先天性核性粉塵樣伴后極性白內(nèi)障相關(guān)侯選基因定位及功能研究
發(fā)布時(shí)間：2019-06-09 11:39
【摘要】： 研究目的： 先天性白內(nèi)障是兒童盲的首要原因。大約1／3的先天性白內(nèi)障由遺傳突變引起,其中最常見(jiàn)的是常染色體顯性遺傳。目前我國(guó)進(jìn)行了大量先天性白內(nèi)障疾病相關(guān)候選基因及其突變方式的研究,研究成果豐富了疾病相關(guān)基因庫(kù)。而有關(guān)突變基因功能的研究尚有待開(kāi)展。本研究擬對(duì)一先天性核性粉塵樣伴后極性白內(nèi)障大家系進(jìn)行疾病相關(guān)候選基因的定位及功能研究。在進(jìn)行遺傳方式分析后,提取家系成員血DNA并克隆疾病相關(guān)侯選基因,通過(guò)DNA測(cè)序明確突變基因及位點(diǎn)。通過(guò)DNA體外重組技術(shù)構(gòu)建攜帶突變基因的真核表達(dá)質(zhì)粒,轉(zhuǎn)染真核細(xì)胞后,在體外水平研究基因突變引起的生物學(xué)效應(yīng),闡明其引起先天性白內(nèi)障的分子機(jī)制。 研究方法： 家系全體成員經(jīng)眼部及全身檢查后,根據(jù)系譜行遺傳方式分析。白內(nèi)障手術(shù)中將先證者晶狀體用灌吸方式吸出,使用透射電鏡觀察晶狀體纖維細(xì)胞結(jié)構(gòu)。提取家系全體成員靜脈血并抽提基因組DNA,使用聚合酶鏈反應(yīng)(PCR)克隆核性白內(nèi)障相關(guān)基因,經(jīng)DNA測(cè)序明確突變基因及位點(diǎn)后,利用高效液相色譜分析(DHPLC)鑒定基因突變。以正常人血基因組DNA為模板,通過(guò)PCR反應(yīng)獲取野生型目的基因并通過(guò)DNA重組技術(shù)克隆至真核表達(dá)載體pEGFPN1。利用定點(diǎn)突變技術(shù)構(gòu)建攜帶突變基因的質(zhì)粒,分別轉(zhuǎn)染至人晶狀體上皮細(xì)胞(HLEB-3)及宮頸癌上皮細(xì)胞(HeLa),激光共聚焦顯微鏡下觀察蛋白定位；免疫熒光技術(shù)檢測(cè)晶狀體上皮細(xì)胞中Connexin43 (Cx43)的表達(dá)分布及縫隙連接形成情況；使用G418抗生素篩選,建立穩(wěn)定表達(dá)突變蛋白的細(xì)胞系；流式細(xì)胞分析術(shù)檢測(cè)轉(zhuǎn)染細(xì)胞的凋亡情況；Western-blot法檢測(cè)凋亡相關(guān)蛋白caspase3的表達(dá)。 研究結(jié)果： 該家系為常染色體顯性遺傳,所有患者晶狀體均表現(xiàn)為特殊的核性粉塵樣伴后極性渾濁。透射電鏡觀察發(fā)現(xiàn),先證者晶狀體纖維細(xì)胞中存在小球樣物質(zhì)沉積。DNA測(cè)序示GJA3基因第2號(hào)外顯子第5個(gè)堿基發(fā)生了G→A的置換(c.5GA),該突變使得GJA3編碼的Connexin46(Cx46)蛋白N端第二個(gè)氨基酸發(fā)生甘氨酸到天冬酰胺的轉(zhuǎn)變(p.G2N)。高效液相色譜分析(DHPLC)驗(yàn)證了該基因突變的存在。分別成功構(gòu)建表達(dá)野生型Cx46蛋白的質(zhì)粒pEGFPN1-wtCx46及表達(dá)突變蛋白的pEGFPN1-Cx46G2N,轉(zhuǎn)染HeLa及HLEB-3細(xì)胞后發(fā)現(xiàn)突變蛋白呈團(tuán)塊樣聚集,這種蛋白的聚集導(dǎo)致縫隙連接形成失敗,并影響了Cx43形成縫隙連接的能力。G418篩選培養(yǎng)一周后,突變基因轉(zhuǎn)染細(xì)胞全部死亡,提示該突變具有致死性。流式細(xì)胞分析證實(shí)了細(xì)胞凋亡比例增多,Western-blot發(fā)現(xiàn)總caspase3表達(dá)降低,而剪切的caspase3增多,提示了細(xì)胞凋亡通路的激活。結(jié)論： 本研究首次發(fā)現(xiàn)GJA3基因N端的一個(gè)新的先天性白內(nèi)障疾病相關(guān)點(diǎn)突變G2N。該突變可以導(dǎo)致蛋白的聚集、縫隙連接形成失敗,進(jìn)而引起細(xì)胞凋亡。本研究拓展了先天性白內(nèi)障的基因突變譜,揭示了縫隙連接蛋白突變導(dǎo)致先天性白內(nèi)障發(fā)病的新機(jī)制。同時(shí)提示Cx46蛋白N端在維持晶狀體正常生理狀態(tài)中擔(dān)當(dāng)重要作用,為先天性白內(nèi)障的預(yù)防和基因治療提供了理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R779.66
本文編號(hào)：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R779.66
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相關(guān)期刊論文 前2條
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2 申屠形超,姚克,孫朝暉,徐雯;特殊表型遺傳性先天性白內(nèi)障的超微結(jié)構(gòu)和基因定位[J];中華眼科雜志;2004年05期
，本文編號(hào)：2495520
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