炎症增强报告基因在角膜内皮的表达

Adeno-associated Virus Vector-delivered Report Gene Expression in Corneal Endothelium

目的研究脂多糖(LPS)诱发的眼内炎症反应能否增强腺相关病毒介导的报告基因在角膜内皮的表达。方法20只SD大鼠前房注射rAAVLacz(1010个病毒颗粒/ml),28d后,取10只大鼠的玻璃体腔注射脂多糖溶液诱发眼内炎症,另10只玻璃体腔注射PBS作为对照,2d后,取角膜行5溴4氯3吲哚βD半乳糖苷(Xgal)组织化学染色,评价报告基因的表达情况;Westernblot检测核因子κB(NFκB)蛋白的表达。结果Xgal组织化学染色证实,报告基因可以在角膜内皮、角膜基质和虹膜细胞表达,玻璃体腔注射LPS2d后,眼内炎症反应达高峰,此时角膜内皮细胞表现出明显的Lacz基因的表达;Westernblot亦证实,角膜组织NFκB蛋白表达与炎症反应成正比。结论rAAV载体能有效携载目的基因到角膜内皮细胞,并且目的基因的表达在炎症情况下明显增强。

Objective To investigate whether recombinant adeno-associated virus (rAAV) vector-mediated report gene expression in corneal endothelium and trans gene expression is induced by inflammation in vivo.Methods Twenty Sprague-Dawley (SD) rats were divided into two groups. The ocular anterior chamber of SD rats was injected with rAAV expressing Escbericbin coli Lacz (rAAV-Lacz 10^(10)Pfu). Twenty-eight days after injection, transient ocular anterior segment inflammation in 10 SD rats was induced by an intravitreal injection of lipopolysaccharide (LPS). The rats in another group were injected with PBS as control. Two days later, the Lacz gene expression in corneal endothelial cells and iris cells were detected by 5-bromo-4-chloro-3-indolyl-β-D-galactosid (X-gal) histochemical staining, and the expression of NF-κB in inflammatory cornea by Western blot.Results Report gene could be expressed in corneal endothelium, stromal, bowman’s membrane and iris cells. Inflammatory reaction peaked at 2 days after LPS treatment and at the same time, most of the endothelial cells showed prominent Lacz gene expression. There was a positive correlation between the expression levels of NF-κB and inflammation in cornea.Conclusion The rAAV vector can deliver genes into corneal endothelial cells and iris cells, and the transgene expression is dramatically induced by inflammation.