RNA interference of GGTA1 physiological and immune functions in immortalized porcine aortic endothelial cells

• Published in: The Journal of surgical research 2013-11, Vol.185 (1), p.410-418
• Main Authors: Han, Wei, MM, Zhou, Jingshi, PhD, Li, Xiao, PhD, Wang, Jianfeng, MM, Li, Junjie, PhD, Zhang, Zhuochao, MM, Yang, Zhaoxu, PhD, Wang, Desheng, PhD, Tao, Kaishan, PhD, MD, Dou, Kefeng, PhD, MD
• Format: Article
• Language: English
• Subjects: Acute Disease; Animals; Aorta - cytology; Apoptosis - immunology; Cell Line, Transformed; Cell Proliferation; Complement; Complement C3 - immunology; Complement Membrane Attack Complex - immunology; Endothelial Cells - cytology; Endothelial Cells - immunology; Galactosyltransferases - genetics; Galactosyltransferases - immunology; Genetic Therapy - methods; Graft Rejection - immunology; Graft Rejection - therapy; Immunoglobulin G - immunology; Immunoglobulin M - immunology; Nature antibody; Primary Cell Culture; RNA interference; RNA, Small Interfering - genetics; Surgery; Swine; Transplantation, Heterologous; Xenotransplantation; α-1,3-Galactosyltransferase
• ISSN: 0022-4804; 1095-8673
• DOI: 10.1016/j.jss.2013.05.080

Abstract Background Pig 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. Methods Immortalized 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. Results After 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. Conclusions RNA interference of GGTA1 in cultured porcine endothelial cells reduces the reaction of immunoglobulin and complement system with the cells. Therefore, this in vitro cell model could be useful for further study of xenotransplantation.