ILK在氧诱导视网膜病变小鼠模型视网膜组织中的表达

背景 研究表明,整合素连接激酶( ILK)在新生血管形成过程中发挥重要作用,目前ILK在其他组织器官新生血管形成过程中的机制已有报道,但少有其与视网膜新生血管形成关系的研究. 目的 探讨ILK在氧诱导视网膜病变(OIR)小鼠模型视网膜新生血管形成和退化过程中的表达及其意义.方法 选用7日龄健康清洁级C57BL/6J小鼠128只,按随机数字表法分为正常对照组和OIR模型组.将小鼠与哺乳母鼠共同置于体积分数(75±2)％氧的玻璃氧箱内饲养5d后转移至正常环境中饲养5d,建立小鼠OIR模型,正常对照组在正常氧环境中饲养21d.取OIR模型组和正常对照组出生后第17天小鼠各5只制备视网膜切片,进行苏木精-伊红染色,检测每张切片中突破视网膜内界膜的血管内皮细胞核数目.取出生后第12、14、17、21天OIR模型组和正常对照组小鼠各4只分别制备视网膜切片和视网膜铺片,应用ADP酶组织化学法动态观察视网膜新生血管形成和退化过程；采用免疫组织化学法、实时定量聚合酶链反应( real-time PCR)法和Western blot法检测ILK蛋白及其mRNA在小鼠视网膜中的表达情况. 结果 OIR模型组突破视网膜内界膜的血管内皮细胞核计数为(45.64±12.17)个,正常对照组为(0.35±0.14)个,两组间差异有统计学意义(t=22.85,P＜0.05).视网膜铺片结果显示,OIR模型组小鼠第14天视网膜新生血管开始出现,与同龄正常对照组小鼠视网膜血管比较,出生后17d小鼠新生血管形成和血管走行异常等达到高峰,至21d时新生血管开始退化.免疫组织化学检测显示,ILK主要表达于视网膜神经节细胞层、内核层、内丛状层和光感受器层.Real-time PCR和Western blot结果表明,OIR模型组第12、14、17、21天ILK mRNA在视网膜中的表达水平为(1.00±0.22)、(1.85±0.17)、(1.58±0.43)、(1.53±0.36),呈先升高后下降的趋势；在第12、14、17、21天与正常对照组比较小鼠OIR模型组ILK/3-actin吸光度值均高于正常对照组,差异均有统计学意义(t=2.97,P＜0.05;t=11.88,P＜0.01；t=16.84,P＜0.01；t=13.00,P＜0.01). 结论 ILK的表达水平与视网膜新生血管的形成存在时空对应关系,ILK的高表达可能与视网膜新生血管形成密切相关.

Expression of integrin linked kinase in retina with oxygen-induced retinopathy

Background It has been well-known that integrin linked kinase（ILK） plays an important role in the pathogenesis of ncorascularization. But there are few researches to elucidate the relationship between ILK and retinal nevascularization. Objective This study was to explore the expression and significance of ILK on retinal neovascularization and new vessels regression in the oxygen-induced retinopathy（OIR） mouse model. Methods One hundred and twenty-eight 7-day-old C57BL/6J mice were randomly divided into the normal group and model group. The mice were rose in（75±2）% O2 environment with mother mice together for 5 days and then in normal environment for 5 days to establish the OIR models. The mice in normal group were rose in the normal environment for 21 days. The 17-day-old mice were sacrificed and retinal sections were prepared for histopathological examination and the numbers of the cellular nuclei of vascular endothelium breaking retinal internal limiting membrane were calculated. Retinal sections and sheeting were prepared in 12,14,17 and 21-day-old mice to examine the formation and regression of new blood vessel using ADP histochemistry staining,and then immunochemistry,real-time PCR and Western-blotting were used to detect the expressions of ILK and its mRNA in retina. Results The numbers of the cellular nuclei of vascular endothelium breaking retinal internal limiting membrane were （45.64±12.17 ） in OIR group, and those of the normal group were（0.35±0. 14）with a significant difference between two groups （t = 22.85, P〈0.05）. Retinal new blood vessel appeared in 14-day mice,and peaked in 17-day mice and then regression in 21-day mice. ILK protein was expressed mainly in retinal ganglion cell layer, inner nuclear layer, inner plexiform layer and photoreceptor layer. Real-time PCR showed that ILK mRNA expressing levels in retina in model group were（ 1.00±0. 22）, （ 1.85±0. 17 ）, （ 1.58±0.43 ） and （ 1.53 ±0.36 ） respectively in 12-, 14-, 17- and 21-day mice. Western- blotting determined that the A value of the ILK/β-actin was increased in 12-, 14-, 17-day mice and decreased in 21- day mice,and the A values were significantly higher in model group than the control group in various aged mice （ t = 2.97,P〈0. 05;t=11.88,P〈0.01;t=16.84,P〈0.01;t=13.00,P〈0.01）. Conclusions These results indicate a space-time eorresponding relation between the expression of ILK and retinal neovascularization. The obvious positive expression of ILK may be highly correlated with retinal neovascularization.