of the GJA3 gene had the substitution of G-A (c. 5GA), which resulted in the second amino acid in the N-terminal of the Connexin46 (Cx46) protein encoded by GJA3. Transformation of the raw glycine to the dearylamine (p. G2N). High performance liquid chromatography The gene mutation was verified by DHPLC. pEGFPN1-wtCx46 and pEGFPN1-Cx46G2N expressing the wild-type Cx46 protein and pEGFPN1-Cx46G2N expressing the mutant protein were successfully constructed. The loss and the ability to form a gap junction with Cx43. After one week of screening by G418, the mutant gene The cell apoptosis was confirmed by flow cytometry, and the expression of total caspase3 was reduced by Western-blot. (e) ase The results showed that GJA3 gene was found for the first time in this study. A new congenital cataract disease-related point mutation G2N at the N end. The mutation can result in The aggregation of the protein, the formation of the gap junction, and the apoptosis of the cells. This study expanded the gene mutation spectrum of the congenital cataract, and revealed that It is suggested that the N-terminal of Cx46 can play an important role in maintaining the normal physiological state of the lens.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R779.66
【参考文献】
相关期刊论文 前2条
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2 申屠形超,姚克,孙朝晖,徐雯;特殊表型遗传性先天性白内障的超微结构和基因定位[J];中华眼科杂志;2004年05期
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