CAJ | 학술논문

目的通过RNA干涉沉默猪内皮细胞内α-1,3-半乳糖转移酶(α-1,3GT)基因,观察基因沉默对猪内皮细胞免疫和生理功能的影响.方法提取原代猪主动脉内皮细胞,并通过SV40LT基因转染构建永生化猪主动脉内皮细胞系.用含猪α-1,3GT干涉基因的慢病毒载体转染细胞,并检测α-1,3GT和α-1,3-半乳糖抗原(α-1,3Gal)的表达情况.将转染后细胞与正常人血清共培养,建立异种移植免疫排斥反应的细胞模型并通过四甲基偶氮唑盐、流式细胞术、免疫组织化学及免疫荧光技术检测基因沉默对内皮细胞免疫和生理功能的影响.结果永生化前后,细胞的免疫和生理功能无明显变化.RNA干涉后,永生化细胞表达α-1,3GT及α-1,3Gal明显减少.与人血清共培养后,转染组细胞增殖速度较高,凋亡率较低,与人免疫球蛋白IgM、IgG及人补体C3和C5b-9结合较对照组及错配组减少.结论通过沉默α-1,3GT基因可降低猪内皮细胞与人免疫球蛋白、补体的结合程度.该基因沉默后的内皮细胞可作为异种移植急性排斥反应细胞模型和研究平台.
목적 통과RNA간섭침묵저내피세포내α-1,3-반유당전이매(α-1,3GT)기인,관찰기인침묵대저내피세포면역화생리공능적영향.방법 제취원대저주동맥내피세포,병통과SV40LT기인전염구건영생화저주동맥내피세포계.용함저α-1,3GT간섭기인적만병독재체전염세포,병검측α-1,3GT화α-1,3-반유당항원(α-1,3Gal)적표체정황.장전염후세포여정상인혈청공배양,건립이충이식면역배척반응적세포모형병통과사갑기우담서염、류식세포술、면역조직화학급면역형광기술검측기인침묵대내피세포면역화생리공능적영향.결과 영생화전후,세포적면역화생리공능무명현변화.RNA간섭후,영생화세포표체α-1,3GT급α-1,3Gal명현감소.여인혈청공배양후,전염조세포증식속도교고,조망솔교저,여인면역구단백IgM、IgG급인보체C3화C5b-9결합교대조조급착배조감소.결론 통과침묵α-1,3GT기인가강저저내피세포여인면역구단백、보체적결합정도.해기인침묵후적내피세포가작위이충이식급성배척반응세포모형화연구평태.
Objective To investigate the effect of α-1,3-galactosyltransferase (α-1,3GT)silencing through RNA interference on the immunological and the physiological functions of porcine endothelial cells.Methods The primary porcine aortic endothelial cells were isolated and immortalized through transfection of SV40LT.The lentivirus carrying the α-1,3GT shRNA was established and transfected into the cells.The expression of α-1,3GT and α-1,3Gal after transfection was detected.The transfected cells were co-cultured with human serum to establish cell model of xenotransplantation.The effects of α-1,3GT) silencing through RNA interference on the immunological and the physiological functions of porcine endothelial cells were observed by using MTT,flow cytometry,immunohistochemistry and immunofluorescence.Results The physiological function of endothelial cells showed no significant difference before and after immortalization,but the expression of α-1,3GT and Gal was decreased greatly after RNA interference.After co-culture,transfected group cells showed higher proliferation rate but lower apoptosis rate,and their binding capability to human IgM,IgG C3 and C5b-9 was decreased as compared with mismatch and control groups.Conclusion Down-regulation of the α-1,3GT could reduce the binding of porcine endothelial cells to human immunoglobulin and complement.The endothelial cells with a-1,3GT silencing by RNA interference can be used for the study on immunology of xenotransplantation.