CCR3拮抗剂对视网膜血管内皮细胞管腔形成的影响及机制  

Effects and mechanism of CCR3 antagonist on tube formation of retinal vascular endothelial cells

作　　者：赫雪飞[1] 刘高勤[1] 陈志刚[2] 周文娟[2] 肖艳辉[1] 陆培荣[1] 

机构地区：[1]苏州大学附属第一医院眼科,江苏省苏州市215006 [2]苏州大学研究生院医学部,江苏省苏州市215006

出　　处：《眼科新进展》2013年第12期1106-1109,1114,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金面上项目(编号:81200727;30972712);江苏省医学重点人才项目(编号:RC2011104)~~

摘　　要：目的探讨CCR3拮抗剂对人视网膜血管内皮细胞(human retinal capillary endothelial cell,HREC)管腔形成能力的影响以及具体机制。方法常规培养HREC细胞系并收集细胞,应用RT-PCR和Western blot检测CCR3在HREC中的表达。按CCR3拮抗剂浓度分为对照组(0 ng·mL-1)和CCR3拮抗剂组(500 ng·mL-1),应用基质胶Matrigel体外三维成型法检测不同浓度CCR3拮抗剂对HREC管腔形成的影响。CCK-8增殖法检测不同浓度CCR3拮抗剂对HREC细胞增殖的影响。RT-PCR和Western blot检测不同浓度CCR3拮抗剂对HREC内VEGF和一氧化氮合酶(nitricoxide synthase,NOS)表达的影响。结果两组HREC在基因水平和蛋白水平均表达CCR3分子。CCR3拮抗剂组HREC管腔形成明显受到抑制:对照组(60.27±6.68)个/视野,CCR3拮抗剂组(31.55±6.81)个/视野,差异有统计学意义(P=0.004)。进一步检测结果发现CCR3拮抗剂抑制HREC的增殖,在0 ng·mL-1、20 ng·mL-1、50 ng·mL-1、100 ng·mL-1、200 ng·mL-1浓度下的增殖抑制率分别为0.10±0.03、0.16±0.08、0.19±0.12、0.87±0.28、1.45±0.31,并呈浓度依赖性,在CCR3拮抗剂浓度为100 ng·mL-1和200 ng·mL-1时,其增殖抑制率与对照组相比差异均有统计学意义(P=0.02、0.00)。RT-PCR和Western blot检测结果显示:CCR3拮抗剂干预后,HREC内VEGF的表达未出现明显变化(P mRNA=0.727、P蛋白=0.793);两组NOS的基因表达(对照组0.89±0.23,CCR3拮抗剂组0.46±0.11)和蛋白表达(对照组0.64±0.12,CCR3拮抗剂组0.35±0.10)差异均有统计学意义(P mRNA=0.048、P蛋白=0.032)。结论 CCR3拮抗剂对HREC的管腔形成有抑制作用,其机制可能通过抑制细胞增殖以及抑制NOS的表达等途径发挥效应。Objective To discuss the effects and mechanism of CCR3 antagonist on tube formation of human retinal vascular endothelial cells(HREC).Methods HREC cell line was normally cultured and harvested for total RNA or protein extraction.CCR3 gene and protein expression in HREC were examined by RT-PCR and Western blot.The cells were divided into control group(0 ng·mL^(-1))and CCR3 antagonist group(500 ng·mL^(-1)). The effects of CCR3 antagonist with different concentrations on tube formation of HREC were detected by three-dimensional matrigel assay.The effects of CCR3 antagonist with different concentrations on HREC proliferation was detected by CCK-8 assay.The effects of CCR3 antagonist with different concentrations on expression of VEGF and NOS in HREC were examined by RT-PCR and Western blot.Results Both CCR3 gene and protein were expressed on HREC of two groups.Tube formation of HREC was markedly inhibited by CCR3 antagonist of CCR3 antagonist group(control group:60.27±6.68 per visual field, CCR3 antagonist group:31.55±6.81 per visual field),there was statistical difference between two groups(P=0.004).The proliferation of HREC was also inhibited by CCR3 antagonist in a dosage-dependent manner.The inhibiting rate with different concentrations of CCR3-antagonist(0 ng·mL^(-1),20 ng·mL^(-1),50 ng·mL^(-1),100 ng·mL^(-1),200 ng·mL^(-1))were 0.10±0.03,0.16±0.08,0.19±0.12,0.87±0.28,1.45±0. 31,respectively.Compared with control group,there were statistical differences in groups of 100 ng·mL^(-1)and 200 ng·mL^(-1)CCR3 antagonist(P=0.02,0.00). RT-PCR and Western blot detection showed that there was no statistical difference on VEGF gene and protein expression between control group and CCR3 antagonist group(P_(mRNA)=0.727,P_(protein)=0.793).But there was significant decrease of NOS gene(control group:0.89±0.23,CCR3 antagonist group:0.46±0.11)and protein expression(control group:0.64±0.12,CCR3 antagonist group:0.35±0.10)in CCR3 antagonist group(P_(mRNA)=0.048,P_(protein)=0.032).Conclusion The inhibitive effects

关 键 词：CCR3拮抗剂 视网膜血管内皮细胞 新生血管 

分 类 号：R96[医药卫生—药理学]