Ret/Cxcr4Signaling Mediated Migration Of Neural Crest-Derived Cells And Its Role In Development Of Hirschsprung’s Disease

PART ONE EXPRESSION PATTERNS OF RET AND CXCR4IN COLON TISSUE FROM PATIENTS WITH HIRSCHSPRUNG’S DISEASEObjective:To investigate the expression patterns and distribution of RET and CXCR4in aganglionic, oligoganglionic and normal ganglionic segments of patients with Hirschsprung’s disease and normal colon tissue, to explore the role of RET and CXCR4in development and of Hirschsprung’s disease (HSCR) and its clinical significance in molecular diagnosis of HSCR.Methods:A total of61HSCR patients’ surgical colon tissue samples were collected and divided into aganglionic, oligoganglionic and normal ganglionic segments,6normal colon tissue samples serve as control; The expression level of RET and CXCR4mRNA in different colon tissue segments of patients with Hirschsprung’s disease was detected by Quantitative real-time PCR; Western blotting were performed to analyze the expression levels of RET and CXCR4protein in different colon tissue segments; Immunohistochemical and immunofluorescence staining were performed to analyze the expression patterns and distribution of RET and CXCR4protein in different colon tissue segments and normal colon tissue.Results:1) The expression levels of RET and CXCR4mRNA in aganglionic colon segments was decreased compared with normal ganglionic colon segments and oligoganglionic colon segments (p<0.01, n=57). However, there were no significant difference between normal ganglionic colon segments and oligoganglionic colon segments from HSCR patients (P>0.05, n=57);2) The expression levels of RET and CXCR4protein in aganglionic colon segments was decreased compared with normal ganglionic colon segments and oligoganglionic colon segments (p<0.01, n=57);3) Immunohistochemical staining showed that intensive RET and CXCR4staining was detected in ganglion cells in the ganglion in control colon specimens and normal ganglionic and oligoganglionic colon segments from HSCR patients, in addition, intensive CXCR4staining was detected in supporting Schwann cells. However, RET and CXCR4staining was significantly decreased in aganglionic colon segments;4) Immunofluorescence staining analysis showed that CXCR4staining was mainly detected in the ganglia where RET-positive ganglion cells were observed in control colon specimens and normal ganglionic and oligoganglionic colon segments from patients with HSCR, RET and CXCR4staining was significantly decreased in aganglionic colon segments in which ganglion cells were absent.Conclusion:Intensive RET and CXCR4staining was detected in the ganglion in human colon specimens, and the expression levels and distribution of RET and CXCR4were significantly decreased in aganglionic segments from HSCR patients compared with normal ganglionic and oligoganglionic colon segments. Elucidating RET and CXCR4expression patterns in different colon segments from HSCR patients demonstrated that RET and CXCR4eould be diagnostic indicators in clinical molecular diagnostic of HSCR. PART TWO CONSTRUCTION OF RECOMBINANT ADENOVIRUS EXPRESSING RET TARGETED SIRNAObjective:To construct recombinant adenovirus vector Ad5-simRET expressing RET targeted siRNA in order to down regulating of RET expression, and to verify the gene silencing efficiency of recombinant adenovirus.Methods:At first, RET targeted siRNA was directed cloned into adenovirus shuttle plasmid pSES-HUS by recombinant DNA technology. Then recombinant adenovirus shuttle plasmid and the backbone plasmid adenovirus type5were cotransfected into competent BJ5183cells to construct the recombinant adenovirus plasmid by homelogens DNA technology. Further, the recombinant adenovirus plasmid carrying RET targeted siRNA was transfected into HEK293cells to harvest high titer recombinant adenovirus. At last, recombinant adenovirus was transfected into SH-SY5Y, and to verify the gene silencing efficiency of recombinant adenovirus by RT-PCR and Western blot analysis.Results:1) Recombinant adenovirus shuttle plasmid pSES-siRET-site1/2/3and pSES-siRET-scrambled were constructed successfully;2) Recombinant adenovirus plasmid Ad5-siRET-site1/2/3were constructed successfully by homelogens DNA technology, and high titer recombinant adenovirus Ad5-simRET was harvested by transfecting recombinant adeno virus plasmid pools into HEK293cells;3)RT-PCR and Western blot confirmed that the harvested high titer recombinant adenovirus Ad5-simRET could be effective to inhibit the expression of RET.Conclusion:Recombinant adenovirus Ad5-simRET that expressing RET targeted siRNA was constructed successfully, which could be effective to inhibit the expression of RET gene, and it would lay the basis for following research. PART THREE THE ROLE OF RET/CXCR4SIGNALING IN MIGRATION AND OTHER BIOLOGICAL BEHAVIOR OF NEURAL CREST-DERIVED CELLS AND ITS POTENTIAL MECHANSIMSObjective:Recombinant adenovirus Ad5-simRET were transfected into NCSCs and SH-SY5Y cell line to down regulate the expression of RET, to investigate the effect of down-regulation of RET on CXCR4expression and inhibiton of RET/CXCR4signaling on migration and other biological behavior of neural crest-derived cells.Methods:Vagal neural crest stem cells (VNCSCs) was obtained by primary culture and characterized by immunofluorescence staining of P75, Nestin, then identified their pluripotency by immunofluorescence staining of TUJ1, GFAP, aSMA; Recombinant adenovirus Ad5-simRET were transfected into VNCSCs and SH-SY5Y cells to down regulate the expression of RET; The expression level of CXCR4and Etv4/5mRNA in VNCSCs and SH-SY5Y cells treated with Ad5-simRET was detected by Quantitative real-time PCR; Western blotting were performed to analyze the expression levels of RET and CXCR4protein; The effects of Ad5-simRET on the proliferation, apoptosis of VNCSCs and SH-SY5Y cells were evaluated by MTT and flow cytometry, respectively; The migration and invasion potential of Ad5-simRET treatment VNCSCs and SH-SY5Y cells were analyzed using Transwell cell migration and invasion assays, respectively.Results:1) VNCSCs was obtained by primary culture and characterized by immunofluorescence staining successfully;2) Transfection of recombinant adenovirus Ad5-simRET were effective to down regulate the RET expression of VNCSCs and SH-SY5Y cells;3) The expression level of CXCR4and Etv4/5mRNA and CXCR4protein in VNCSCs and SH-SY5Y cells treated with Ad5-simRET was decreased compared to the control group and negative control group;4) Compared with the control group and negative control group, the proliferation of VNCSCs and SH-SY5Y cells were decreased after transfected with Ad5-simRET. However, the apoptosis of VNCSCs and SH-SY5Y cells were no significant difference between the control group, negative control group and Ad5-simRET group;5) Our data showed that transfected with Ad5-simRET result in inhibition of the migration of NCSCs and SH-SY5Y cells, respectively. In addition, AMD3100, a highly specific chemokine receptor CXCR4antagonist, inhibit the migration of NCSCs and SH-SY5Y cells to SDF-1;6) After transfected with Ad5-simRET, the invasion of VNCSCs and SH-SY5Y cells were markedly decreased compared with the control group and negative control group.Conclusion:Down-regulation of RET expression could be inhibit the expression CXCR4, Etv4/5in VNCSCs and SH-SY5Y cells which derived from neural crest. Inhibition of RET/CXCR4signaling give rise to inhibit proliferation, migration, invasion of neural crest-derived cells. PART FOUR THE ROLE OF RET/CXCR4SIGNALING MEDIATED MIGRATION OF NEURAL CREST-DERIVED CELLS IN DEVELOPMENT OF ENTERIC NERVOUS SYSTEMObjective:To investigate the expression patterns and distribution of Ret and Cxcr4in intestinal tissue from mouse embryos at different development stages (E11.5～PN10). To down regulate the expression of Ret via tail vein injection of plasmid vector expressing Ret targeted siRNA, to evaluate the role of Ret/Cxcr4signaling mediated migration of neural crest-derived cells in development of enteric nervous system.Methods:qPCR and Western blot were performed to analyze the expression levels of Ret and Cxcr4mRNA and protein in intestinal tissue from mouse embryos at different development stages; Immunofluorescence staining were performed to analyze the expression distribution of Ret and Cxcr4protein in intestinal tissue from mouse embryos; The expression of Ret was down regulated via tail vein injection of plasmid vector expressing Ret targeted siRNA; Neural crest-derived cells were labeled by P75/Ret immunofluorescence staining and cell count was used to evaluate the role of Ret/Cxcr4signaling mediated migration of neural crest-derived cells in development of enteric nervous system.Results:1) Our data showed that the expression levels of Ret and Cxcr4mRNA in intestinal tissue from mouse embryos at early ENS development stages that is critical for migration, proliferation, colonization of enteric neural crest cells in intestinal tract were significant higher than that at later ENS development stages, and The expression level showed a decreasing trend with ENS development;2) Western blot showed that the expression patterns of Ret and Cxcr4protein is similar to that of Ret and Cxcr4mRNA;3) Transplacental RNAi performed via tail vein injection of plasmid vector expressing Ret targeted siRNA was effective to down regulate the expression of Ret in intestinal tissue from mouse embryos;4) Cell count of P75/Ret immunofluorescence staining labeled neural crest-derived cells demonstrated that suppression of Ret/Cxcr4signaling via tail vein injection of plasmid vector expressing Ret targeted siRNA could be inhibit migration of enteric neural crest cells in intestinal tract.Conclusion:The expression levels of Ret and Cxcr4in intestinal tissue from mouse embryos at early ENS development stage that is critical for migration of enteric neural crest cells in intestinal tract were significant higher than that at later ENS development stage. Suppression of Ret/Cxcr4signaling could inhibit migration of enteric neural crest cells in intestinal tract. Ret/Cxcr4signaling may play an important role in development of enteric nervous system.