P2X_7受体在RGC-5的表达及有效下调P2X_7基因的siRNA序列筛选  

Expression of P2X_7 receptor in RGC-5 cell line and selection of an effective siRNA sequence down-regulating P2X_7 receptor

作　　者：黄永霞 方敏[2] 侯飞[2] 胡慧玲[2] Huang Yongxia;Fang Min;Hou Fei;Hu Huiling(Guangdong-Hongkong-Macao Institute of CNS Regeneration, Jinan University, Guangzhou 510632, China;Shenzhen Eye Hospital (Jinan University Affiliated Eye Hospital), Shenzhen 518040, China)

机构地区：[1]暨南大学粤港澳中枢神经再生研究院,广州510632 [2]深圳市眼科医院(暨南大学附属深圳眼科医院),深圳518040

出　　处：《中国医师杂志》2018年第12期1782-1786,共5页Journal of Chinese Physician

基　　金：广东省自然科学基金项目(2014A030310081);深圳医疗卫生三名工程(SZSM201812091)~~

摘　　要：目的检测嘌呤受体P2X_7信使RNA(mRNA)及其蛋白在RGC-5细胞系的表达。设计合成针对P2X_7受体的小型干扰RNA(siRNA),并筛选出其中能高效抑制其表达的序列。方法采用RT-PCR、Western blot及免疫荧光检测P2X_7受体mRNA及蛋白在RGC-5细胞系中的表达情况,并与小鼠视网膜及其他组织进行比较。设计针对P2X_7受体的3对干扰序列(P2X_7-siRNA-1～3),使用ribo FECTTMCP转染RGC-5细胞,60 h后检测细胞中P2X_7受体mRNA及蛋白表达变化。结果RGC-5细胞系与小鼠视网膜组织均能检测到P2X_7受体mRNA及蛋白的表达,且该受体主要表达于RGC-5的细胞核和细胞质。P2X_7-siRNA-3可同时有效下调P2X_7的mRNA和蛋白的表达。结论RGC-5可作为体外研究P2X_7受体作用的细胞模型。成功筛选出的针对P2X_7受体基因的siRNA将为后续下调该受体、减轻青光眼性视神经病变的基因治疗奠定基础。Objective This study aims to investigate the gene expression of P2X7 receptor in retinal ganglion cell line (RGC-5), and to design and select the small interfering RNA (siRNA) which can specifically and effectively downregulate P2X7 receptor expression.Methods The mRNA and protein expressions of P2X7 receptor in RGC-5 cells were examined by real-time polymerase chain reaction (qRT-PCR), Western blot and Immunofluorescence, and compared with those of mouse retina and other tissues. Three P2X7 receptor-specific double stranded siRNAs (P2X7-siRNA-1-3) were designed and delivered into RGC-5 cells by riboFECT?CP. 60 hours after transfection, the expression level of P2X7 receptor was analyzed by qRT-PCR, Western blot and Immunofluorescence.Results The expression of P2X7 receptor was detected in both RGC-5 cell line and mouse retina, and the receptor was mainly expressed in the nucleus and cytoplasm of RGC-5 cells. P2X7-siRNA-3 can effectively down-regulate the mRNA and protein expression of P2X7 at the same time.Conclusions RGC-5 cell line can serve as a good model for studying the biological function of P2X7 receptor in retinal ganglion cells in vitro. We have managed to design and synthesize the siRNA pair that specifically and effectively lowering P2X7 receptor expression. The current study has laid the foundation for future investigations on treating glaucomatous retinopathy by downregulation of P2X7 receptor.

关 键 词：受体 嘌呤能P2X7 视网膜神经节细胞 RNA 小分子干扰 体外研究 

分 类 号：R775[医药卫生—眼科]