刀豆蛋白A条件化培养基对体外培养的兔角膜内皮细胞生长的影响

目的研究刀豆蛋白A(concanavalin A,ConA)条件化培养基对体外培养的兔角膜内皮细胞(rabbit corneal endothelial cells,RCECs)生长的影响。方法采用揭取后弹力层及内皮细胞联合组织块法进行RCECs的体外原代培养,分别用含有体积分数为5%、10%、15%、20%ConA的活化大鼠脾细胞条件上清液的RPMI1640培养基进行RCECs原代培养,并用含10%(体积分数)胎牛血清(fetal bovine serum,FBS)的RPMI1640培养基作为对照。神经烯醇化酶免疫组织化学染色法进行细胞鉴定;噻唑蓝比色法观察各组RCECs生长的状况;SPSS11.0One-way ANOVA及LSD检验进行统计分析。结果成功进行了RCECs的体外培养,培养细胞内神经烯醇化酶阳性表达。ConA条件化培养基各组细胞增殖均高于对照组(P<0.001),且10%、15%ConA条件化培养基组的RCECs细胞数量明显高于其他各实验组(P<0.001)。结论ConA条件化培养基具有促进RCECs生长的作用,可作为培养辅佐剂。

Effect of conditioned medium with concanavalin A-activated spleen cells supernatant on cultured rabbit corneal endothelial cells

Objective To evaluate the effect of conditioned medium made up of ConA-activated spleen cells supernatant on cultured rabbit corneal endothelial cells (RCECs).Methods RCECs were primarily cultured in vitro by revealing descemet membrane and endothelium combining tissues masses.Culture medium was RPMI 1640 with 10% FBS and varied concentrations of conA-activated spleen cells supernatant.Experimental groups were divided by different concentrations of ConA-conditioned supernatant:5%,10%,15%,and 20%.In the control group only RPMI1640 and 10% FBS were used.Methyl thiazolyl tetrazolium colorimetry analysis was used to evaluate the proliferation of RCECs cellular population.The cultured cells were identified by immunohistochemical staining for neuronal specific enolase (NSE).Results The RCECs were cultured successfully.RCECs presented positive expression in cytoplasm with NSE immunohistochemical assay.There were significant differences in the experimental groups compared with the control group (P<0.001).The effects were optimal in 15% and 20% groups.Conclusion Conditioned medium with ConA-activated spleen cells supernatant can enhance proliferation of cultured RCECs,and thus can be used as adjutant in RCECs culture.