Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA.
Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA; Second Affiliated Hospital, University of South China, Hengyang City, Hunan, China.
Int J Surg. 2020 Nov;83:184-188. doi: 10.1016/j.ijsu.2020.09.034. Epub 2020 Sep 25.
There are many causes of systemic complement activation, which may have detrimental effects on a pig xenograft. Transgenic expression of one or more human complement-regulatory proteins (hCRPs), e.g., hCD46, provides some protection to the xenograft, but it is not known whether it protects the xenograft from the effects of systemic complement activation. We used wild-type (WT) pig aortic endothelial cells (pAECs) to activate complement, and determined whether the expression of hCD46 on a1,3galactosyltransferase gene-knockout (GTKO) pAECs protected them from injury.
CFSE-labeled and non-labeled pAECs from a WT, a GTKO, or a GTKO/hCD46 pig were separately incubated with heat-inactivated pooled human serum in vitro. Antibody pre-bonded CFSE-labeled and non-labeled pAECs were mixed, and then incubated with rabbit complement. The complement-dependent cytotoxicity was measured by flow cytometry.
There was significantly less lysis of GTKO/CD46 pAECs (6%) by 50% human serum compared to that of WT (91%, p<0.001) or GTKO (32%, p<0.01) pAECs. The lysis of GTKO pAECs was significantly increased when mixed with WT pAECs (p<0.05). In contrast, there was no significant change in cytotoxicity of GTKO/CD46 pAECs when mixed with WT pAECs.
The expression of hCD46 protected pAECs from systemic complement activation.
全身性补体激活有多种原因，可能对猪异种移植物产生有害影响。一种或多种人补体调节蛋白（hCRP）的转基因表达，例如 hCD46，可为异种移植物提供一定程度的保护，但尚不清楚其是否能防止异种移植物受到全身性补体激活的影响。我们使用野生型（WT）猪主动脉内皮细胞（pAEC）激活补体，以确定 GTKO 猪 pAEC 上 hCD46 的表达是否能保护它们免受损伤。
将 CFSE 标记和非标记的 WT、GTKO 或 GTKO/hCD46 猪的 pAEC 分别与体外热失活的人血清孵育。预先结合抗体的 CFSE 标记和非标记 pAEC 混合，然后与兔补体孵育。通过流式细胞术测量补体依赖性细胞毒性。
与 WT（91%，p<0.001）或 GTKO（32%，p<0.01）pAEC 相比，50%人血清对 GTKO/CD46 pAEC 的裂解明显减少（6%，p<0.001）。当与 WT pAEC 混合时，GTKO pAEC 的裂解明显增加（p<0.05）。相比之下，当与 WT pAEC 混合时，GTKO/CD46 pAEC 的细胞毒性没有明显变化。
hCD46 的表达可保护 pAEC 免受全身性补体激活的影响。
