刀豆蛋白A条件化培养基对体外培养兔角膜内皮细胞Na^+-K^+-ATP酶的作用

Distribution and activity of Na^+-K^+-ATPase in cultured rabbit corneal endothelial cells with or without ConA-conditional cultured medium

目的研究刀豆蛋白A(ConA)条件化培养基对体外培养兔角膜内皮细胞膜表面Na+-K+-ATP酶的分布及其酶活性的影响。方法超微量ATP酶试剂盒测定原代培养兔角膜内皮细胞细胞膜表面Na+-K+-ATP酶的活性,并对ConA条件化培养基作用下不同生长周期(5、7、10d)细胞Na+-K+-ATP酶的活性进行测定;免疫酶组织化学电镜下观察不同培养基作用下体外培养角膜内皮细胞膜表面Na+-K+-ATP酶的分布。结果ConA条件化培养基组兔角膜内皮细胞Na+-K+-ATP酶活性明显高于无ConA组(P<0.01),原代细胞生长7～10d时Na+-K+-ATP酶活性最高(P<0.01)。单层兔角膜内皮细胞膜外侧可见Na+-K+-ATP酶阳性反应。在ConA条件化培养基处理组,其阳性反应物较多、致密;而在无ConA组则阳性反应物较少、疏松。结论体外原代培养角膜内皮细胞在7～10d时,Na+-K+-ATP酶活性最高。ConA条件化培养基可提高兔角膜内皮细胞Na+-K+-ATP酶活性。

Objective The activity of Na^＋-K^＋-ATPase on the corneal epithelial cells is an important index in evaluating the metabolism and function of cell.Na^＋-K^＋-ATPase play potential role in maintaining the transparency of cornea.Concanavalin A（ConA）-conditioned medium contain many types of cytokines and can promote the growth of cultured cells.The aim of this paper was to evaluate the distribution and activity of Na^＋-K^＋-ATPase in cultured corneal endothelial cells of rabbit with or without concanavalin A（ConA）-conditioned medium.MethodsThe corneal endothelial sheet and Descemet membrane was isolated from the healthy mature New Zealand albino rabbit eyes under the anatomic microscope and cultured in RPMI1640 medium containing 5,10,15 and 20 mL of ConA（10% ConA-activated spleen cells supernatant）,10 mL of fetal bovine serum（10%） and 60 mL of IMDM using mass culture method.A video system connected to the inverted phase-contrast microscope was used to observe the morphologic change of cultured cells.The activity of Na^＋-K^＋-ATPase on the cultured cells was evaluated with Bradford,trypan blue dyeing and ATPase reagent kit in the 5th,7th and 10th day.The distribution of Na^＋-K^＋-ATPase on primarily cultured corneal endothelial cells was examined under the electron microscopic cytochemistry.ResultsBy tearing corneal endothelial sheet and Descemet membrane,The corneal endothelial cells of rabbit were successfully cultured using mass cultivation.Na＋-K＋-ATPase showed the higher density on the surface of cellular membrane in ConA-conditioned supernatant group.Na＋-K＋-ATPase presented the highest activity in primarily cultured corneal endothelial cells in the 10th day,showing a significant differences in different time points（P〈0.01）.ConclusionActivity of Na^＋-K^＋-ATPase in RCECs is highest at 7th to 10th day.Medium with ConA-conditioned can improve the activity of Na^＋-K^＋-ATPase in RCECs.The activity of Na^＋-K^＋-ATPase can be used to evaluate the function of cultured corneal endothelial cells.