U73122和SQ22536对视网膜色素上皮细胞增生及分泌TGF-β_2的影响  

Effects of U73122 and SQ22536 on proliferation and TGF-β_2 secretion of RPE cells in vitro

作　　者：张未娟[1] 赵少贞[1] 邓志宏 

机构地区：[1]天津医科大学眼科中心,300070

出　　处：《眼科研究》2010年第6期519-523,共5页Chinese Ophthalmic Research

摘　　要：目的观察磷脂酶C(PLC)抑制剂U73122和腺苷酸环化酶(AC)抑制剂SQ22536对豚鼠视网膜色素上皮(RPE)细胞增生及分泌转化生长因子-β2(TGF-β2)的影响。方法胰蛋白酶消化法培养原代豚鼠RPE细胞,角蛋白免疫组织化学染色鉴定为阳性后,传2代细胞用于实验。实验组加入含不同浓度U73122或SQ22536的培养液,设溶剂对照。24h后观察RPE细胞形态,噻唑蓝(MTT)法检测加入不同浓度U73122后RPE细胞的增生情况。酶联免疫吸附实验(ELISA)检测1×10-5mol/LU73122及SQ22536作用2、4、6、8、16h后细胞培养液中TGF-β2的分泌量。结果培养的RPE细胞通过角蛋白染色后呈棕黄色阳性反应。MTT法检测显示,U73122实验组中除5×10-6mol/L浓度组外,其余各浓度组OD值与对照组比较均显著下降,且随着浓度的增加,OD值下降越明显(P<0.05);SQ22536各浓度组与对照组OD值相比差异均无统计学意义(F=1.316,P>0.05)。ELISA结果显示,加入1×10-5mol/LU73122后2h实验组TGF-β2的分泌量与对照组比较,差异无统计学意义(P>0.05),4、6、8、16h后,实验组较对照组及2h组均明显增加(P<0.05);而SQ22536组RPE细胞TGF-β2的分泌量在2、4、6、16h比较差异无统计学意义(P>0.05),8h时分泌量降低。结论 PLC抑制剂U73122能抑制RPE细胞的增生,并可使RPE细胞分泌TGF-β2增加。AC抑制剂SQ22536对RPE细胞的生长和TGF-β2的分泌无明显影响。提示RPE细胞分泌TGF-β2的调控可能与PLC信号转导途径有关。BackgroundPrevious studies highlight the importance of TGF-β2in the development of myopia.It was known that RPE cells is associated with secretion of the TGF-β2.However,how the RPE cells to regulate the secretion of TGF-β2 is unclear.ObjectiveThis study was to evaluate the roles of phospholipase C inhibitor,U73122,and adenylate cyclase inhibitor,SQ22536,on the morphology,proliferation and function of secreting transfer growth factor-β2（TGF-β2）in cultured retinal pigment epithelial cells（RPE）.MethodsRetinal pigment epithelial cells were isolated from the eyes of guinea pig and digested and cultured in the DMEM/F12 medium containing 20% fetal bovine serum.The second generation of RPE cells were identified by keratin and plated into 96-well and 24-well dishes at a density of 1×105/well.The medium was shifted into DMEM/F12 medium without fetal bovine serum after cells nearly grew platefully.Different concentrations of U73122 or SQ22536 were added in medium respectively in 24 hours after the first medium change.The same dose of DMSO or ddH2O was used in control group.After another 24 hours,the proliferation of RPE cells was evaluated by using MTT assay.The TGF-β2 secreted by RPE cells was detected using ELISA method at 2,4,6,8,16 hours after the addition of 1×10-5 M U73122 or SQ22536.ResultsCultured cells showed the positive response for keratin.MTT result revealed that the OD value in 5×10-6 mol/L U73122 24 hours group was 0.277±0.020 and that in control group was 0.271±0.005,showing an insignificant difference between them（t=0.771,P0.05）.In 24 hours after action of 1×10-5,1.5×10-5,2×10-5 mol/L U73122,the OD values were 0.214±0.011,0.181±0.012 and 0.136±0.009 respectively,showing statistically significant differences in comparison with control group（t=9.008,t=16.050,t=25.483,P0.01）.In 5×10-6,1×10-5,2×10-5,5×10-5 mol/L SQ22536 groups,the OD values were 0.133±0.011,0.139±0.009,0.134±0.009 and 0.148±0.013 respectively,and no significant differences were seen among various group�

关 键 词：视网膜色素上皮细胞 转化生长因子-Β2 磷脂酶抑制剂 腺苷酸环化酶抑制剂 

分 类 号：R774.1[医药卫生—眼科]