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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 investigate the role of α1,2 fucosyltransferase (HT) gene in overcoming xenotransplantation hyperacute rejection (HAR). Methods pcDNA3-HTcDNA recombinant plasmids were constructed and cultured in vitro. Porcine endothelial cells (PAECs) were cultured in vitro. The pcDNA3-HTcDNA was transfected into PAEC by lipofection method. Resistant cell clones were screened by neomycin (G418) The expression of H-antigen and xenoantigen α-Gal in transfected cells was detected by flow cytometry (FCM). Untransfected PAEC and human umbilical vein endothelial cells (HUVEC) were used as controls respectively. The transfected cells and normal cells were incubated with 20%, 40% and 60% human serum for 2 h, compared with the percentage of lysate cells. Results The recombinant plasmids were transfected into PAEC and the resistant cell clones were screened. The human HT gene fragment was amplified by PCR. The normal and transfection PAECs were detected by FCM. The average fluorescence intensity of α-Gal decreased from 1346.81 to 194.44. The H antigen The average fluorescence intensity increased from 9.10 to 215.93, the α-Gal M2 region decreased from 99.98% to 37.180A, and the H antigen M2 region increased from 13.80% to 78.54%. The percent of cells in the transfected cells after incubation with human serum was lower than that of the normal cells by (32.32 ± 2.37)%, (59.54 ± 4.56)% and (71.46 ± 5.94)%, respectively % To (15.78 ± 2.69)%, (30.16 ± 1.46)% and (40.48 ± 3.77)%, respectively, with statistical significance (P <0.05) ). Conclusion The expression of PAECα-Gal in transfected HT gene is obviously decreased, the expression of H antigen is increased, and the tolerance to human serum is enhanced. Translocation of HT gene can inhibit HAR to a certain extent.