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目的 研究晶状体特异性表达的制瘤素 (oncostatin M ,OSM)对眼发育的影响。 方法 将去除部分序列的小鼠 OSM c DNA [6 6 1碱基对 (base pair,bp) ]连接到αA-晶状体蛋白 (αA- crystallin)启动子上 ,然后用显微注射的方法将其导入单细胞胚胎内 ,建立转基因鼠。常规组织学及免疫组织化学方法对转基因鼠进行特性鉴定 ,末端脱氧核苷酸转移酶介导的脱氧尿嘧啶末端标记 (terminal deoxynucleotidyltransferase- mediated d UTP nick- end labelling,TUNEL )试验检测细胞凋亡 ,原位杂交检测 caspase- 3m RNA的表达 ,兔抗活化的 caspase- 3抗体检测活化的 caspase- 3蛋白质。 结果 胚胎期 14 .5、17.5 d,转基因蛋白 OSM特异性表达于晶状体纤维细胞内 ,从胚胎期 17.5 d开始 ,转基因鼠视网膜开始发生变性 ,出生时 ,5 0 %以上的视网膜细胞丢失。TUNEL试验显示转基因鼠视网膜细胞凋亡。转基因鼠视网膜细胞中caspase- 3被激活。 结论 晶状体特异性的 OSM表达激活 caspase- 3,导致了眼的异常发育、细胞凋亡及广泛的视网膜变性。
Objective To study the effects of lens-specific oncostatin M (OSM) on ocular development. Methods Mouse OSM cDNA with partial sequence deletion (6 6 1 base pair (bp)] was ligated to the αA-crystallin promoter and then introduced by microinjection Single-cell embryos, the establishment of transgenic mice. The histological and immunohistochemical methods were used to characterize the transgenic mice and detect the apoptosis by terminal deoxynucleotidyltransferase-mediated d UTP nick-end labeling (TUNEL) The expression of caspase-3 mRNA was detected by in situ hybridization, and the activated caspase-3 protein was detected by rabbit anti-activated caspase-3 antibody. Results At embryonic stage 14.5 and 17.5 d, OSM was expressed specifically in lens fibroblasts. From the embryonic day 17.5 d, the retinas of transgenic mice began to degenerate. At birth, more than 50% of retinal cells were lost. TUNEL assay showed retinal cell apoptosis in transgenic mice. Transgenic mouse retinal cells caspase-3 is activated. Conclusion Lens-specific OSM expression activates caspase-3, leading to abnormal eye development, apoptosis and extensive retinal degeneration.