人血栓调节蛋白转基因在猪内皮细胞中的稳定表达与炎症反应的减轻有关。
Stable expression of the human thrombomodulin transgene in pig endothelial cells is associated with a reduction in the inflammatory response.
机构信息
Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA; Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA; Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA.
出版信息
Cytokine. 2021 Dec;148:155580. doi: 10.1016/j.cyto.2021.155580. Epub 2021 Jun 4.
BACKGROUND
Xenotransplantation is associated with an inflammatory response. The proinflammatory cytokine, TNF-α, downregulates the expression of thrombomodulin (TBM), and induces coagulation dysfunction. Although human (h) TBM-transgenic pigs (p) have been developed to reduce coagulation dysfunction, the effect of TNF-α on the expression of hTBM and its functional activity has not been fully investigated. The aims of this study were to investigate (i) whether the expression of hTBM on pig (p) cells is down-regulated during TNF-α stimulation, and (ii) whether cells from hTBM pigs regulate the inflammatory response.
METHODS
TNF-α-producing T, B, and natural killer cells in blood from baboons with pig heart or kidney xenografts were investigated by flow cytometry. TNF-α staining in the grafts was detected by immunohistochemistry. Aortic endothelial cells (AECs) from GTKO/CD46 and GTKO/CD46/hTBM pigs were stimulated by hTNF-α, and the expression of the inflammatory/coagulation regulatory protein, TBM, was investigated.
RESULTS
After pig organ xenotransplantation, there was a trend to increases in TNF-α-producing T and natural killer cells in the blood of baboons. In vitro observations demonstrated that after hTNF-α stimulation, there was a significant reduction in the expression of endogenous pTBM on pAECs, and a significant increase in the expression of inflammatory molecules. Blocking of NF-κB signaling significantly up-regulated pTBM expression, and suppressed the inflammatory response induced by hTNF-α in pAECs. Whereas the expression of pTBM mRNA was significantly reduced by hTNF-α stimulation, hTBM expression on the GTKO/CD46/hTBM pAECs was not affected. Furthermore, after hTNF-α stimulation, there was significant suppression of expression of inflammatory molecules on GTKO/CD46/hTBM pAECs compared to GTKO/CD46 pAECs.
CONCLUSIONS
The stable expression of hTBM in pig cells may locally regulate the inflammatory response. This will help suppress the inflammatory response and prevent coagulation dysregulation after xenotransplantation.
背景
异种移植会引起炎症反应。促炎细胞因子 TNF-α下调血栓调节蛋白（TBM）的表达，并诱导凝血功能障碍。尽管已经开发出人类（h）TBM 转基因猪（p）来减少凝血功能障碍，但 TNF-α对 hTBM 的表达及其功能活性的影响尚未得到充分研究。本研究的目的是探讨（i）TNF-α刺激期间猪（p）细胞上 hTBM 的表达是否下调，以及（ii）来自 hTBM 猪的细胞是否调节炎症反应。
方法
通过流式细胞术研究了具有猪心脏或肾脏异种移植物的狒狒血液中的 TNF-α 产生 T、B 和自然杀伤细胞。通过免疫组织化学检测移植物中的 TNF-α 染色。用 hTNF-α刺激 GTKO/CD46 和 GTKO/CD46/hTBM 猪的主动脉内皮细胞（AEC），并研究炎症/凝血调节蛋白 TBM 的表达。
结果
在猪器官异种移植后，狒狒血液中 TNF-α 产生的 T 和自然杀伤细胞呈上升趋势。体外观察表明，hTNF-α刺激后，pAEC 上内源性 pTBM 的表达显著降低，炎症分子的表达显著增加。阻断 NF-κB 信号显著上调 pTBM 表达，并抑制 hTNF-α诱导的 pAEC 炎症反应。虽然 hTNF-α刺激显著降低了 pTBM mRNA 的表达，但 GTKO/CD46/hTBM pAEC 上的 hTBM 表达不受影响。此外，与 GTKO/CD46 pAEC 相比，hTNF-α 刺激后 GTKO/CD46/hTBM pAEC 上炎症分子的表达明显受到抑制。
结论
hTBM 在猪细胞中的稳定表达可能局部调节炎症反应。这有助于抑制异种移植后的炎症反应和防止凝血功能障碍。
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