Study On The Mechanism Of Doc2a Gene Down-expression In Hirschsprung’s Disease

PART ONE The expression of Doc2 a is down-regulated in aganglionic segments of Hirschsprung’s diseaseObjective: The study is to observe the expression of Doc2 a in aganglionic segments of HSCR.Methods: We collected the colon tissues of HSCR patients and HSCR model mice.Western-blot was to show the protein expression of Doc2 a gene;RT-q PCR was to show the RNA expression of Doc2 a gene;immunofluorescence(IF)and histochemical(HE)staining were help for verifying the expression change of Doc2 a gene.Results: 1.Ednrb-/Ednrb-mice are typical model mice of HSCR.The phenotypes of Ednrb+/Ednrb+ and Ednrb+/Ednrb-mice are the same.The genotype of Ednrb+/Ednrb+ and Ednrb+/Ednrb-mice needs identification,while the Ednrb-/Ednrb-mice can be distinguished by the white patches.2.The results of Western blot showed that the protein expression of Tuj1 and Doc2 a was hardly expressed in narrowed segments of HSCR;the result of RT-q PCR showed that the RNA expression of Tuj1 and Doc2 a was hardly expressed in narrowed segments of HSCR;the results of HE showed that the narrowed segments of HSCR were aganglionic,and few neurons were found in transition zone;the result of IF showed that Doc2 a expressed hardly in narrowed segments of HSCR and Tuj1 was the marker of neurons.Conclusion: The expression of Doc2 a in aganglionic segments of HSCR patients and model mice is down-regulated obviously.PART TWO Phenotypic study of knockdown Doc2 a gene in Neuro-2a cell lines and Enteric neural crest cellsObjective: To investigated the effects of Doc2 a down-regulation on Neuro-2a cell line and Enteric neural crest cells in vitro.Methods: Model construction: We knocked down the expression of Doc2 a gene in neuro-2a cell line and ENCCs to observe the changes about the neuronal polarity and nerve fiber connections,as well as the corresponding changes in cell cycle,proliferation,apoptosis and migration via sh RNA lentivirus transfection,IF,CCK8 proliferation experiment,flow cycle experiment,flow apoptosis experiment and Transwell migration experiment.Result: 1.The Neuo-2a cell line is a good tool for studying neuronal diseases in vitro,and ENCCs are progenitor cells of enteric neurons.Sh RNA lentivirus transfection technique was used to knock down the expressionof Doc2 a gene.2.In the experiments of knocking down the Doc2 a gene: the results of IF showed that the number of neurites increased and the neuronal polarity enhanced in the knock down group;the results of CCK8 proliferation experiments showed that the proliferation of knock down group slowed down;the results of flow cycle and flow apoptosis experiments showed that the apoptosis rate of the knock down group increased;and the results of transwell migration experiments showed that the migration abliity of the knock down group was significantly weaked.3.In the experiments of knocking down the ENCCs: the knock down group showed accelerated differentiation and enhanced polarity of nerve balls,which were manifested by the connection and interlacing of nerve fibers network in the process of culture;the results of IF showed that blank control group and negative control group rarely adhered to the wall,which means low-differentiation with the fibers extending only inside,and the knock down group was high-differentiation with the fibers extending intricately outside.Conclusion: The down regulation of Doc2 a gene can enhance the neuronal polarity,increase the number of neurites and nerve fiber connections,which is the similar to the phenomenon that parasympathetic and sympathetic fibers proliferate largely and distribute intricately in narrowed segments of HSCR.PART THREE Study on the mechanism of Doc2 a and its protein interaction molecules in the aganglionic segments of HSCRObjective: To preliminarily study the mechanism of Doc2 a and its protein interaction molecules in the aganglionic segments of HSCR.Methods: In the knock down group of Neuro-2a cell line and ENCCs,HSCR patients and HSCR model mice,the Western blot and RT-q PCR were to show the expression change of the interacting proteins with Doc2 a,and the COIP was to explore and verify the downstream molecules interacting with Doc2 a.Results: 1.In the Neuo-2a cells and ENCCs,the down-regulation of Doc2 a could down-regulate the expression of Snap25 and Unc13 a by half,while the expression of Unc13 b was double approximately.2.In the narrowed segments of HSCR patients and model mice,the results of Western blot showed that the protein expression of Unc13 b were up-regulated obviously;and the results of RT-q PCR showed that the RNA expression of Unc13 b were double.3.In the experiments of COIP,the protein interaction experiments between Doc2 a and Unc13 b were verified positively and negatively.We found that the same protein bands exhibited in both the experimental group and the INPUT group,indicating that Doc2 a could interact with Unc13 b protein,and there was a direct or indirect effect between them.Conclusion: The down-regulated expression of Doc2 a can up-regulate the expression of Unc13 b,and there is a protein interaction between Doc2 a and Unc13 b.Therefore,we hypothesize that the down-regulation of Doc2 a gene in the aganglionic segments of HSCR due to genetic and environmental factors activates a large release of excitatory neurotransmitters via the up-regulation of Unc13 b,which may induce the occurrence of HSCR.