Repression of allo-cell transplant rejection through CⅡTA ribonuclease P^+hepatocyte

AIM: Allo-cell transplant rejection and autoimmune responses 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 MHCII molecules, on repressing the expression of MHCII molecules on hepatocyte.METHODS: M1-RNA is the catalytic RNA subunit of RNase P from Escherichia coli. It were constructed that M1-RNA with guide sequences (GS) recognizing the 452, 3408 site of CIITA by PCR from pTK117 plasmid, then were cloned into the EcoRI/Bg/II or EcoR//SalIsite of vector psNAV (osNAV-M1-452-GS, psNAV-M1-3408-GS) respectively. The target mould plate (3176-3560) of CIITA was obtained from Raji cell by RT-PCR, and then inserted into the XhoI/EcoRIof pGEM-7zf(+) plasmid (pGEM-3176). These recombinant plasmids were screened out by sequence analysis. psNAV-M 1-452-GS, psNAV-M1-3408-GS and its target RNA pGEM-3176 were transcribed and then mixed up and incubated in vitro. It showed that M1-3408-GS could exclusively cleave target RNA that formed a base pair with the GS. Stable transfectants of hepatocyte cell line with psNAVl-M1-3408-GS were tested for expression of class Ⅱ MHC through FCM, for mRNA abundance of MHCII, Ii and CIITA by RTPCR., for the level of IL-2 mRNA on T cell by mixed lymphocyte reaction.RESULTS: When induced with recombinant human interferon-gamma (IFN-γ), the expression of HLA-DR, -DP,-DQ on psNAV-M1-3408-GS+ hepatocyte was reduced 83.27 %, 88.93 %, 58.82 % respectively, the mRNA contents of 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 tolerance through cleaving CIITA. These results provided insight into the future application of M1-3408-GS as a new nucleic acid drug against allo-transplantation rejection and autoimmune 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 CⅡTA,which was a major regulator of MHCII molecules,on repressing the expression of MHCⅡ molecules on hepatocyte. METHODS:M1-RNA is the catalytic RNA subunit of RNase P from Escherichia coll.It were constructed that M1-RNA with guide sequences (GS) recognizing the 452,3408 site of CⅡTA by PCR from pTK117 plasmid,then were cloned into the EcoRⅠ/BgⅢ or EcoR//SalIsite of vector psNAV (psNA V-M1-452-GS,psNAV-M1-3408-GS) respectively.The target mould plate (3176-3560) of CⅡTA was obtained from Raji cell by RT-PCR,and then inserted into the XhoI/EcoRIof pGEM-7zf(+) plasmid (pGEM-3176).These 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 vitro.It showed that M1-3408-GS could exclusively cleave target RNA that formed a base pair with the GS.Stable transfectants of hepatocyte cell line with psNAV-M1-3408- GS were tested for expression of class Ⅱ MHC through FCM,for mRNA abundance of MHCⅡ,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-γ),the expression of HLA-DR,-DP, -DQ on psNAV-M1-3408-GS^+ hepatocyte was reduced 83.27%,88.93%,58.82% respectively,the mRNA contents of 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 tolerance through cleaving CⅡTA.These results provided insight into the future application of M1-3408-GS as a new nucleic acid drug against allo-transplantation rejection and autoimmune diseases.