Src-家族酪氨酸激酶对皮质性白内障晶状体中缝隙连接蛋白的影响

目的探讨抑制Src-家族酪氨酸激酶的异常激活在皮质性白内障的形成中对晶状体缝隙连接蛋白43（Cx43）的影响。方法分离10d的鸡胚胎全晶状体并在M199培养液中培养10d为对照组;加入0.1nmol/LSFK特异性抑制剂PP1作为PP1组。观察2组晶状体的混浊程度、计算晶状体的混浊面积百分比。对培养1、5、10d的晶状体细胞进行WGA、DAPI免疫荧光染色,观察晶状体组织学改变及Cx43分布和表达量的变化。在晶状体前囊膜上进行Lucifer Yellow染料负荷试验,测量染料弥散距离,评价缝隙连接的功能。结果对照组晶状体的混浊面积百分比在4d、10d分别为26%和50%,而PP1组为20%和14%（P〈0.01）。培养5d、10d对照组晶状体上皮细胞（LECs）出现死亡,Cx43主要在LECs间表达;PP1组LECs死亡明显减少,Cx43的表达在上皮与纤维的交界面明显增强。在培养后1d、10d,对照组染料弥散距离与PP1组比较差异有统计学意义（P〈0.01）。结论在体外培养鸡胚晶状体模型中,PP1可能通过保护Cx43缝隙连接功能以保持晶状体上皮组织结构正常,减缓皮质性白内障的发生。

Effects of Src family kinases on gap junction Cx43 in formation of cortical cataract

Objective This study was designed to investigate the inhibition effects of Src family kinases（SFK） activation on gap junction protein connexin43 （Cx43） in a cataract model . Methods The lenses were isolated from the chicken embryo aged day 10 （E10） and cultured in M199 medium containing 10% fetal bovine serum for 10 days. The PP1 , a specific inhibitor of SFK,was added in medium in the initial culture time. The degree of lenses opacification was quantified using image-analysis software. The morphological changes of cell membranes and cell nuclei were detected by DAPI immunofluorescence staining. The expression and distribution of Cx43 protein in cultured lenses were detected using immunofluorescence staining. Lucifer Yellow dye uptake on scraped anterior capsules of cultured lenses was employed for the functional evaluation of Cx43 gap junction of lens epithelium. Results The opacification area of cultured lenses was obvious different at the 4th and 10th day respectively between control group and PP1-treated group （ P 〈 0. 01, P 〈 0. 01 ）. There were no obvious changes on histomorphology and expression of Cx43 protein in both groups. At day 5 and 10, the lens epithelium was detached from the lens fibers, and the dead ceils were found within or beneath the lens epithelium in control lenses. Cx43 was detected to distribute on the interface between lens epithelial cells. But in PP1-treated lenses,the lens epithelium was attached to fibers of the lens,and the lens epithelial cells combined tightly each other. The dead cells decreased dramatically, and the structure of lens epithelium maintained normal. The expression of Cx43 was detected at the interfaces between lens epithelium and lens fibers and on the anterior capsules. The dye transfer distances in Lucifer Yellow dye uptake analysis in control lenses was 50. 93±7.75,66.67±7.29,99. 52±12.67 μm respectively at day 1,5 and 10, and that in PP1-treated lenses was 89.54 ± 9.60,69.18 ±5.49,130. 63±10.52 μm respectively, showing the significa