RNA interference of GGTA1 physiological and immune functions in immortalized porcine aortic endothelial cells.
RNA interference of GGTA1 physiological and immune functions in immortalized porcine aortic endothelial cells.
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DOI：
10.1016/j.jss.2013.05.080
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发表时间：
2013-11
期刊：
影响因子：
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通讯作者：
Wei Han;Jing-shi Zhou;Xiao Li;Jian-feng Wang;Junjie Li;Zhuo-chao Zhang;Zhao-xu Yang;De-sheng Wang;K. Tao;K. Dou
中科院分区：
文献类型：
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作者：
Wei Han;Jing-shi Zhou;Xiao Li;Jian-feng Wang;Junjie Li;Zhuo-chao Zhang;Zhao-xu Yang;De-sheng Wang;K. Tao;K. Dou
猪器官是异种移植中常用的器官，α-1，3-半乳糖是引起超急性排斥反应的主要原因。缺乏α-1，3-半乳糖基转移酶的转基因猪的开发在一定程度上克服了这一问题，但转基因猪的维护困难，限制了其在基础研究中的应用。为此，我们建议建立一种研究超急性排斥反应的细胞模型。方法将针对GGTA1的短发夹状RNA导入永生化猪主动脉内皮细胞。检测细胞增殖、细胞凋亡、补体C3活化以及人免疫球蛋白与补体系统成分IgM、Ig G、C3和C5b-9的结合。结果经RNA干扰后，定量聚合酶链式反应和流式细胞术分别检测到GGTA1在转录水平和蛋白水平上的降低。在含人血清的条件下，转染组细胞的增殖率高于未转染组，凋亡率低于未转染组。此外，C3的激活以及人免疫球蛋白IgM和Ig G与补体成分C3和C5b-9的结合低于永生化组，但高于未转染组。结论GGTA1对猪内皮细胞的RNA干扰降低了免疫球蛋白和补体系统与细胞的反应。因此，该体外细胞模型可用于异种移植的进一步研究。
BackgroundPig organs are commonly used in xenotransplantation, and α-1,3-galactose has been shown to be the main cause of hyperacute rejection. The development of transgenic pigs that lack α-1,3-galactosyltransferase (GGTA1) has overcome this problem to a certain extent, but transgenic pigs are difficult to maintain, making their usefulness in basic research limited. For this reason, we propose to establish a cell model to study hyperacute rejection.MethodsImmortalized primary porcine aortic endothelial cells were transfected with a short hairpin RNA targeted to GGTA1. Cell proliferation, apoptosis, complement C3 activation, and the binding of human immunoglobulins and components of the complement system, including IgM, IgG, C3, and C5b-9, were examined.ResultsAfter RNA interference, GGTA1 was found to be reduced at both the transcript and protein level as assessed by quantitative polymerase chain reaction and flow cytometry, respectively. When cultured in the presence of human serum, the proliferation rate of the transfected cells was higher than that of untransfected cells, and the apoptosis rate was lower. Additionally, activation of C3 and the binding of human immunoglobulins IgM and IgG and complement component C3 and C5b-9 to the transfected cells were lower than in the immortalized group but higher than in untransfected cells.ConclusionsRNA interference of GGTA1 in cultured porcine endothelial cells reduces the reaction of immunoglobulin and complement system with the cells. Therefore, thisin vitrocell model could be useful for further study of xenotransplantation.