CAJ | 학술논문

目的使用慢病毒介导的RNA干扰(RNAi)方法抑制小鼠血管内皮细胞异种抗原半乳糖α1,3-半乳糖(Galα1,3-Gal)的表达.方法经体外设计合成针对α1,3-半乳糖基转移酶(α1,3-GT)mRNA的序列特异性小发夹RNA(shRNA),并构建携带α1,3-GT shRNA的重组慢病毒载体,以慢病毒感染小鼠血管瘤内皮细胞系EOMA细胞.采用荧光实时定量聚合酶链反应检测转染后的EOMA细胞α1,3-GT mRNA表达水平的变化;免疫荧光法和流式细胞术检测转染后的EOMA细胞异种抗原Galα1,3-Gal表达水平的变化.并将转染后的EOMA细胞与人血清混合培养,用四甲基偶氮唑盐比色法测定细胞存活率.结果成功获得携带α1,3-GT shRNA的成熟重组慢病毒颗粒,转染EOMA细胞效率可达75%.与空白对照组、空转染组以及阴性siRNA对照组比较,重组慢病毒转染EOMA细胞后,能有效抑制α1,3-GT的表达,抑制率为88%(P＜0.05);Galα1,3-Gal抗原表达水平明显降低(P＜0.05);而空白对照组、空转染组和阴性siRNA对照组间的差异无统计学意义(P＞0.05).重组慢病毒转染后的EOMA细胞与人血清混合培养后的存活率较各对照组明显提高(P＜0.05).结论成功构建靶向α1,3-GT基因的重组慢病毒载体,通过RNAi沉默α1,3-GT基因,抑制EOMA细胞异种抗原Galα1,3-Gal的表达,在体外实验中可减轻异种超急性排斥反应.
목적 사용만병독개도적RNA간우(RNAi)방법억제소서혈관내피세포이충항원반유당α1,3-반유당(Galα1,3-Gal)적표체.방법 경체외설계합성침대α1,3-반유당기전이매(α1,3-GT)mRNA적서렬특이성소발협RNA(shRNA),병구건휴대α1,3-GT shRNA적중조만병독재체,이만병독감염소서혈관류내피세포계EOMA세포.채용형광실시정량취합매련반응검측전염후적EOMA세포α1,3-GT mRNA표체수평적변화;면역형광법화류식세포술검측전염후적EOMA세포이충항원Galα1,3-Gal표체수평적변화.병장전염후적EOMA세포여인혈청혼합배양,용사갑기우담서염비색법측정세포존활솔.결과 성공획득휴대α1,3-GT shRNA적성숙중조만병독과립,전염EOMA세포효솔가체75%.여공백대조조、공전염조이급음성siRNA대조조비교,중조만병독전염EOMA세포후,능유효억제α1,3-GT적표체,억제솔위88%(P＜0.05);Galα1,3-Gal항원표체수평명현강저(P＜0.05);이공백대조조、공전염조화음성siRNA대조조간적차이무통계학의의(P＞0.05).중조만병독전염후적EOMA세포여인혈청혼합배양후적존활솔교각대조조명현제고(P＜0.05).결론 성공구건파향α1,3-GT기인적중조만병독재체,통과RNAi침묵α1,3-GT기인,억제EOMA세포이충항원Galα1,3-Gal적표체,재체외실험중가감경이충초급성배척반응.
Objective To investigate the feasibility of inhibiting Galα (1,3)-Gal expression in mouse vascular endothelial cells by lentivirus-mediated RNAi.Methods The shRNA specified to α1,3-GT mRNA was designed and synthesized in vitro and cloned into the lentivirus vector.EOMA cells were infected by recombinant lentivirus.Real-time RT-PCR was used to detect mRNA transcriptional levels of αl,3-GT as well as immunofluorescence and flow cytometry were applied to detect Galα(1,3)-Gal antigen level after gene transfection.Co-culture of infected EOMA and serum of human was done and the survival rate was measured by MTT.Results The αl,3-GT shRNA sequences were cloned into the recombinant lentivirus vector correctly and the lentivirus was produced successfully.The transfection efficiency to EOMA was 75 %.Real-time PCR revealed that the mRNA transcription of α1,3-GT was obviously inhibited by α1,3-GT shRNA recombinant lentivirus with the rate of 88 % (P＜0.05),while there were no obvious differences among control group,no shRNA lentivirus group and negative-shRNA lentivirus group (P＞ 0.05).Immunofluorescence and flow cytometry demonstrated the same results that Galα(1,3)-Gal antigen expression in EOMA transfected by α1,3-GT shRNA lentivirus was less than that of control group,no shRNA lentivirus group and negative-shRNA lentivirus group (P＜0.05),but there were no obvious differences among the later three groups (P＞0.05).After co-culture with serum of human,MTT showed the survival rate of EOMA infected by α1,3-GT shRNA lentivirus was obviously increased (P＜ 0.05).Conclusion Recombinant α2,3-GT shRNA 1entivirus is constructed successfully,which can inhibit the expression of α1,3-GT and Galα1,3-Gal in EOMA by RNAi and control hyperacute rejection in vitro.