Lentivirus-mediated RNAi against xenoantigen in mouse vascular endothelial cells
10.3760/cma.j.issn.0254-1785.2010.10.013
- VernacularTitle:靶向慢病毒载体RNA干扰小鼠血管内皮细胞异种抗原的研究
- Author:
Feng QI
;
Yachuan GU
;
Chengyu LU
;
Zhixiang ZHANG
;
Xianghui HE
;
Liwei ZHU
- Publication Type:Journal Article
- Keywords:
Xenotransplantation;
RNA interfere;
Lentivirus;
Hyperacute rejection
- From:
Chinese Journal of Organ Transplantation
2010;31(10):621-625
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the feasibility of inhibiting Galα (1,3)-Gal expression in mouse vascular endothelial cells by lentivirus-mediated RNAi.Methods The shRNA specified to α1,3-GT mRNA was designed and synthesized in vitro and cloned into the lentivirus vector.EOMA cells were infected by recombinant lentivirus.Real-time RT-PCR was used to detect mRNA transcriptional levels of αl,3-GT as well as immunofluorescence and flow cytometry were applied to detect Galα(1,3)-Gal antigen level after gene transfection.Co-culture of infected EOMA and serum of human was done and the survival rate was measured by MTT.Results The αl,3-GT shRNA sequences were cloned into the recombinant lentivirus vector correctly and the lentivirus was produced successfully.The transfection efficiency to EOMA was 75 %.Real-time PCR revealed that the mRNA transcription of α1,3-GT was obviously inhibited by α1,3-GT shRNA recombinant lentivirus with the rate of 88 % (P＜0.05),while there were no obvious differences among control group,no shRNA lentivirus group and negative-shRNA lentivirus group (P＞ 0.05).Immunofluorescence and flow cytometry demonstrated the same results that Galα(1,3)-Gal antigen expression in EOMA transfected by α1,3-GT shRNA lentivirus was less than that of control group,no shRNA lentivirus group and negative-shRNA lentivirus group (P＜0.05),but there were no obvious differences among the later three groups (P＞0.05).After co-culture with serum of human,MTT showed the survival rate of EOMA infected by α1,3-GT shRNA lentivirus was obviously increased (P＜ 0.05).Conclusion Recombinant α2,3-GT shRNA 1entivirus is constructed successfully,which can inhibit the expression of α1,3-GT and Galα1,3-Gal in EOMA by RNAi and control hyperacute rejection in vitro.
