Basic Research
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. May 15, 2003; 9(5): 1077-1081
Published online May 15, 2003. doi: 10.3748/wjg.v9.i5.1077
Repression of allo-cell transplant rejection through CIITA ribonuclease P+ hepatocyte
Rong Guo, Ping Zou, Hua-Hua Fan, Feng Gao, Qing-Xin Shang, Yi-Lin Cao, Hua-Zhong Lu
Rong Guo, Ping Zou, Institute of Hematology, the Union Hospital, Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China
Hua-Hua Fan, Feng Gao, Hua-Zhong Lu, The Department of Blood Engineering, Shanghai Municipal Blood Center, Shanghai 200051, China
Qing-Xin Shang, Yi-Lin Cao, Shanghai Municipal Tissue Engineering Research and Developing Center, Shanghai 200011, China
Author contributions: All authors contributed equally to the work.
Supported by the Science and Technology Development Foundation of Shanghai municipality, No. 0143nm068 and the Great Branch Project of the Science and Technology Development Foundation of Shanghai Municipality, No. 00DJ14001-8
Correspondence to: Dr. Rong Guo, Institute of Hematology, the Union Hospital, 1127 Jiefang Dadao, Wuhan 430022, Hubei Province China. gh7311@yahoo.com.cn
Telephone: +86-27-85730575
Received: November 6, 2002
Revised: November 23, 2002
Accepted: December 16, 2002
Published online: May 15, 2003
Abstract

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 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 EcoR I/Bgl II or EcoR I/Sal I site 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 Xho I/EcoR I of 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.

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