[The role RNA interference of alpha1, 3GT plays in resistance to complement mediated cytotoxicity of porcine endothelial cells].
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.
Zhu
Min
M
Institute of Organ Transplantation, Key Laboratory of Organ Transplantation Appointed by Ministry of Education and Ministry of Health, P.R. China.
Xia
Zhen-xiong
ZX
Wang
Shu-sen
SS
Cao
Rong-hua
RH
Qi
Hong-gang
HG
Chen
Dong
D
Liu
Bin
B
Zhang
Wei-jie
WJ
Chen
Shi
S
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
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D003165:Complement System Proteins; D005700:Galactosyltransferases; C045097:UDP-galactose-lactosylceramide alpha 1-4-galactosyltransferase