| Section 1 Expression of mBD-2 on the Mouse Gastric Mucosa[Objective]The mBD-2 was an important member of defense, which was mainly expressed in the epithelium of the digestive tract, respiratory tract and genital tract. The objective of this study was showed the status of mBD-2 expression on the mouse gastric mucosa.[Methods]1. Immunohistochemistry:The expression of mBD-2 in the gastric mucosa of mouse was observed by in vivo experiments. We made a slice of the mouse gastric mucosa by immunohistochemical method, and observed the expression of mBD-2 in the gastric mucosa of mouse.2. Polymerase chain reaction:The expression of mRNA in the gastric mucosa of mouse was detected by PCR reaction.3. Western blot:The expression of mBD-2 in gastric mucosa of mouse was detected by Western blot.[Results]1. The expression of mBD-2 in the gastric mucosa of mouse:There were many brown granules in the cytoplasm of gastric mucosal epithelial cells of mouse, which indicated that mBD-2 was expressed in the mouse gastric mucosa.2. The expression of mRNA in gastric mucosa of mouse:The expression level of mRNA of mBD-2 in mouse gastric mucosa was significantly.3. The expression of mBD-2 protein in the gastric mucosa of mouse:The expression level of protein of mBD-2 in mouse gastric mucosa was significantly.[Conclusions]The expression of mBD-2 in mouse gastric mucosa studied by immunohistochemistry, PCR and Western blot. The expression of mBD-2 in mRNA level and protein level in gastric mucosa of mouse were detected.Section 2 Expression of mBD-2 on the Mouse Gastric Mucosal Epithelial Cells[Objective]The expression of mBD-2 in the gastric mucosa of mouse was studied by the experiment. In order to further investigate the mechanism of mBD-2 expression, we will conduct experiments in vitro to study the expression of mBD-2 in mouse gastric epithelial cells. In order to lay the foundation for further study on the mechanism of mBD-2 expression.[Methods]1. Immunofluorescence method:Cells cultured and cultured in vitro were cultured and cultured. The expression of mBD-2 in mouse gastric epithelial cells was observed by immunohistochemistry.2. Polymerase chain reaction:RNA was extracted from the mouse gastric mucosal epithelium cells, and the expression of mRNA was detected by PCR reaction.3. Western blot:The expression of mBD-2 protein in the mouse gastric mucosa epithelial cells was detected by Western blot.[Results]1. Expression of mBD-2 in mouse gastric mucosa epithelial cells:The expression of mBD-2 in mouse gastric mucosa epithelial cells was detected by immunofluorescence assay, and the expression of green fluorescence was detected, and no fluorescent protein was detected in the negative control group;2. The expression of mRNA in mouse gastric mucosa epithelial cells:The expression level of mRNA of mBD-2 in mouse gastric mucosa epithelial cells was significantly.3. The expression of mBD-2 protein in mouse gastric mucosa epithelial cells: The expression level of protein of mBD-2 in mouse gastric mucosa epithelial cells was significantly.[Conclusions]These experiments in vitro showed that mBD-2 was expressed in mouse gastric epithelial cells, and expressed in the mRNA level and the protein level. In order to lay a foundation for further study on the mechanism of mBD-2 in vitro.Section 3 the Effect on the Mouse Gastric Mucosal Epithelial Cells Growth and mBD-2 Expression by Escherichia coli[Objective]E. coli was one of the main causes to induce acute gastritis. In this study, the E. coli effected on the mouse gastric mucosal epithelial cells, and the mBD-2 expression level was detected. This provided the basic to the mBD-2 produced mechanism。[Methods]1. MTT method:MTT method for the detection of the optimal concentration and action time of E. coli in the proliferation of mouse gastric epithelial cells in vitro.2. TUNEL method:The apoptosis of the cells was detected by TUNEL assay after E. coli acts on mouse gastric mucosa epithelium cells in vitro.3. Polymerase chain reaction:Different concentrations of Escherichia coli in mice gastric mucosa epithelial cells, collecting the cells after 4,8 and 12h, using quantitative mBD-2 to detect the expression level of in mRNA.4. Western blot:Different concentrations of E. coli in mouse gastric mucosa epithelial cells, collecting the cells after 4,8 and 12h, using Western blot to detect the expression of mBD-2 at protein level.[Results]1. The relationship between the proliferation inhibition rate of E. coli and the concentration of E. coli and the action time of the mouse gastric epithelial cells:The inhibition effect of different concentrations of E. coli on gastric mucosal epithelial cells in mouse was observed in 12,24 and 48h. The inhibition rate of cell proliferation was increased with the increase of the concentration of E. Coli (P<0.05)2. Effect of E. coli on apoptosis of mouse gastric epithelial cells:The apoptosis of cells was observed after 103/ml of E. coli was used to mouse gastric mucosal epithelial cells. It can be seen that the cells with the action of E. coli were found to be in the presence of apoptosis, and the number of green fluorescent cells in the experimental group was more than that in the control group (P<0.05)3. Effect of different concentrations of E. coli on the mRNA expression of mBD-2 in mouse gastric mucosa epithelial cells:The expression levels of 103/ml and 104 mBD-2 mRNA in 4h were significantly higher than those in 10/ml and 102/ml groups (P<0.05), and 103/ml and 104 mBD-2 were significantly higher than 10 and 102/ml in 8h(P<0.05). However, there was no significant difference between different concentrations of E. coli in 12h cells.4. Effect of different concentrations of E. coli on the expression of mBD-2 protein in gastric epithelial cells of mouse:The expression levels of 103/ml and 104 mBD-2 protein in 4h were significantly higher than those in 10/ml and 102/ml groups (P<0.05), and 103/ml and 104 mBD-2 were significantly higher than 10 and 102/ml in 8h(P<0.05). However, there was no significant difference between different concentrations of E. coli in 12h cells.[Conclusions]1. E. coli can inhibit the proliferation of mouse gastric mucosal epithelial cells, and the inhibition rate was positively correlated with the concentration of Escherichia coli and the time of action.2. E. coli can result in the apoptosis of mouse gastric mucosa epithelial cells, and the apoptosis rate is increased gradually with the prolonging of time.3. After E. Coli affected the mouse gastric mucosa epithelial cells, the expression level of mBD-2 was higher in both mRNA and protein levels, and was positively correlated with the time and the concentration of bacteria.Section 4 Mechanism of MAPK on mBD-2 Expression Induced by E. coli in Mouse Gastric Mucosal Epithelial Cells[Objective]In section 3, mBD-2 was induced by E. coli in mouse gastric mucosal epithelial cells. In this study, the mBD-2 production mechanism of MAPK Induced by E. coli was detected.[Methods]The Phosphorylation of p-ERK1/2, p-p38, p-JNK, p-Rsk90 and ERK1/2, p38, JNK and Rsk90 on the mouse gastric mucosal epithelial cells induced by 103/ml E. coli at 0,4,8 and 12h was detected by Western blot.[Results]1. Expression of ERK1/2 in gastric epithelial cells of mouse:After E. coli acts on mouse gastric mucosa epithelium cells, p-ERK1/2 protein phosphorylation levels of 8 and 12h were significantly higher than those in the 0 and 4h groups (P<0.05); p-Rsk90 protein phosphorylation levels of 8 and 12h were significantly higher than those in the 0 and 4h groups (P<0.05).2. Expression of JNK in gastric epithelial cells of mouse:After E. coli acts on mouse gastric mucosa epithelium cells, the expression level of JNK and the phosphorylation level of p-JNK were very low, and no significant difference was expressed (P>0.05).3. Expression of p38 in gastric epithelial cells of mouse:After E. coli acts on mouse gastric mucosa epithelium cells, the expression level of p38 and the phosphorylation level of p-p38 were very low, and no significant difference was expressed (P>0.05).[Conclusions]E. coli can activate ERK1/2 pathway in mouse gastric mucosal epithelial cells.Section 5 mBD-2 and MAPK Protein Expression and Cell Growth After ERK inhibited on Mouse Gastric Mucosal Epithelial Cells[Objective]In section 5 showed E. coli effected mouse gastric mucosal epithelial cells by ERK pathway. In this study, the ERK 1/2 was silenced by RNA interference, and the MAPK and mBD-2 was detected by Western blot. And the mouse gastric mucosal epithelial cells growth status was detected by MTT.[Methods]Rsk90, JNK and p38 was detected by Western blot after ERK silenced. With 103 /ml of E. coli on mouse gastric mucosal epithelial cells at 4,8 and 12h. And the mBD-2 was detected by PCR and Western blot, MTT assay for detection of cell growth at 12,24 and 48h.[Results]1. Detection results of plasmid transfection effect:Cells of blank control, negative plasmid transfection, transfection of plasmid vector were collected, and extracted RNA and total protein. After PCR and Western blot We can found the interference group had no expression of the target gene and protein, and the negative control group and blank control group of the target gene and protein were expressed. It showed that the expression of ERK1 gene was significantly inhibited.2. The expression of mBD-2 after ERK was silenced:Cells were collected after E.coli acted on the mouse gastric mucosal epithelial cells at 0,4,8 and 12h. We found The expression of mBD-2 at mRNA and protein levels were not significantly different, and the expression was lower.3. The expression of other proteins in the MAPK pathway after ERK was silenced:Rsk90, JNK and p38 protein phosphorylation levels were not significantly different at all time points (P>0.05).4. The growth of the mouse gastric mucosal epithelial cells after ERK was silenced:The cell inhibition rate of the interference group was significantly higher than that in the blank group and the negative control group at 12 and 24h (P<0.05). The three groups of cells had the same rate at 48h, and this time almost all of the cells were dead. For the interference group, the inhibition rate was significantly higher than the other two groups, in the 24 hours has reached 97.7%.[Conclusions]After ERK was silenced, the expression of mBD-2 was decreased, which makes the mBD-2 of the mouse gastric mucosal epithelial cells decreased, and the cell growth was more decreased when it was attacked by E. coli. These results further indicate that the expression of mBD-2 in mouse gastric epithelial cells is regulated by ERK. |
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