Functional Analysis Of A Novel Development Related Gene Mcpr1 During Maxillafacial Development

Embryonic development is a process with fast celluar dividision and highly differentiation, during which there is cell death companied with cell proliferation and differentiation. And cell death is very important for keeping cell dynamic balance and remodeling embryonic construction, which happens during specific periods and places according to rigorous programme. And Gene expression regulation is involved in the whole process and developes a regulation net to play a role during embryonic development. mcpr1 was a novel gene cloned by subtractive hybridization method at our development, which was obtained by comparing palatal shelves of normal murine embryo with ones of cleft palate models induced by retinoic acid. It was registered in GenBank as AYO74887, and named mouse cleft palate related gene. mRNA length of mcprl gene had been identified, and expressions in some tissues had been observed. However there has been little to be revealed about mcprl. This study includes three parts involved in revealing functions of mcprl gene initially. So is the following. 1. Preparation and Identification of MCPR1 Polyclonal AntibodiesThe coding region of mcprl was obtained by PCR, and an expression vector pGEX-4T- mcprl was constructed by inserting fragment of the coding region of mcprl into a prokaryotic expression vector pGEX-4T-1. Then the recombinant plasmid was transferred into E. coli to prepare GST-MCPR1 fusion protein.GST-MCPR1 fusion protein was obtained after the induction of IPTG The fusion protein was extracted and purified by SDS-PAGE, and the new protein band was cut as antigens, injected into the rabbit to produce immunoserum. The immunoserum was purified initially and identified by Western blotting and ELISA, then we obtained the MCPR1 polyclonal antibodies with high titers and specificity. The antibodies made it possible to analyze the role of MCPR1 on the protein level, such as the interaction between MCPR1 and other proteins with immunological methods, and laid the foundation to elucidate its function during the embryonic development.2. The establishment of tissue expression profiles and tempo-spatial expression during maxillafacial developmentIt was observed that mcprl expressed in liver, heart, brain, spleen, kidney, lung and muscle on the level of transcription and translation. The results of immunohistochemistry showed that MCPR1 expressed extensively in different patterns during murine embryo development, however there was no tissue specification or obvious tendency. To investigate the expression of mcprl gene, murine embryonic palatal mesenchymal (MEPM) cells, ectomesenchymal cells of human mandibular process, dental follicle cells, dental pulp cells and periodontal ligament cells were detected in polyclonal antibodies of MCPR1 by immunohistochemistry. The results showed that MCPR1 expressed in cytoplasm not nuclear, and it expressed in MEPM cells and periodontal ligament cells strongly. MCPR1 expressed mainly in cytoplasm of different cells in different levels, which inferred that MCPR1 might take part in the development of many organs during different periods of embryonic development.To observe the tempo-spatial expression of MCPR1 during murine tooth germ development and evaluate the effect of MCPR1 on tooth germ development, Immunohistochemical stain was used . MCPR1 was detected to express at all stages of tooth germ, but the distribution patterns at various stages were different. It was inferred that MCPR1 might play an important role in modulating the differentiation and mature of enamel organ.3. The effect of AS-ODN and GST-MCPR1 on biological characterization of MEPM cellsRT-PCR and immunohistochemistry analysis showed that mcprl expressed stronger in murine palate induced by retinoic acid than normal...