1、中国一先天性无虹膜症家系临床表型分析和PAX6基因突变检测 2、人皮肤成纤维细胞的原代培养、传代、纯化及鉴定

发布时间：2018-06-23 09:47

本文选题：先天性无虹膜症 + PAX6基因　；参考：《华中科技大学》2014年博士论文

【摘要】：目的通过对中国一先天性无虹膜症(congenital aniridia)家系进行PAX6基因突变检测及其临床特点的分析,寻找突变位点并探讨其致病机制。方法对该家系中5名患者和2名表型正常的家系成员进行临床资料收集,绘制遗传家系图,分析其发病特点。采集该家系5名患者和2名表型正常的家系成员及100名正常对照者的外周静脉血,提取基因组DNA,针对PAX6基因的14个外显子设计合成引物,进行聚合酶链式反应后,扩增产物采用Sanger双脱氧链末段终止法测序。对每个外显子,将患者的测序结果、表型正常的家系成员测序结果与正常对照者的测序结果进行比对,寻找突变位点。借助生物信息学技术对突变基因进行结构和功能的分析。结果1.该家系中5名患者均表现为无虹膜,并不同程度的上睑下垂、眼球震颤,伴或不伴角膜混浊、晶状体混浊,提示家系内临床表型具有多样性。随年龄增加症状加重。其遗传特点为垂直连续传递,与性别无关,遗传方式符合常染色体显性遗传。2.在该家系5名患者中均检测到PAX6基因第7外显子杂合c.393-396delTAGC(p.Ser132LysfsX14)突变,表型正常的家系成员及正常对照者中均未检测到该突变。该突变可导致开放读码框中编码提前终止,编码的蛋白质中富谷氨酸-甘氨酸连接域、高度保守的同源DNA结合域和富含脯氨酸／丝氨酸/苏氨酸的区域缺失,PAX6基因单倍剂量不足,最终导致眼发育异常。结论本研究对中国一先天性无虹膜症家系进行了临床表型分析,发现了家系中某些症状的严重程度呈遗传性的现象并分析了可能的原因；发现了家系中的杂合突变c.393-396delTAGC(p.Ser132LysfsX14),为该家系的遗传咨询和产前诊断奠定了基础。该突变国内外尚未见报道,本研究结果亦拓展了中国人群PAX6基因突变谱。目的建立人皮肤成纤维细胞原代培养、传代及纯化的方法,并对其进行鉴定,为之后的实验提供足够纯度和数量的人皮肤成纤维细胞。方法采用组织块贴壁法体外培养人皮肤成纤维细胞,用胰蛋白酶消化法进行传代,传代时用差别消化和贴壁法进行纯化,倒置显微镜下观察其形态及生长状况；运用间接免疫荧光技术对纯化后的成纤维细胞进行波形蛋白免疫荧光染色,荧光显微镜下观察染色情况并进行阳性细胞计数。结果1.组织块贴壁法体外培养人皮肤成纤维细胞,约一周后开始有成纤维细胞自组织块边缘爬出,向外生长。成纤维细胞贴壁生长,呈长梭形、扁平星形。随培养天数增加,细胞数量增多,逐渐形成漩涡状、栅栏状排列,第四周左右细胞可爬满25cm2细胞培养瓶瓶底。2.原代培养时镜下可见梭形的成纤维细胞和扁平多边形的角质细胞贴壁共存。传代时通过差别消化和贴壁法纯化,传代后成纤维细胞纯度高,镜下不可见角质细胞。3用兔抗人波形蛋白一抗和结合FITC的驴抗兔二抗及DAPI对传代后的成纤维细胞进行免疫荧光染色,可见蓝色椭圆形的细胞核与绿色长梭形的细胞质一一对应,成纤维细胞纯度98%。结论组织块贴壁法可成功进行人皮肤成纤维细胞的原代培养,传代时用差别消化和贴壁法可对成纤维细胞进行有效纯化,满足下一步实验要求。
[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 .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R773.1

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