分子遗传学分析-海洋生物学专业论文.docx

PAGE PAGE VI 摘要 此外，还发现 5 例患者分别在第 8 和 12 外显子上均有两个位点的同义单核苷酸 多态性 （single nucleotide polymorphism, SNP），分别是 c.981GA(V327V)和 c.1626TC (F542F)，而正常成员没有。 3．通过比对包括人类在内的六个物种 TGFβI 同源蛋白的序列发现，新突变 编码的氨基酸所在位置在所有正常野生型蛋白中均一致，表明该氨基酸位点进 化上高度保守。并有可能在蛋白的结构和功能中有重要的作用。 4. 成功构建了野生型/突变型 TGFβI 真核表达载体，并成功转染到正常人角 膜上皮细胞，进行表达水平检测。Western Blot 结果显示，重组质粒转染组有重 组蛋白的表达，而阴性对照组和空质粒转染组无 TGFβI 的表达。以上结果为下 一步对 TGFβI 基因突变致 Avellino 型角膜营养不良的可能分子机制研究打下前 期基础。 5. 在一定浓度范围内，雌激素会刺激内源性 TGFβI 基因在人角膜上皮细胞 中的表达。 结论： 1．研究表明，该遗传性 Avellino 型角膜营养不良家系的疾病发生与位于人 5 号染色体 5q31 上的 TGFβI 基因的杂合错义突变（c.371GA）有关。 2．c.371GA 突变所在的密码子编码的氨基酸在 TGFBI 蛋白的进化上高度 保守，并有可能在蛋白的结构和功能中发挥着重要作用。 3．该家系的分子遗传学研究进一步证明 Avellino 型角膜营养不良的遗传异 质性，即同一基因的同一位点突变会导致临床表型的明显差异。 4．无法检测到 TGFβI 基因在人角膜上皮细胞的表达情况，可能其表达水平 较低或不表达。 5. 该家系女性患者表型较重可能与雌激素有关。 关键词：Avellino 型角膜营养不良；杂合突变；转化生长因子 β 诱导基因；遗传 AB ABSTRACT ABSTRACT Purpose: To characterize the clinical features of a Chinese family with Avellino corneal dystrophies（ACD）from Hubei Province, to screen the causative gene for the disease and to explore the molecular mechanisms underlying the disease. To analyze phenotype-genotype correlations on the ACD from the molecular genetic point of view, and provide the basis for prevention and treatment of the disease in the future. Methods: Eighteen people from a Chinese family of three generations were included in this study. All members underwent complete ophthalmic and systemic clinical examination, to rule out the possibility of systemic disease and syndrome, to determine the clinical phenotype. Molecular genetic analysis was performed on all subjects included in the study. Genomic DNA was extracted from peripheral blood collected from all affected and unaffected participants. All exons of human TGFβI gene were amplified by polymerase chain reaction （PCR）, sequenced and compared with reference sequence. Evolutional conservation of both mutation sites were analyzed by homologous sequence alignment among ten species. The potential damage of the missense mutation on the encoded