Trial using pig cells with the H–D antigen knocked down
Trial using pig cells with the H–D antigen knocked down
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DOI：
10.1007/s00595-012-0274-x
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发表时间：
2013-07
期刊：
影响因子：
2.5
通讯作者：
A. Yamamoto;K. Ikeda;Dandan Wang;S. Nakatsu;Y. Takama;T. Ueno;H. Nagashima;A. Kondo;M. Fukuzawa;S. Miyagawa
中科院分区：
文献类型：
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作者：
A. Yamamoto;K. Ikeda;Dandan Wang;S. Nakatsu;Y. Takama;T. Ueno;H. Nagashima;A. Kondo;M. Fukuzawa;S. Miyagawa
目的利用猪胞苷单磷酸- n -乙酰神经氨酸羟化酶（pCMAH）小干扰RNA （siRNA）降低H-D抗原的表达水平。方法采用猪内皮细胞（PEC）系，α1,3半乳糖转移酶敲除仔猪的PEC和成纤维细胞。Real-time PCR检测siRNA对mRNA的降解作用。将H-D抗原染色，然后将细胞与人血清孵育进行FACS分析。用乳酸脱氢酶测定法计算人血清中酶解的程度。结果首次测定了各siRNA对pCMAH mRNA的抑制作用。pCMAH sirna混合物在相当程度上降低了PEC和成纤维细胞上H-D抗原的表达。然后证实了GalT-KO细胞在人血清中的异种抗原性进一步降低。此外，PEC细胞系显示sirna对补体依赖性细胞毒性的显著下调，这表明人血清中的抗h -d抗原能够引起猪细胞的裂解。结论siRNA沉默pcmah可降低H-D抗原的表达及其抗原性，从而证实H-D抗原是这种情况下主要的非gal抗原之一。
PurposeThis 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).MethodsA 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.ResultsSuppression 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.ConclusionpCMAH 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.