|
Barrett's esophagus (Barrett's esophagus, BE) refers to the junction of esophageal squamous columnar epithelium migrate to the proximal gastroesophageal junction and associated with intestinal metaplasia of the disease. BE is a precancerous lesion, and adenocarcinoma of the esophagus (esophageal adenocarcinoma, EAC) are closely related. In Western countries, by the BE transformed from the incidence of esophageal adenocarcinoma was significantly increased, becoming the fastest growing of all malignant one. Asian countries such as BE and esophageal adenocarcinoma Western countries, though not common, but in recent years, epidemiological data also show an increasing trend, so China has accelerated and in-depth research in this field, and through interventions for prevention of great significance. Gastrin (Gastrin) G is a gastrointestinal peptide hormone secreted by cells, stimulate gastric acid secretion, and promote the growth of gastrointestinal mucosa. Gastrin receptors are present in the stomach CCK 2 receptor (CCK2R), belonging to the G protein-coupled receptor superfamily, it regulates the secretion of gastric parietal cells, enterochromaffin-cadmium cells and smooth muscle cells release histamine contraction. Gastrin binding to its receptor, by activating a plurality of signal transduction pathways, the mitogenic signal to the nucleus, causing cell proliferation. In recent years, more and more studies show that gastrin is also involved in a number of cancer development and progression. In the pancreas and stomach, it was found CCK2R regulate the growth of, and in many tumors have been found in CCK2R existence. Recent studies have found abroad also exist in Barrett metaplasia CCK2R, and Barretts esophagus is a precancerous lesion of esophageal adenocarcinoma. This prompts us: in esophageal adenocarcinoma may also have CCK2R presence and in the occurrence and development of esophageal adenocarcinoma play a role. At present, for CCK2R signaling mechanism is not clear from the existing research results, in addition to the classic inositol phospholipids, cAMP signaling pathway, may also be a mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) of participation. MAPK is the late 1980s, growth factor phosphorylated product research and found a class of serine / threonine protein kinase family, MAPK pathway is the regulation of inflammation, proliferation, differentiation, apoptosis and other cell biological effects of key cell signaling pathway. Its members ERK1 / 2 is p42/44MAPK, the signal passes the cell mitogen key kinase signal after activation can activate various nuclear transcription factors, affect gene expression, cell growth, differentiation and proliferation regulation close . Therefore, this study started from gastrin, through its specific blocking receptors studied gastrin and its receptor in human esophageal adenocarcinoma cells (SEG-1) proliferation role in the process, in order to explore The mechanism for the future clinical prevention provide a theoretical basis. Objective: To investigate gastrin and its receptor in human esophageal adenocarcinoma cell line SEG-1 proliferation in the mechanism for future prevention and treatment work to provide a theoretical basis. Method: 1 in vitro cell culture techniques, the application of MTT assay effect of different concentrations of gastrin SEG-1 cell proliferation; 2 RT-PCR was used to detect CCK2R mRNA in gastrin and gastrin Proglumide proglumetacin amine the expression level of the three groups; 3 with the same concentration of gastrin (10-7 mol / L) at time gradient stimulated cells, Western blot determined at each time point total p42/44 MAPK, phosphorylated p42/44 MAPK protein expression to determine the phosphorylation of p42/44 MAPK maximum point in time. 4 Western blot assay, the intervention group total p42/44 MAPK, phosphorylated p42/44 MAPK phosphorylation of proteins in the maximum expression of time. Results: ① gastrin (G-17) to SEG-1 cell proliferation :10-9mol / L ~ 10-5mol / L G17 acts on the SEG-1 cells after 24h, the proliferation rate was 108.79%, 117.23 %, 125.38%, 138.53%, 150.33%, compared with the control group, was significantly higher, statistically significant (P lt; 0.05). ② proglumide (PGL) for SEG-1 cell proliferation :10-8mol / L ~ 10-5mol / L of PGL role in SEG-1 cells after 24h, the growth inhibition rates were 14.23%, 18.37%, 34.24%, 55.97%, significantly inhibited the proliferation of SEG-1 cells were statistically significant (P lt; 0.05). ③ gastrin (G-17) proglumide (PGL) the SEG-1 cell proliferation :10-8mol / L ~ 10-5mol / L acts on the PGL containing 10-7mol / L G-17 in SEG -1 cells after 24h, the growth inhibition rates were 0.57%, 3.50%, 5.00%, 12.22%, significantly inhibited the G-17 对 SEG-1 cell growth-promoting effect. ④ human esophageal adenocarcinoma cell line SEG-1CCK2R gene expression: detected by RT-PCR analysis showed that human esophageal adenocarcinoma cell lines SEG-1 gene expression in CCK2R, G-17 acts on the SEG-1 cells CCK2R/GAPDH ash ratio of 0.78 degrees, decreased expression; PGL role in cell ratio was 1.59 after the gray that appear upregulated; G-17 with PGL also act on cells grayscale ratio of 1.37, the ratio is still higher than basal expression gray 1.03. ⑤ gastrin (G-17), proglumide (PGL) acting on the SEG-1 cells p42/44 MAPK phosphorylation changes: Western blot showed that, G-17 acts on the SEG-1 cells p42/44 MAPK phosphorylation was increased; and p42/44 MAPK phosphorylation with the G-17 action time and change, 0min, 5min, 10min, 20min, 40min of p42/p44 MAPK phosphorylation percentages were 3.92% , 19.62%, 39.58%, 22.41%, 14.93%, showing that the p42/44 MAPK phosphorylation 10min highest level, so that the maximum time point p42/44 MAPK phosphorylation point in time. PGL role in cell after p42/44 MAPK phosphorylation decreased (1.74%); using PGL G17 act on the cells, p42/44 MAPK phosphorylation (21.68%) was significantly higher than G-17 group (41.32%) is low, only slightly higher than the control group (5.94%), indicating that the G-17 to make p42/44 MAPK phosphorylation could be inhibited by PGL. Conclusion: a gastrin can promote human esophageal adenocarcinoma cell line SEG-1 growth in a dose-dependent manner. Gastrin receptor antagonist proglumide can inhibit gastrin on SEG-1 cell growth-promoting effect. Two human esophageal adenocarcinoma cell line SEG-1 gene expression in CCK2R, gastrin acts on cells expressing declined Proglumide role in cell after having been upregulated, gastrin and Proglumide simultaneously on cells and its expression is still higher than basal expression. 3 under the action of the same concentration of gastrin, gastrin receptors CCK2R caused by p42/44MAPK a time-dependent phosphorylation. 4 gastrin can induce human esophageal adenocarcinoma cell line SEG-1 phosphorylation within p42/44MAPK, suggesting that gastrin on MAPK pathway was activated.
|