转基因鼠晶状体特异性表达的制瘤素对眼发育的影响

The effects of lens-specific overexpression of OSM on the eye development in the transgenic mice

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 determine the effects of lensspecific overexpression of OSM on the eye development. Methods A truncated mouse OSM cDNA (661 bp) was linked to the αA-crystallin promoter. Transgenic mice were characterized by routine histological and immunohistochemical techiniques. TUNEL assays were used to detect cell death. The mRNA expression of caspase-3 was detected by in situ hybridization, Rabbit anti-cleavage caspase-3 antibody was used to detect active capase-3. Results At embryonic day (E) 14.5 and 17.5, expression of the OSM transgenic protein was detected specifically in lens fiber cells. The onset of retinal degeneration in the mid portion of the transgenic retinae was observed started from E17.5. By the time of birth 50% or more of the retinal cells were missing. The OSM transgenic retinal cells underwent apoptosis indicated by TUNEL assays. Most strikingly, activation of caspase-3 protein were observed throughout the transgenic retinas. Conclusions Lens-specific overexpression of OSM activate caspase-3, leading to abnormal eye development, apoptosis and widespread retinal degeneration.