摘要
目的研究重组腺相关病毒(rAAV)能否介导报告基因(Lacz)转移至大鼠的角膜内皮细胞;以及眼内炎症反应能否增强报告基因在大鼠角膜内皮细胞的表达。方法20只SD大鼠前房注射rAAV-Lacz(109Pfu),28d后,随机抽取10只SD大鼠为实验组,玻璃体腔注射脂多糖溶液(LPS)诱发眼内炎症;对照组(10只)玻璃体腔注射PBS溶液。2d后,分别取两组大鼠的角膜行5-溴-4-氯-3-吲哚-β-D-半乳糖苷(X-gal)组织化学染色,评价报告基因的表达情况。结果X-gal组织化学染色证实,经前房注射,报告基因可以在角膜内皮细胞表达,对照组表达效率为5.12%±0.03%;玻璃体腔注射LPS溶液2d后,眼内出现明显的炎症反应,此时报告基因在角膜内皮细胞的表达效率为46.03%±2.12%,较对照组明显增强,差异有显著统计学意义(P<0.01)。结论rAAV载体能有效携载目的基因至角膜内皮细胞,炎症可增强rAAV载体介导的报告基因在角膜内皮的表达。
Objective Recombinant adeno-associate virus vector （rAAV） is unpathogenic and replication-defect virus vector, and its potable gene can be expressed in multiple tissue. This study was designed to investigate the expression of rAAV-mediated report gene in corneal endothelium. Methods rAAV-Lacz （10^9Pfu） was injected in the ocular anterior chamber of 20 Sprague-Dawley（SD） rats. The transient ocular anterior segment inflammation was induced in 10 of 20 SD rats by an intravitreal injection of lipopolysaccharide （LPS） 28 days after rAAV-Lacz injection. PBS was injected in 10 control rats. Two days later, the Lacz gene expression in corneal endothelial cells was evaluated by 5-bromo-4-chloro-3-indolyl-β-D-galactosid （X-gal） histochemical staining. Corneal spaceman was prepared for histological observation to assess the influence of rAAV on endothelial cell function. Results The corneal endothelial cell density was （2 272±87.1）/mm^2 in 2 days and （2 382±111.3）/mm^2 in 14 days after rAAV-Lacz injection without significant difference compared with before injection（2 370±94.5/mm^2, P 〉 0.05）. The Lacz-positive cells from corneal endothelium were stained into blue, showing a 5.12%±0.03% expressing rate in control group 30 days after rAAV injection, Inflammatory reaction peaked at 2 days after intravitreal injection of LPS, at the same time, Lacz-positive cells in corneal endothelial cells were 46.03%±2.12%, showing a prominent Lacz gene expression. There is a statistically significant difference in Lacz gene expression between two groups （P 〈 0.01）. Conclusion The rAAV vector can deliver genes into corneal endothelial cells, and the transgene expression is dramatically induced by inflammation.
出处
《眼科研究》
CAS
CSCD
北大核心
2006年第2期169-171,共3页
Chinese Ophthalmic Research
基金
湖北省科技攻关计划项目资助(2002AA301C38)
关键词
重组腺相关病毒
角膜内皮
报告基因
recombinant adeno-associated virus （rAAV）
corneal endothelium
report gene
