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AIM:Allo-cell transplant rejection and autoimmuneresponses were associated with the presence of class Ⅱmajor histocompatibility complex (MHC Ⅱ) molecules on cells.This paper studied the effect of Ribonuclease P (RNase P)against CⅡTA,which was a major regulator of MHCIImolecules,on repressing the expression of MHCⅡ moleculeson hepatocyte.METHODS:M1-RNA is the catalytic RNA subunit of RNaseP from Escherichia coll.It were constructed that M1-RNAwith guide sequences (GS) recognizing the 452,3408 siteof CⅡTA by PCR from pTK117 plasmid,then were clonedinto the EcoRⅠ/BgⅢ or EcoR//SalIsite of vector psNAV(psNA V-M1-452-GS,psNAV-M1-3408-GS) respectively.Thetarget mould plate (3176-3560) of CⅡTA was obtained fromRaji cell by RT-PCR,and then inserted into the XhoI/EcoRIofpGEM-7zf(+) plasmid (pGEM-3176).These recombinantplasmids were screened out by sequence analysis,psNAV-M1-452-GS,psNAV-M1-3408-GS and its target RNA pGEM-3176 were transcribed and then mixed up and incubatedin vitro.It showed that M1-3408-GS could exclusively cleavetarget RNA that formed a base pair with the GS.Stabletransfectants of hepatocyte cell line with psNAV-M1-3408-GS were tested for expression of class Ⅱ MHC throughFCM,for mRNA abundance of MHCⅡ,Ii and CIITA by RT-PCR.,for the level of IL-2 mRNA on T cell by mixedlymphocyte reaction.RESULTS:When induced with recombinant humaninterferon-gamma (IFN-γ),the expression of HLA-DR,-DP,-DQ on psNAV-M1-3408-GS~+ hepatocyte was reduced83.27%,88.93%,58.82% respectively,the mRNA contentsof CⅡTA,HLA-DR,-DP,-DQ and Ii decreased significantly.While T cell expressed less IL-2 mRNA in the case of psNAV-M1-3408-GS~+ hepatocyte.CONCLUSION:The Ribonuclease P against CⅡTA-M1- 3408-GS could effectively induce antigen-specific tolerancethrough cleaving CⅡTA.These results provided insightinto the future application of M1-3408-GS as a new nucleicacid drug against allo-transplantation rejection andautoimmune diseases.
AIM: Allo-cell transplant rejection and autoimmune responses were associated with the presence of class II major histocompatibility complex (MHC II) molecules on cells. This paper studied the effect of Ribonuclease P (RNase P) against C II TA, which was a major regulator of MHC II molecules, on repressing the expression of MHC II molecules on hepatocyte. METHODS: M1-RNA is the catalytic RNA subunit of RNaseP from Escherichia coll. It was constructed that M1-RNAwith guide sequences (GS) recognizing the 452,3408 site of CIITA by PCR from pTK117 plasmid, (3176-3560) of CTATA was obtained from Raji cell by The recombinant plasmids were screened out by sequence analysis, psNAV-M1-452-GS, psNAV-M1-3408-GS and its target RNA pGEM-3176 were transcribed and then mixed up and incubated in vit ro.It showed that M1-3408-GS could exclusively cleavetarget RNA that formed a base pair with the GS.Stabletransfectants of hepatocyte cell line with psNAV-M1-3408-GS were tested for expression of class II MHC throughFCM, for mRNA abundance of MHC II, Ii and CIITA by RT-PCR., For the level of IL-2 mRNA on T cell by mixed lymphocyte reaction .RESULTS: When induced with recombinant human interferon-gamma (IFN-?), The expression of HLA- DR, , The mRNA contents of CⅡTA, HLA-DR, -DP, -DQ and Ii significantly decreased .While T cell expressed less IL-2 mRNA in the case of psNAV-M1-3408-GS ~ + hepatocyte. CONCLUSION: The Ribonuclease P against CIITA-M1- 3408-GS could induce induce-specific tolerance through cleaving CIITA.These results provide insightinto the future application of M1-3408-GS as a new nucleicacid drug against allo-transplantation rejection and autoimmune diseases.