ABSTRACT
Objective:To construct a RNAi lentiviral vector targeting rat CD80 gene and detect its effect of gene silencing in NRK and IEC6 cells.Methods:The effective sequence of siRNA targeting rat CD80 gene was confirmed in our previous work.Oligo-DNA fragment containing short hairpin frame was synthesized and reannealed,and then cloned into pGCSIL-GFP lentiviral expression vector.PCR and sequencing analysis were made for verifying the positive clones.The virus packaging plasmids were transfected into 293T cells to harvest shRNA lentivirus.After infection in NRK and IEC6 cells,Real-time PCR was performed to determine the expressing level of CD80.Results:PCR and sequencing revealed that shRNA plasmids was correctly constructed.Virus with a titer of 4×10~8 TU/ml was successfully packaged.CD80 expression in NRK and IEC6 cells could be knockdown by virus infection as characterized by 66.9% and 63.5% decrease of CD80 mRNA in NRK and IEC6 cells respectively,compared with negative control lentivirus.Conclusion:The recombinant lentiviral shRNA expressing vector targeting rat CD80 gene has been successfully constructed and packaged.CD80 mRNA could be down-regulated availably in NRK and IEC6 cells.
ABSTRACT
Objective To construct an RNAi lentiviral vector targeting rat CD86 gene and detect its effect of gene silencing in dendritic cells. Methods The effective sequence of siRNA targeting rat CD86 gene was confirmed in our previous work. DNA oligo containing short hairpin frame was synthesized and re-annealed, and then cloned into pGCL-GFP lentivind expression vector. PCR and sequencing analysis were made for verifying the positive clones. The virus packaging plasmids were transfected into 293T cells to har-vest shRNA lentivirus. After infection in dendritic cells, real-time PCR and Western blot were performed to determine the expression level of CD86 at mRNA and protein of NC( negative control virus). Results PCR and sequencing analysis revealed that shRNA plasmid was correctly constructed. Virus with a titer of 2×108 TU/ml was successfully packaged. CD86 expression in dendritic cells can be knockdown at both mRNA and protein level by virus infection as characterized by 90.6% decrease of mRNA and significant inhibition of protein compared with NC. Conclusion The recombinant lentiviral shRNA expressing vector targeting rat CD86 gene has been successfully constructed and packaged. CD86 mRNA and protein can be effectively down-regulated in dendritic cells.
ABSTRACT
Objective:To investigate the effect of ischaemia preconditioning (IP) on function of the transplanted lungs.Methods:Rat orthotopic left lung transplantation (OLT) was performed using the cuff anastomosis technique.The rats were divided into two groups ① CONT (n=16):OLT without IP.② IP (n=15):OLT plus IP.Results:In IP group blood gas exchange and recipient survivals were significantly improved and extravascular water content decreased compared with CONT group.Conclusion:Ischaemia preconditioning not only ameliorates graft injury but inhibits graft dysfunction after OLT.