The Establishment Of Allogeneic Grafted Tumor Mice Model With MICA Alleles

MICA gene is functional gene of MIC family. It is an important immune molecule which is polymorphic in the genome. The NKG2D receptor recognizes MICA specific ligand. MICA antigens for alloreactive responses have been appearing continuously in the organ transplant scenario. It also plays an important role in tumor immunity, autoimmune diseases, immune surveillance and immune escape. MICA gene is a polymorphic gene family and related to immune responses. There is a wide range of geographical and ethnic differences and 68 alleles have been found. Researchers show growing interests in the study of MICA as new target molecules. MICA antigens are able to elicit the synthesis of alloantibody in transplant recipients which have been found in association with GVHR and HVGR. Currently on the MICA alleles and anti-MICA Abs produced in organ transplantation have not been reported. We hope to establish allogeneic grafted tumor model which making MICA alleles and MICA matching a significant marker to evaluate from rejected grafts. AimsWe hope to establish BLCL with specific HLA and MICA alleles typing by EB virus-transformed PBMC and using PCR-SSP. We also hope to establish vector plasmid containing MICA alleles and human PBL-engrafted NOD/SCID mice to obtain the foundation of study and evidence of different capability of diverse alleles on MICA-mediated allograft rejection.MethodsPart one, BLCL was transformed by EB virus。Part two,we analyzed MICA alleles typing using PCR-SSP mixed primers and provided experimental basis by tested MICA specific sites. Part three,the MICA alleles specific sites was analyzed to pro-selected cell lines of our laboratory. We established plasmid containing cDNA extracted from the cell lines. Part four, by injected PBMC and allogeneic tumor cells of one body. We established PBL-engrafted NOD/SCID mice with consistent HLA and MICA matching.Results1. BLCL of specific MICA alleles transformed by EV virus was obtained successfully.2. In this study we developed a high-resolution polymerase chain reaction-sequence-specific primer that detected specific sites of MICA alleles. The method was recognized 4 cell lines of our laboratory. BLCL was typed as MICA*008 and 7901 cell line was also tested as MICA*008.3. We established plasmid named pcDNA3(+)/MICA*008 containing MICA*008 cDNA after 7901 cell line was recognized MICA alleles typing by PCR-SSP. Plasmid pcDNA3(+)/MICA*008 was transfected into BLCL and MICA molecule was detected in transfected BLCL.4. The volume of grafted tumor in the group of hu-PBL-NOD/SCID mice were less than the control group.ConclusionThis study identified MICA gene specific sites of BLCL and cell lines kept in our laboratory and established plasmid pcDNA3(+)/MICA*008. We obtained hu-PBL-NOD/SCID mice model with good HLA matching using the methods of injecting PBMC and grafted tumor cell which from the same body. This model can offer a good platform to research the role and mechanisms of different MICA alleles, anti-MICA Abs and MICA matching in allogeneic transplantation.