流式细胞术检测供体猪α-1,3-Gal和人源CD46的表达

摘要

Objective To detect the reliability of α-1,3-galactosyl(α-Gal) antigen knockout and the expression of human protein CD46(hCD46) on the surface of genetically modified pigs at the protein level, to provide reliable donors for xenotransplantation. Methods A total of 500 μl blood from wild type (WT), α-1,3-galactosyl transferase knockout (GTKO), hCD46 and GTKO/hCD46 Bama miniature swine were collected for peripheral blood mononuclear cell (PBMC)isolation. Expression of α-Gal antigen was detected by flow cytometry after coincubation with fluorescein isothiocyanate-Griffonia simplicifolia isolectin B4 (FITC-GSIB4) and PBMC. Expression of hCD46 protein was detected by flow cytometry after co-incubation of PBMC with fluorescein isothiocyanate-CD46 membrane protein antibody (anti-CD46-FITC). Results The α-Gal expression on the surface of PBMC were negative in GTKO and GTKO/hCD46 Bama pigs and positive in WT and hCD46 Bama pigs, consistent with the sequencing results. The hCD46 protein was detected positively on the surface of PBMC of hCD46 and GTKO/hCD46 Bama pigs and was negative in WT and GTKO Bama pigs, which was consistent with the results of genotype identification after birth. Conclusion A method for visually detecting the expression of α-Gal antigen and hCD46 protein in donor porcine using microscale blood was established.%目的 检测基因修饰猪细胞表面α-1,3-半乳糖(α-Gal)抗原敲除情况和人源蛋白CD46 (hCD46)的表达情况,为异种器官移植提供可靠的供体.方法 采集500 μl野生型(wild type,WT)、α-1,3-半乳糖基转移酶基因敲除 (GTKO)、hCD46和GTKO/hCD46巴马小型猪血液,分离外周血单核细胞 (peripheral blood mononuclear cell,PBMC).异硫氰酸荧光素标记的植物凝集素(FITC-GSIB4)与PBMC共孵育后采用流式细胞术检测α-Gal抗原敲除情况,异硫氰酸荧光素标记的hCD46膜辅蛋白抗体(anti-CD46-FITC)与PBMC共孵育后采用流式细胞术检测hCD46蛋白表达情况.结果 GTKO和GTKO/hCD46巴马猪PBMC表面α-Gal检测呈阴性,WT和hCD46巴马猪检测呈阳性,与测序结果一致;hCD46和GTKO/hCD46巴马猪PBMC表面hCD46蛋白检测呈阳性,WT和GTKO巴马猪检测呈阴性,与PCR鉴定结果一致.结论 建立了微量血液流式细胞术直观检测供体猪α-Gal抗原和人源CD46蛋白表达的方法.