The Research Of The Bone Marrow Mesenchymal Stem Cells Differentiate Into The Intestinal Epithelioid Cells Through ERK1/2 Pathway

Objective:The aim of this paper is to discuss the mechanism of intestinal epithelial cells in inducing differentiation of bone marrow mesenchymal stem cells,and to provide reference for differentiation mechanism of mesenchymal stem cells.Methods:1.We extracted the bone marrow mesenchymal stem cells from the femur and tibia of rats by the whole bone marrow adherent culture method,and identified them with the cell morphology,surface antigens,and cell adipogenesis induction.2.The experimental group was co-cultivation group(BMSCs+ IEC-6)and blank control group(BMSCs+BMSCs).IEC-6 and BMSCs were respectively placed in upper and lower rooms of Transwell for 3,7 and 10 days to induce the differentiation of bone marrow mesenchymal stem cells,and then we detected the expression of CK and Villin by immunofluorescence technique.3.The experimental group was co-cultivation group(BMSCs+IEC-6)?the inhibitor group(BMSCs+ IEC-6 +U0126)and the blank control group(BMSCs+BMSCs).Co-cultivated in the Transwell for 36h and 72h,then we detected the expression of ERK1/2 pathway related protein p-ERK1/2 and p-MEK1/2 with the Western Blot.Results:1.We extracted the bone marrow mesenchymal stem cells with the whole bone marrow adherent culture method.The cells we got were spindle-shaped and radial arrangement,then the cell surface antigen marker CD29 was positive,the positive rate was 99.77%,the cell surface antigen marker CD34 was positive,and the negative rate was 9.71%.It also could be induce to differentiate into adipose cell,and the oil red O dye was red,so we thought it met the criterion of BMSCs.2.We co-cultivated the bone marrow mesenchymal stem cells and the intestinal epithelial cells in Transwell for 3?7 and 10 days,and the result showed that the cells what we induced express epithelial cell characteristics of cytokeratin(CK)and villous protein(Villin protein).With the extension of co-culture time,the morphology of cells changed significantly.At the 3 days of co-cultivation,there were very few cells morphology changed;when co-cultured for 7 days,some cells became round-shaped;and when co-cultivated for 10 days,most cells changed from long shuttle to round.3.After detection of ERK1/2 pathway related proteins by Western Blot,we found that the simple stem cell group did not express p-mek1/2 and p-ERK1/2;The expression levels of p-mek1/2 and p-ERK1/2 were highly expressed in the experimental co-culture group without ERK1/2 pathway inhibitor U0126,and the expression quantity of the co-culture group of 72h was higher than that of the co-culture group of 36h,and the difference was statistically significant(p<0.05).The expression levels of the inhibition group of 36h p-mek1/2 and p-ERK1/2 were significantly lower than those in the experimental co-culture group,and the difference was statistically significant(p<0.05).There was no statistically significant difference in the expression of p-mek1/2 and p-ERK1/2 in the inhibition group of 72h(p>0.05).After adding ERK1/2 pathway inhibitor U0126(the inhibition group for 36h),phosphorylation of MEK1/2 was inhibited,and its downstream ERK1/2 could not be activated,resulting in that the downstream ERK1/2 could not be phosphorylated to affect the next step of cell differentiation.Conclusion:1.BMSCs of rats could be isolated and cultured in vitro with the whole bone marrow adherent culture method,and the cell activity was good and could be used for follow-up experiments.2.After differentiation of BMSCs induced by IEC-6 in Transwell,BMSCs could be induced to differentiate into intestinal epithelial-like cells.Not only the changes of the cell morphology,but also the CK and Villin proteins of intestinal epithelial cells could be expressed.3.The expression of p-MEK1/2 and p-ERK1/2 was significantly decreased in the inhibitory group of E01K1/2 pathway inhibitor U0126(inhibition group 36h),and the phosphorylation of MEK1/2 was inhibited,so its downstream ERK1/2 could not be activated,resulting in that the downstream ERK1/2 could not be phosphorylated to affect the next step of cell differentiation.It is indicated that the branch pathway ERK1/2 pathway of MAPK pathway plays an important role in the differentiation of BMSCs to intestinal epithelioid cells.