| Purpose(1)To explore the origin of duodenal gastrinoma and its association with Men1.(2)To establish the method of culturing primary enteric glial cells(EGCs)from duodenum of mice.(3)To evaluate the effect of EGF on the expression of Men1 and analyze the possible mechanisms in primary EGCs from mice.(4)To analyze the effect of Men1 in the gastrinoma of mice.Methods(1)Test the expression of GFAP and S100 B in specimens from human duodenal gastrinoma by using immunofluorescence,which are the markers of neural crest;and compare the expression level of menin in tissues from tumor with that from para tumor.(2)Culture primary EGCs from duodenal lamina propria of mice,test the expression of GFAP,SMA,E-cadherin and Pgp9.5 of primary EGCs by using immunofluorescence and western blot,and detect the purity of primary EGCs by flow cytometry.(3)EGF was added into the media for culturing primary EGCs.The Men1 m RNA level of primary EGCs was tested by Q-PCR after the effect of EGF,the expression and distribution of menin were tested by immunofluorescence and western blot,and the gastrin level was tested by ELISA.The possible mechanisms of EGF on primary EGCs were predicted by using bioinformatics,and were verified by using western blot and immunoprecipitation.(4)Construct the mouse model with conditional deletion of Men1(Gastrin Cre ERT2)and whole body deletion of Somatostatin(Gastrin Cre ERT2 Men1 fl/fl Sst-/-),treat the mice with omeprazole,and then observe the occurrence of duodenal gastrinoma of the mice.Results(1)Both GFAP and S100 B were positive in specimens from human duodenal gastrinoma.Compared with the expression of tissues from para tumor,the menin level was lower in gastrinoma,and menin located in the cytoplasm mainly.(2)We cultured primary EGCs with positive GFAP from duodenum of mice sucessfully.And the primary EGCs were negative for SMA,E-cadherin and Pgp9.5 by using immunofluorescence and western blot.About 96% of primary cells were positive for GFAP by using flow cytometry.(3)After the effect of EGF,menin of primary EGCs was translocated into cytoplasms from nucleus firstly,and then degradated in the cytoplasm.But Men1 m RNA level did not change significantly.The gastrin secreted by EGCs increased significantly.Erk2 and Akt might take part in the translocation and degradation of menin,which was predicted by bioinformatics.After the effect of EGF,Erk2 and Akt were phosphorylated,and binded to menin in the nucleus and cytoplasm,respectively,which were confirmed by western blot and immunoprecipitation.In addition,the ubiquitinated menin increased after the effect of EGF,and inhibitors of Erk2 and Akt can block the translocation and degradation of Menin induced by EGF.(4)After treated with omeprazole for 6 months,no duodenal gastrinoma was found in mice with conditional deletion of Men1(Gastrin Cre ERT2)and whole body deletion of Somatostatin(Gastrin Cre ERT2 Men1 fl/fl Sst-/-).In addition,no significant increase of serum gastrin level or the number of cells with positive gastrin in the duodenum was tested.Conclusions(1)Human duodenal gastrinoma originates from neural crest cells(enteric glial cells).Men1 is closely associated with duodenal gastrinoma.(2)Primary EGCs were cultured from duodenum of mice successfully,with a purity of 96%,which can be used for in vitro studies.(3)EGF can induce the nuclear translocation and cytoplasmic degradation of menin in primary EGCs through ubiquitin-proteasome system,which is mediated by Erk2 and Akt.The degradation of menin stimulated the release of gastrin.These suggested that EGF accelerated the delevelopment of gastrinoma from EGCs by decreasing the expression of menin.(4)Conditional deletion of Men1(Gastrin Cre)combined with whole body deletion of Somatostatin and short-term treatment with omeprazole can not induce the formation of duodenal gastrinoma in mice.Long term observation and mouse model with other kinds of conditional deletion of Men1(such as GFAP Cre)are needed in future.Part 1 The origin of duodenal gastrinoma and its association with Men1Purpose: To explore the origin of duodenal gastrinoma and its association with Men1.Methods: Test the expression of GFAP and S100 B in specimens from human duodenal gastrinoma by using immunofluorescence,which are the markers of neural crest;and compare the expression level of menin in tissues from tumor with that from para tumor.Results: Both GFAP and S100 B were positive in specimens from human duodenal gastrinoma.Compared with the expression of tissues from para tumor,the menin level was lower in gastrinoma,and menin located in the cytoplasm mainly.Conclusions: Human duodenal gastrinoma originates from neural crest cells(enteric glial cells).Men1 is closely associated with duodenal gastrinoma.Part 2 The culturing and identification of primary glial cells from the duodenum of micePurpose: To establish the method of culturing primary enteric glial cells(EGCs)from duodenum of mice.Methods: Culture primary EGCs from duodenal lamina propria of mice,test the expression of GFAP(the marker of neural crest cells),SMA(the marker of myofibroblast),E-cadherin(the marker of epithelial cell)and Pgp9.5(the marker of neuron)of primary EGCs by using immunofluorescence and western blot,and detect the purity of primary EGCs by flow cytometry.Results: We cultured primary EGCs with positive GFAP from duodenum of mice successfully.And the primary EGCs were negative for SMA,E-cadherin and Pgp9.5 by using immunofluorescence and western blot.About 96% of primary cells were positive for GFAP by using flow cytometry.Conclusions: Primary EGCs were cultured from duodenum of mice successfully,with a purity of 96%,which can be used for in vitro studies.Part 3 The effect of EGF on Men1 in the primary glial cells from the duodenum of mice and its possible mechanismPurpose: To evaluate the effect of EGF on the expression of Men1 and analyze the possible mechanisms in primary enteric glial cells(EGCs)from mice.Methods: EGF was added into the media for culturing primary EGCs.The Men1 m RNA level of primary EGCs was tested by Q-PCR after the effect of EGF,the gastrin level was tested by ELISA,the expression and distribution of menin were tested by immunofluorescence and western blot,and the gastrin level was tested by ELISA.The possible mechanisms of EGF on primary EGCs were predicted by using bioinformatics,and were verified by using western blot and immunoprecipitation.All the experiments were repeated by using STC1 cell line,which were derived from intestinal tumor of mice.Results: After the effect of EGF,menin of primary EGCs was translocated into cytoplasms from nucleus firstly,and then degradated in the cytoplasm.But Men1 m RNA level did not change significantly.The gastrin secreted by EGCs increased significantly.Erk2 and Akt might take part in the translocation and degradation of menin,which was predicted by bioinformatics.After the effect of EGF,Erk2 and Akt were phosphorylated,and binded with menin in the nucleus and cytoplasm,respectively,which were confirmed by western blot and immunoprecipitation.In addition,the ubiquitinated menin increased after the effect of EGF,and inhibitors of Erk2 and Akt can block the translocation and degradation of Menin induced by EGF.All the experiments were repeated in STC1 cells successfully.Conclusions: EGF can induce the nuclear translocation and cytoplasmic degradation of Menin in primary EGCs through ubiquitin-proteasome system,which is mediated by Erk2 and Akt.The degradation of menin stimulated the release of gastrin.These suggested that EGF accelerated the delevelopment of gastrinoma from EGCs by decreasing the expression of menin.Part 4 The effect of conditional deletion of Men1 on the serum gastrin level and development of duodenal gastrinoma in micePurpose: To analyze the effect of Men1 in the gastrinoma of mice.Methods: Construct the mouse model with conditional deletion of Men1(Gastrin Cre ERT2)and whole body deletion of Somatostatin(Gastrin Cre ERT2 Men1 fl/fl Sst-/-),treat the mice with omeprazole,and then observe the occurrence of duodenal gastrinoma of the mice.Results: After treated with omeprazole for 6 months,no duodenal gastrinoma was found in mice with conditional deletion of Men1(Gastrin Cre ERT2)and whole body deletion of Somatostatin(Gastrin Cre ERT2 Men1 fl/fl Sst-/-).In addition,no significant increase of serum gastrin level or the number of cells with positive gastrin in the duodenum was tested.Conclusions: Conditional deletion of Men1(Gastrin Cre)combined with whole body deletion of Somatostatin and short-term treatment with omeprazole can not induce the formation of duodenal gastrinoma in mice.Long term observation and mouse model with other kinds of conditional deletion of Men1(such as GFAP Cre)are needed in future. |
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