Division of Organ Transplantation (E9), Department of Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
Surg Today. 2013 Jul;43(7):782-6. doi: 10.1007/s00595-012-0274-x. Epub 2012 Aug 5.
This report describes an attempt to reduce the expression level of Hanganutziu-Deicher (H-D) antigens by small interfering RNA (siRNA) for pig cytidine monophospho-N-acetylneuraminic acid hydroxylase (pCMAH).
A pig endothelial cell (PEC) line, and PEC and fibroblasts from an α1,3galactosyltransferase knockout (GalT-KO) piglet were used. Real-time PCR was used to evaluate the degradation of mRNA by siRNA. The H-D antigen was stained, and then the cells were incubated with human serum for the FACS analysis. The extent of lysis in human serum was next calculated using an LDH assay.
Suppression of the mRNA of pCMAH by each siRNA was first determined. The mixture of siRNAs for pCMAH reduced the expressions of the H-D antigen on the PEC and fibroblasts to a considerable extent. The further reduction in the xenoantigenicity for human serum of the GalT-KO cells was then confirmed. In addition, the PEC line showed a significant downregulation in complement-dependent cytotoxicity by the siRNAs, thus indicating that the anti-H-D antigen in human serum is capable of causing lysis of the pig cells.
pCMAH silencing by siRNA reduced the expression of the H-D antigen and its antigenicity, thus confirming that the H-D antigen is one of the major non-Gal antigens in this situation.
本报告描述了一种通过小干扰 RNA（siRNA）降低猪胞苷单磷酸-N-乙酰神经氨酸羟化酶（pCMAH）中 Hanganutziu-Deicher（H-D）抗原表达水平的尝试。
使用猪内皮细胞（PEC）系以及来自α1,3半乳糖基转移酶敲除（GalT-KO）仔猪的 PEC 和成纤维细胞。实时 PCR 用于评估 siRNA 对 mRNA 的降解作用。用 H-D 抗原染色，然后用人类血清孵育进行 FACS 分析。接下来使用 LDH 测定法计算人血清中的裂解程度。
首先确定了 pCMAH 的每个 siRNA 对 mRNA 的抑制作用。pCMAH 的 siRNA 混合物将 PEC 和成纤维细胞上 H-D 抗原的表达大大降低。然后进一步证实了 GalT-KO 细胞对人血清中异种抗原性的降低。此外，PEC 系显示出 siRNA 引起的补体依赖性细胞毒性的显著下调，表明人血清中的抗 H-D 抗原能够导致猪细胞的裂解。
siRNA 对 pCMAH 的沉默降低了 H-D 抗原及其抗原性，从而证实 H-D 抗原是这种情况下主要的非 Gal 抗原之一。
