title: [The role RNA interference of alpha1, 3GT plays in resistance to complement mediated cytotoxicity of porcine endothelial cells].
abstract: OBJECTIVE
  To evaluate whether RNA interference can protect porcine endothelial cells from complement mediated cytotoxicity.
  
  
  METHODS
  Immortalized porcine aortic endothelial cells of the line PED were cultured and transfected with alpha1,3-galactosyltransferase (alpha1, 3-GT) specific siRNAs. Cells transfected with mismatch SiRNA was used as negative controls. Forty-eight hours later the cells were collected. The expression of alpha1, 3-GT mRNA was examined by RT-PCR. The expression of alpha-Gal was examined by flow cytometry. PED cells ere labeled with (51)Cr and mixed with normal human serum (NHS). The release of (51)Cr was measured by gamma-ray counter. Heat inactivated NHS (HINHS) was used as control.
  
  
  RESULTS
  Two isoforms (isoform 1 and isoform 2) were amplified from the PED cells. The expression of alpha1, 3-GT in the PED cells transfected with SiRNA-1 was lower by 70% in comparison with the mock group (69% for the isoform 1 and 72% for the isoform 2, both P < 0.05). However, the expression of alpha1, 3-GT in the PED cells transfected with SiRNA21 was not different from those in the mock group and mismatch group (both P > 0.05). Flow cytometry showed that the average fluorescence intensity of the PED cells transfected with SiRNA-1 was 52, 9, significantly lower than that of the mismatch group and mock group (493.9 and 5-5.7 respectively, both P < 0.0). Fluorescence microscopy observed the "silence effect" of alphaGal after SiRNA-1 transfection. Added with 20% and 40% NHS, the cell dissolution rate of the SiRNA transfection group was lower than that of the mock group by 70% and 60% respectively.
  
  
  CONCLUSIONS
  alpha1, 3GT gene silencing actually occurs following transfection of SiRNA-1. Porcine endothelial cells can be the targets of RNAi.
authors: Institute of Organ Transplantation, Key Laboratory of Organ Transplantation Appointed by Ministry of Education and Ministry of Health, P.R. China.
authors: Min
authors: M
authors: Zhu
authors: Zhen-xiong
authors: ZX
authors: Xia
authors: Shu-sen
authors: SS
authors: Wang
authors: Rong-hua
authors: RH
authors: Cao
authors: Hong-gang
authors: HG
authors: Qi
authors: Dong
authors: D
authors: Chen
authors: Bin
authors: B
authors: Liu
authors: Wei-jie
authors: WJ
authors: Zhang
authors: Shi
authors: S
authors: Chen
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology; D003165:Complement System Proteins / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D042783:Endothelial Cells / Q000276:immunology* / Q000378:metabolism; D005700:Galactosyltransferases / Q000096:biosynthesis* / Q000235:genetics; D020868:Gene Silencing; D034622:RNA Interference / Q000502:physiology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000502:physiology; D013552:Swine; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D003165:Complement System Proteins; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase