本文選題：先天性無虹膜癥 + PAX6基因��； 參考：《華中科技大學》2014年博士論文

【摘要】：目的通過對中國一先天性無虹膜癥(congenital aniridia)家系進行PAX6基因突變檢測及其臨床特點的分析,尋找突變位點并探討其致病機制。 方法對該家系中5名患者和2名表型正常的家系成員進行臨床資料收集,繪制遺傳家系圖,分析其發(fā)病特點。采集該家系5名患者和2名表型正常的家系成員及100名正常對照者的外周靜脈血,提取基因組DNA,針對PAX6基因的14個外顯子設計合成引物,進行聚合酶鏈式反應后,擴增產(chǎn)物采用Sanger雙脫氧鏈末段終止法測序。對每個外顯子,將患者的測序結(jié)果、表型正常的家系成員測序結(jié)果與正常對照者的測序結(jié)果進行比對,尋找突變位點。借助生物信息學技術(shù)對突變基因進行結(jié)構(gòu)和功能的分析。 結(jié)果1.該家系中5名患者均表現(xiàn)為無虹膜,并不同程度的上瞼下垂、眼球震顫,伴或不伴角膜混濁、晶狀體混濁,提示家系內(nèi)臨床表型具有多樣性。隨年齡增加癥狀加重。其遺傳特點為垂直連續(xù)傳遞,與性別無關,遺傳方式符合常染色體顯性遺傳。2.在該家系5名患者中均檢測到PAX6基因第7外顯子雜合c.393-396delTAGC(p.Ser132LysfsX14)突變,表型正常的家系成員及正常對照者中均未檢測到該突變。該突變可導致開放讀碼框中編碼提前終止,編碼的蛋白質(zhì)中富谷氨酸-甘氨酸連接域、高度保守的同源DNA結(jié)合域和富含脯氨酸／絲氨酸/蘇氨酸的區(qū)域缺失,PAX6基因單倍劑量不足,最終導致眼發(fā)育異常。 結(jié)論本研究對中國一先天性無虹膜癥家系進行了臨床表型分析,發(fā)現(xiàn)了家系中某些癥狀的嚴重程度呈遺傳性的現(xiàn)象并分析了可能的原因；發(fā)現(xiàn)了家系中的雜合突變c.393-396delTAGC(p.Ser132LysfsX14),為該家系的遺傳咨詢和產(chǎn)前診斷奠定了基礎。該突變國內(nèi)外尚未見報道,本研究結(jié)果亦拓展了中國人群PAX6基因突變譜。 目的建立人皮膚成纖維細胞原代培養(yǎng)、傳代及純化的方法,并對其進行鑒定,為之后的實驗提供足夠純度和數(shù)量的人皮膚成纖維細胞。 方法采用組織塊貼壁法體外培養(yǎng)人皮膚成纖維細胞,用胰蛋白酶消化法進行傳代,傳代時用差別消化和貼壁法進行純化,倒置顯微鏡下觀察其形態(tài)及生長狀況；運用間接免疫熒光技術(shù)對純化后的成纖維細胞進行波形蛋白免疫熒光染色,熒光顯微鏡下觀察染色情況并進行陽性細胞計數(shù)。 結(jié)果1.組織塊貼壁法體外培養(yǎng)人皮膚成纖維細胞,約一周后開始有成纖維細胞自組織塊邊緣爬出,向外生長。成纖維細胞貼壁生長,呈長梭形、扁平星形。隨培養(yǎng)天數(shù)增加,細胞數(shù)量增多,逐漸形成漩渦狀、柵欄狀排列,第四周左右細胞可爬滿25cm2細胞培養(yǎng)瓶瓶底。2.原代培養(yǎng)時鏡下可見梭形的成纖維細胞和扁平多邊形的角質(zhì)細胞貼壁共存。傳代時通過差別消化和貼壁法純化,傳代后成纖維細胞純度高,鏡下不可見角質(zhì)細胞。3用兔抗人波形蛋白一抗和結(jié)合FITC的驢抗兔二抗及DAPI對傳代后的成纖維細胞進行免疫熒光染色,可見藍色橢圓形的細胞核與綠色長梭形的細胞質(zhì)一一對應,成纖維細胞純度98%。 結(jié)論組織塊貼壁法可成功進行人皮膚成纖維細胞的原代培養(yǎng),傳代時用差別消化和貼壁法可對成纖維細胞進行有效純化,滿足下一步實驗要求。
[Abstract]:Objective To investigate the mutation detection and clinical characteristics of PAX6 in a family of congenital aniridia in China , to find mutation sites and to explore its pathogenic mechanism .

Methods Five patients and two normal family members of the family were collected by clinical data , and their characteristics were analyzed . Five patients and two normal family members of the family and 100 normal controls were collected from peripheral venous blood . Genomic DNA was extracted . The results of sequencing were compared with the sequencing results of normal controls . The results of sequencing were compared with those of normal controls .

Results 1 . Five patients in the family showed no iris , and different degrees of ptosis , eyeball tremor , accompanied with or without corneal opacity , lens opacity , suggesting that the clinical phenotype in the family was varied . The mutation could result in the deletion of the exon 7 of PAX6 gene in the open reading frame .

Conclusion This study has performed a clinical phenotype analysis of a family of congenital non - iridosia in China , and found that the severity of some symptoms in the family is hereditary and the possible causes are analyzed .
The heterozygous mutation c . 393 - 396delTAGC ( p . Ser132 Lys fsX14 ) was found in the family , which laid the foundation for the genetic counseling and prenatal diagnosis of the family . The mutation at home and abroad has not been reported at home and abroad . The results of this study have also extended the gene mutation spectrum of PAX6 in Chinese population .

Objective To establish a method for primary culture , passage and purification of human skin fibroblasts , and to identify them and provide sufficient purity and quantity of human skin fibroblasts for subsequent experiments .

Methods Human skin fibroblasts were cultured in vitro by tissue patch wall method . The morphology and growth of human skin fibroblasts were observed by trypsin digestion , and the morphology and growth of human skin fibroblasts were observed by differential digestion and adherent method .
The purified fibroblasts were stained with the indirect immunofluorescence technique , and the staining was observed under the fluorescence microscope and the positive cells were counted .

Results 1 . Human skin fibroblasts were cultured in vitro by tissue patch wall method . After about one week , the fibroblasts were grown from the edge of the tissue mass .

Conclusion The tissue patch wall method can be used for primary culture of human skin fibroblasts , and the fibroblasts can be effectively purified by differential digestion and adherent method when passaging , which can meet the next experimental requirements .
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R773.1

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