| Background and purpose:Glucagon-like peptide-2 is a specific growth factor acted on intestinal epithelium. It is the product of glucagon genes of a kind of endocrine cells called L cells. Previous studies suggested that GLP-2 could promote the growth of intestinal epithelium, stabilize gut-barrier function in animal models, and promote repair and healing of damaged intestinal tissues caused by several chronic bowel diseases and intestinal mucosa injuries. Therefore, GLP-2 had value in clinical applications of reducing gut injuries caused by possible kinds of diseases.In recent years, the functions of GLP-2 on the protection and repair of the small intestine were investigated in vivo by using animal models. Our early experiments studied the promoting and protective functions of GLP-2 on transplantated small intestine and mesenteric ischemia-reperfusion injury. However, researches failed to go forward at the cellular level because of the difficulties in separation and counting of intestinal epitheliums. This study will investigate the GLP-2's functions and its mechanism of actions at the primary cultivated intestinal epithelial cells.Methods:We used the 24-hours-old SD rats. The small intestine was taken out and the intestinal fossae cells were separated and cultured. Cultivating for six to eight days, the intestinal epithelial cells grew and showed a cobblestone appearance and then were used for the experiments. In this experiment, we choosed six wells as a group. We would analyse the results of the experiment between groups.1. The cells were divided into four groups including control group, 2nmol/L GLP-2 group,20nmol/L GLP-2 group and 200nmol/L GLP-2 group. Then we could observe the function of GLP-2 in different doses on intestinal epithelial cells respectively at the fourth hour, second day, fourth day, sixth day and eighth day.2. The growth curve of intestinal epitheliums was obtained by the MTT colorimetric assay at the fourth hour, second day, fourth day, sixth day and eighth day to grope for the specific dose of GLP-2 which had the most obvious promoting effect on small intestinal epithelial cells. PCNA assay was used to observe the action time of GLP-2 dose group at the second day, fourth day and sixth day.3. The distribution of GLP-2 receptors were detected in early, middle and late periods of primary culture. GLP-2 receptors could be observed in epitheliums, fibroblast and endocrine cells.4. Injury models were made by hydrogen peroxide The activity of LDH, SOD and MDA were detected to study the protective effect of GLP-2 on intestinal epithelial cells.Results:1. The primary culture of small intestinal epithelial cells.Morphological observations indicated that the crypt cells of small intestinal mucosa formed pseudopodium, adhered to the wall and individual or formed a pile distributing in 2 to 4 days. They formed cell clusters in 6 to 8 days and appeared like cobblestones in 8 to 10 days of cell culture. The growth stopped after 11 days.2.20nmol/L GLP-2 has the best effect of proliferation on the intestinal epithelial cells.Results of MTT showed that:Compared with the control group, GLP-2 groups all significantly promoted proliferation (p<0.05).20nmol/L GLP-2 had the strongest effect on cells proliferation (p<0.01). Results of PCNA indicated that on the fourth day of cell culture GLP-2 began to act, and the most obvious effect of proliferation was observed on the sixth day.3. GLP-2 receptors were detected by immunocytochemistry.GLP-2 receptors could be observed in epitheliums,fibroblast and endocrine cells.4. GLP-2 has the protective effect against the injury caused by hydrogen peroxide on intestinal epithelial cells.The cells were divided into three groups which were control group, injury group and 20nmol/L GLP-2 protective group. Hydrogen peroxide was applied as a poisoning agent at the sixth day since planting. The activity of LDH, SOD and MDA were detected. Results showed that the LDH activity in supernatant of GLP-2 group decreased by 19.36% obviously (P<0.01) compared with injury group, the SOD activity of GLP-2 group increased by 76.67%(p<0.01) compared with injury group, and the MDA activity decreased by 28.16%(P<0.01).Conclusion:The experimental results mentioned above suggested that GLP-2 significantly promoted the proliferation of primary cultured intestinal epithelial cells, and this action might be mediated through GLP2 receptors. GLP-2 played a protective role on intestinal epitheliums against H2O2 injury in mice.
|
PDF Full Text Request