title: Repression of allo-cell transplant rejection through CIITA ribonuclease P+ hepatocyte.
abstract: OBJECTIVE
  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 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/BglII or EcoR//SalIsite of vector psNAV (psNAV-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-gamma), 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Hematology, the Union Hospital, Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China. gh7311@yahoo.com.cn
authors.forename: Rong
authors.initials: R
authors.lastname: Guo
authors.forename: Ping
authors.initials: P
authors.lastname: Zou
authors.forename: Hua-Hua
authors.initials: HH
authors.lastname: Fan
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.forename: Qing-Xin
authors.initials: QX
authors.lastname: Shang
authors.forename: Yi-Lin
authors.initials: YL
authors.lastname: Cao
authors.forename: Hua-Zhong
authors.initials: HZ
authors.lastname: Lu
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006084:Graft Rejection / Q000201:enzymology / Q000276:immunology / Q000517:prevention & control*; D022781:Hepatocytes / Q000201:enzymology / Q000276:immunology / Q000637:transplantation*; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D007376:Interleukin-2 / Q000235:genetics; D019164:Liver, Artificial; D007959:Lymphocyte Culture Test, Mixed; D009687:Nuclear Proteins; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P; D023822:Tissue Engineering; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014184:Transplantation, Homologous
chemical_list: D029968:Escherichia coli Proteins; D000949:Histocompatibility Antigens Class II; D007376:Interleukin-2; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D015534:Trans-Activators; D007371:Interferon-gamma; D004247:DNA; D004722:Endoribonucleases; C468368:RPP14 protein, human; D043262:Ribonuclease P; C470865:ribonuclease P, E coli