流式细胞分析测定猪外周血单个核细胞Gal α1,3 Gal水平(英文)

Detection of Gal α1,3 Gal Level in Porcine Peripheral Blood Mononuclear Cells Using Flow Cytometry Analysis

在考虑以猪器官作为供体对人进行异种器官移植时,α1,3半乳糖被认为是引起超急性免疫排斥的主要异种抗原.人们建立了各种方法以降低猪α1,3半乳糖水平,但是也有可能筛选得到在自然情况下α1,3半乳糖表达水平比较低的猪.为了研究在正常猪单个核细胞中α1,3半乳糖浓度分布的差异,利用鸡免疫球蛋白Y(ck-IgY)抗体通过流式细胞分析对正常猪的α1,3半乳糖水平的差异进行检测.取3～8周龄猪的全血,用肝素抗凝处理后经密度梯度离心获取外周血单个核细胞(PBMCs),与FITC标记的ck-IgY(10μg/ml)孵育,经流式细胞仪检测α1,3半乳糖水平.结果显示,相同周龄猪的α1,3半乳糖水平可有0.4～2.6倍差异,同一猪的水平在不同周龄有1.3～5.6倍差异.ck-IgY的特异性由棉籽糖和α1,3半乳二糖测定,棉籽糖(100μmol/L)可抑制70%ck-IgY结合,而α1,3半乳二糖(6.25μmol/L)可完全取消ck-IgY的结合,说明ck-IgY与猪单个核细胞是特异性结合.上述发现说明,ck-IgY是检测猪单个核细胞表面α1,3半乳糖的特异试剂,不同猪或是同一猪在不同时间的α1,3半乳糖水平有着明显的差异.

Gal α 1,3 Gal has been considered as a major antigen to cause hyperacute rejection in porcine to human xenotransplantation.Some strategies have been developed to reduce or eliminate Gal α1,3 Gal in porcines.However,it is also possible to select normal porcines with low level of Gal α1,3 Gal.This study is to investigate possible variations in the levels of Gal α1,3 Gal in normal porcines by widely used flow cytometry analysis with chicken-IgY（ck-IgY） antibody.Porcine peripheral blood mononuclear cells（PBMCs） were isolated from heparinised whole blood of 3～8 week-old porcines and then were incubated with FITC labelled ck-IgY for flow cytometry analysis.The level of Gal α 1,3 Gal was measured from 3 week-old porcines at weekly intervals until 8 week-old.Porcines of the same age were found to display 0.4～2.6 folds variations in the levels of Gal α 1,3 Gal.Individual animals displayed 1.3～5.6 folds variation between samples taken on different weeks.The specificity of ck-IgY was determined by raffinose and α 1,3 galactobiose application.Raffinose inhibited ck-IgY binding up to 70% at a concentration of 100 μmol/L;while α 1,3 galactobiose completely diminished ck-IgY binding at a concentration of 6.25 μmol/L,indicating that ck-IgY binding to porcine PBMCs was specific binding.These findings demonstrate that ck-IgY is a specific reagent to detect Gal α 1,3 Gal on porcine PBMCs.The levels of Gal α 1,3 Gal between individual porcines or in a same individual at different age vary significantly.