Relation between human decay-accelerating factor (hDAF) expression in pig cells and inhibition of human serum anti-pig cytotoxicity: value of highly expressed hDAF for xenotransplantation
Relation between human decay-accelerating factor (hDAF) expression in pig cells and inhibition of human serum anti-pig cytotoxicity: value of highly expressed hDAF for xenotransplantation
复制标题
DOI：
10.1111/j.1399-3089.2006.00365.x
复制
发表时间：
2007-01-01
影响因子：
3.9
通讯作者：
Nakao, Akimasa
中科院分区：
文献类型：
--
作者：
Liu, DaGe;Kobayashi, Takaaki;Nakao, Akimasa
背景资料：尽管α 1，3-半乳糖基转移酶敲除（GT-KO）猪的成功生产增加了临床异种移植的期望，但仍在探索基因工程猪的其他修饰，因为即使是GT-KO猪也不能完全抑制宿主的免疫反应。其中一个潜在的候选者是补体调节蛋白，如人衰变加速因子（hepatocyte）。然而，关于猪细胞中抑制补体激活需要多高的hkD表达水平的报道很少。本研究的目的是研究hepatocyte的表达水平和其对人血清cytotoxicity.Methods的抑制作用之间的关系：表达载体（pCAGGS）含有hepatocyte基因转染到猪成纤维细胞使用电穿孔系统（Gene Pulser II）。转染后48至52小时，用FITC标记的抗-hepatocyte抗体染色细胞，然后应用于细胞分选仪。通过对荧光强度设门来收集具有各种表达水平的hDAF转染的细胞。相对于人对照内皮细胞中的表达水平，测定hkD表达水平。收集表达x1、x5、x10、x15和x30 hTERT的细胞，置于96孔板中孵育16 h，然后进行MTT法检测。在猪细胞中比在人内皮细胞中高5倍的HSP 70表达可有效抑制大多数人血清的补体依赖性细胞毒性。然而，15- 30倍的表达hepatocytes需要有效抑制人血清的最高细胞毒性capability.Conclusions;一个更高水平的hepatocytes在猪细胞中的表达比以前认为必要的可能需要提供额外的好处，在抑制抗体介导的排斥反应。表达非常高水平的hlc的基因工程猪将有利于异种移植。
Background: Although the successful production of alpha 1,3-galactosyltransferase-knockout (GT-KO) pigs has increased expectations of clinical xenotransplantation, additional modifications of genetically engineered pigs are still being explored, because even GT-KO pigs are incapable of inhibiting the host's immunological response completely. One of the potential candidates is a complement-regulatory protein, such as human decay-accelerating factor (hDAF). However, there are few reports on how high the expression level of hDAF in pig cells would be required for suppression of complement activation. The purpose of this study was to examine the relationship between the level of hDAF expression and its inhibitory effect on human serum cytotoxicity.Methods: An expression (pCAGGS) vector containing the hDAF gene was transfected into pig fibroblasts using an electroporation system (Gene Pulser II). Forty-eight to fifty-two hours after transfection, the cells were stained with FITC-labeled anti-hDAF antibody and then applied to the cell sorter. hDAF-transfected cells with various expression levels were collected by gating on fluorescence intensity. The level of hDAF expression was determined relative to that in human control endothelial cells. Collected cells expressing x1, x5, x10, x15 and x30 hDAF were incubated into 96-well plates for 16 h, and the cells were subjected to 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay.Results: hDAF expression levels in transfected cells at the time of MTT assay (16 h after sorting) were comparable to those immediately after sorting. hDAF expression in pig cells five times higher than in human endothelial cells was effective in inhibiting complement-dependent cytotoxicity of most human sera. However, 15- to 30-fold expression of hDAF was required for effective inhibition of human sera with the highest cytotoxic capacity.Conclusions; A much higher level of hDAF expression in pig cells than previously considered necessary might be required to provide additional benefit in inhibiting antibody-mediated rejection. Genetically engineered pigs that express very high levels of hDAF would be beneficial for xenotransplantation.