靶向慢病毒载体RNA干扰小鼠血管内皮细胞异种抗原的研究  

Lentivirus-mediated RNAi against xenoantigen in mouse vascular endothelial cells

作　　者：戚峰[1] 谷雅川[1] 吕承育[1] 章志翔[1] 何向辉[1] 朱理玮[1] 

机构地区：[1]天津医科大学总医院普通外科,300052

出　　处：《中华器官移植杂志》2010年第10期621-625,共5页Chinese Journal of Organ Transplantation

基　　金：基金项目：国家自然科学基金（30500489,30940066）

摘　　要：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.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.

关 键 词：异种移植 RNA干扰 慢病毒 超急性排斥反应 

分 类 号：R686[医药卫生—骨科学]