目的:研究与先天性白内障相关的连接蛋白Cx50 S258F突变体对斑马鱼晶状体发育的影响.方法:将野生型和携带S258F突变的Cx50编码区分别构建到pcDNA3.1 myc/his真核表达载体上,通过体外转录获得相应的RNA产物,将RNA产物分别显微注射到1细胞期的斑马鱼胚胎,通过显微镜观察斑马鱼晶状体表型,并利用全胚原位杂交技术分析过表达野生型和突变型Cx50对晶状体标志基因Cx23表达的影响.结果:与野生型胚胎相比,S258F突变型Cx50过表达引起斑马鱼胚胎白内障表型,过表达野生型Cx50也引起白内障表型,但是畸形数量明显少于突变型,野生型和突变型Cx50的过表达均下调了Cx23的表达.结论:连接蛋白Cx50 S258F突变体可引起斑马鱼白内障表型,提示Cx50的S258F突变体是造成先天性白内障的原因.%Objective; To explore the effect of missense mutation S258F of connexin 50 (Cx50) associated with autosomal dominant congenital cataract on zebrafish lens development. Methods; The fragment encoding wild - type or mutant - type was subcloned into pcDNA3. lmyc - His. The 5'capped mRNA of both wild - type and mutant was synthesized from pcDNA3. lmyc - His plasmids. A total of SO - lOOpg of RNA was injected into one - cell stage embryos. Living embryos were observed under a microscope. Whole - mount in situ hybridization on embryos were performed to detect the expression of lens marker Cx23. Results: Compared with wild -type embryos, overexpression of CxSO S2S8F led to cataract formation in zebrafish lens. Although overexpressing wild -type CxSO displayed similar phenotype, the percent of defect phenotype decreased significantly. Conclusion: Overexpressing Cx50 S258F leads to cataract formation in zebrafish lens, suggesting that the missense mutation S2S8F could be causative for congenital cataract formation.
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