本文選題：斑狀角膜營養(yǎng)不良 + CHST6基因�。� 參考：《大連醫(yī)科大學》2011年碩士論文

【摘要】：目的:斑狀角膜營養(yǎng)不良(Macular Corneal Dystrophy,MCD)是一種少見的角膜基質層營養(yǎng)不良,屬于常染色體隱性遺傳病。Groenouw于1980年首次闡述,故又稱Groenouw II型角膜營養(yǎng)不良。臨床表現(xiàn)為進行性視力喪失、畏光和眼表不適感等,裂隙燈檢查可見雙眼進行性角膜基質彌漫霧狀混濁,間(或)有局灶性斑片狀白色混濁,由中央向周邊及深層發(fā)展,最終需要進行角膜移植手術。2000年Akama等[1]首次發(fā)現(xiàn)斑狀角膜營養(yǎng)不良的致病基因為一種新的碳水化合物磺基轉移酶基因即CHST6基因,并將其定位于染色體16q22上。CHST6基因編碼角膜N-乙酰葡萄糖胺-6-磺基轉移酶(corneal N-acetylglucosamine-6-sulphotransferase,GlcNAc6ST),這種酶把硫酸根離子傳遞給N-乙酰葡萄糖胺,該物質參與角膜中硫酸角質素蛋白多糖的生物合成。CHST6基因突變導致這種酶活性的降低或消失,使角膜中硫酸角質素(keratan sulphate,KS)等粘多糖合成異常,異常物質沉積于角膜,破壞角膜細胞及纖維結構,最終導致角膜混濁[2]。自Akama后眾多學者相繼在其所在國家的MCD患者CHST6基因中發(fā)現(xiàn)了突變,我國對斑狀角膜營養(yǎng)不良也有較多研究,但是多數(shù)僅限于病例報告及組織病理學研究,較少有關于基因突變研究的報告。本實驗對我國東北地區(qū)一斑狀角膜營養(yǎng)不良家系三個陽性體征患者進行研究:抽取外周靜脈血提取DNA并進行CHST6基因序列正反雙向測序;對穿透性角膜移植術后的患者角膜進行組織病理學檢查,進一步探討斑狀角膜營養(yǎng)不良與CHST6基因突變的關系及研究其組織病理學改變。 方法:選取我國東北地區(qū)一斑狀角膜營養(yǎng)不良家系中已發(fā)現(xiàn)的同代三名患者,其中男性2人,女性1人。分別抽取外周靜脈血2ml,提取基因組DNA。CHST6基因共有四個外顯子,編碼區(qū)位于第三個外顯子上,應用聚合酶鏈反應(PCR)對CHST6基因的編碼區(qū)進行基因擴增,然后進行基因測序。分別取兩名患者角膜移植術后的1/2片病變角膜行4%甲醛溶液固定,石蠟包埋、切片,進行阿辛藍(AB)和阿辛藍-過碘酸雪氏(AB-PAS)染色,于光學顯微鏡下進行觀察;對另1/2角膜片行2.5%戊二醛溶液固定,環(huán)氧樹脂包埋,染色后進行透射電鏡觀察并攝像。 結果:對該家系三名患者的CHST6基因編碼區(qū)進行基因正反雙向測序,發(fā)現(xiàn)的共同突變點為:第三外顯子第62個堿基插入堿基G,原第62個堿基T→A,造成開放閱讀框的改變;AB染色陽性:可見角膜基質中細胞質及彈性纖維藍色著染;AB-PAS染色中可見角膜上皮變薄,上皮下大片的紅藍著染的斑塊狀物質;透射電鏡觀察到角膜上皮下可見大量圓形物質。 結論:本研究證實了CHST6編碼區(qū)的基因突變c.61_62insG是導致這一斑狀角膜營養(yǎng)不良家系角膜混濁的直接原因,迄今為止該突變尚未被發(fā)現(xiàn)。
[Abstract]:Objective: macular Corneal dystrophy (Macular Corneal dystrophy) is a rare corneal stromal dystrophy. It is an autosomal recessive hereditary disease. Groenouw was first described in 1980, so it is also called Groenouw II corneal dystrophy. The clinical manifestations were progressive loss of vision, photophobia and ocular surface discomfort. Slit-lamp examination showed diffuse haze opacity of the corneal stroma in both eyes, focal flaky white opacity in the middle and / or local areas, which developed from the center to the peripheral and deep layers. In 2000, Akama et al first found a novel carbohydrate sulfotransferase gene (CHST6 gene), which is a novel carbohydrate sulfotransferase gene. The CHST6 gene was located on the chromosome 16q22 and encoded the keratoacetal N-acetylglucosamine-6-sulphotransferase (GlcNAc6STN), which transfered sulfate ion to N-acetylglucosamine-6-sulphotransferase (N-acetylglucosamine-6-sulphotransferase). This substance involved in the biosynthesis of keratin sulfate proteoglycan. CHST6 gene mutation resulted in the decrease or disappearance of the enzyme activity, the abnormal synthesis of mucopolysaccharide such as keratan sulphate KSin in the cornea, and the deposition of abnormal substances in the cornea. Destruction of corneal cells and fiber structure, eventually leading to corneal opacity [2]. Since Akama, many scholars have found mutations in the CHST6 gene of MCD patients in their country. In China, there are many studies on corneal speckle dystrophy, but most of them are limited to case reports and histopathological studies. There are few reports on gene mutation. In this study, we studied three patients with positive signs in a pedigree with corneal dystrophy in northeast China: DNA was extracted from peripheral venous blood and CHST6 gene sequence was sequenced in both positive and negative directions. Histopathological examination was performed on cornea after penetrating keratoplasty to investigate the relationship between speckle corneal dystrophy and mutation of CHST6 gene and its histopathological changes. Methods: three patients of the same generation were selected from a pedigree with corneal dystrophy in Northeast China, including 2 males and 1 female. There were four exons of genomic DNA.CHST6 gene extracted from peripheral venous blood. The coding region was located on the third exon. The coding region of CHST6 gene was amplified by polymerase chain reaction (PCR) and then sequenced. After keratoplasty, 1 / 2 of the lesions were fixed with 4% formaldehyde solution, embedded in paraffin wax, sectioned, stained with ABA and AB-PASs, and observed under optical microscope. Another 1 / 2 cornea was immobilized in 2.5% glutaraldehyde solution, embedded with epoxy resin, then stained with transmission electron microscope and photographed. Results: the CHST6 gene coding region of three patients from this family was sequenced in both directions. The common mutation sites were found as follows: the 62nd base of exon 3 was inserted into base Gand the original 62 base of T _ (2) A, which caused the change of open reading frame to be positive for AB staining. The results showed that the cytoplasm and elastic fibers of corneal stroma were blue stained with AB-PAS. In the staining, the corneal epithelium became thinner, A large area of red and blue stained plaques; a large number of circular substances were observed under transmission electron microscope. Conclusion: this study confirmed that c.61_62insG, a gene mutation in the CHST6 coding region, was the direct cause of corneal opacity in this pedigree with corneal dystrophy. So far, this mutation has not been found.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R772.2

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