转人α1,2岩藻糖苷转移酶基因猪动脉内皮细胞抗人血清溶破实验  

Transgenic expression of human al ,2-fucosyltransferase (HT) in porcine arterial endothelial cells to confer resistance to human serum-mediated cytolysis

作　　者：马志方[1] 李胜芝[1] 刘秉乾[1] 张明[1] 王广有[1] 马腾骧[1] 

机构地区：[1]天津医科大学第二医院泌尿外科天津市泌尿外科研究所

出　　处：《中华泌尿外科杂志》2006年第8期537-540,共4页Chinese Journal of Urology

基　　金：天津市科委科技发展计划重点项目基金资助项目(043803411)

摘　　要：目的研究转入α1,2岩藻糖苷转移酶(HT)基因克服异种移植超急排斥反应(HAR)的作用。方法构建pcDNA3-HTcDNA重组质粒,体外培养猪动脉内皮细胞(PAEC),脂质体转染法将pcDNA3-HTcDNA转染PAEC,新霉素(G418)筛选具有抗性的细胞克隆,PCR检测重组基因的整合,流式细胞术(FCM)检测转染细胞H抗原和异种抗原α—Gal表达。分别以未转染的PAEC和人脐静脉内皮细胞(HUVEC)作对照。将转染细胞和正常细胞分别以20％、40％和60％人血清孵育2 h,比较溶破细胞百分数。结果重组质粒转染PAEC经筛选得到抗性细胞克隆,PCR扩增出人HT基因片段,FCM检测正常PAEC与转染PAEC,α-Gal平均荧光强度由1346．81降至194．44,H抗原平均荧光强度由9．10增至215．93;α—Gal M2区由99．98％降至37．180A,H抗原M2区由13．80％升至78．54％。转染细胞以人血清孵育后溶破细胞百分数较正常细胞降低,分别由(32．32±2．37)％、(59．54±4．56)％和(71．46±5．94)％降至(15．78±2．69)％、(30．16±1．46)％和(40．48±3．77)％,差异均有统计学意义(P值均<0．05)。结论转人HT基因PAECα—Gal表达明显降低,H抗原表达升高,对人血清溶破的耐受性增强。转人HT基因可以一定程度抑制HAR。Objective To study the role of human α1 ,2-fucosyhransferase （HT） transgene in overcoming hyperacute rejection （HAR）. Methods pcDNA3-HTcDNA recombinant plasmid was constructed. Porcine arterial endothelial cells （PAEC） were cultured in vitro. The recombinant plasmid was transfected into PAEC by lipofectinnmine mediated method. Stable transfectants were selected using G418. The integration and expression of transgene was assayed by PCR and flow cytometry （FCM）. Normal PAECs and human umbilical vein endothelial cells （HUVEC） were used as controls. Transgenic cells and normal PAECs were exposed to 20% ,40% and 60% human serum for 2 h,then the percentages of the lyzed cells were compared. Results The transfected cells were selected by G418, The HTcDNA fragment was produced by PCR in survival cells,which expressed lower α-Gal and higher H antigen. The mean fluorescence intensity of α-Gal decreased from 1346.81 to 194.44,while that of H antigen increased from 9.10 to 215.93 by FCM. The M2 gate of α-Gal decreased from 99.98% to 37.18% ,while that of H antigen increased from 13.80% to 78.54%. The percentage of the lyzed cells of transgenic PAEC decreased from （32. 32 ±2. 37）%, （59.54±4.56）% and （71.46 ±5.94）% to （15.78 ±2.69）%,（30. 16 ± 1.46）% and （40.48 ± 3.77） % ,respectively, compared with that of normal PAECs after exposure to human serum （ all P 〈 0.05）. Conclusions Transgenic PAEC expression of human HT α-Gal decreases and H antigen increases,which can resist human serum lysis more efficiently. Human HT transgene can inhibit HAR to some degree.

关 键 词：移植 异种 岩藻糖基转移酶类 

分 类 号：R392[医药卫生—免疫学]