The Effects Of CX3CR1 On BM MSCs To Repair The Tight Junctions Between Injured Intestinal Epithelial Cells

Objective: In vitro isolated, cultivated and identificated rat bone marrow mesenchymal stem cells(BM MSCs). Using TNF-? to stimulate Caco-2 cells in order to establish the vitro intestinal mucosal epithelial injured model. On the basis of the above cell and model, we investigated the effects of CX3CR1 on BM MSCs to repair the tight junctions between injured intestinal epithelial cells.Methods: 1. BM MSCs were isolated using adherence screening method, cultured to the third generation, identified by the microscope to observe the morphology of BM MSCs, flow cytometry to detect the typical cell phenotype on BM MSCs, the differentiation method to detect the ability of BM MSCs to differentiate into adipocytes and osteoblasts. 2. Caco-2 cells were used to imitate intestinal epithelial cells, using the immune fluorescence technique, RT-PCR and Western blot to assay Occludin and ZO-1 which represented the degree of the intestinal barrier function. We used TNF-a in different concentrations 0, 50, 100 and 200 ng/m L to treat Caco-2 cells so that we can explore the best injured concentration of TNF-a for Caco-2 cells; then we used the best injured concentration stimulate Caco-2 cells 0, 12, 24 and 48 h so that we can explore the best injured hour of TNF-a for Caco-2 cells, by this way we can establish the model of stable injured intestinal epithelial. 3. We used transwell to put BM MSCs co-culture with Caco-2. Then we detected the expressions of tight junctions on Caco-2 and CX3CR1 on BM MSCs through RT-PCR and Western blot. 4.We used CX3CR1 antibody to incubate the receptor on BM MSCs, next put the anti-CX3CR1-BM MSCs co-culture with impaired Caco-2 cells through transwell, continued to detect the expressions of tight junctions on Caco-2 with RT-PCR and Western blot, further clarified the role of CX3CR1 on BM MSCs to repair injured intestinal epithelial cells.Results: 1. We succeeded to extract and culture BM MSCs in vitro. Under the microscopic, we can see BM MSCs attachment growth, cluster growth, like the shape of fibroblas, a swirl or chrysanthemum shaped which is the typical shape characteristics of the MSCs. Flow cytometry detected the third generation of BM MSCs to find that 93.0% of the cells expressed CD90 but negative in CD45, 97.5% of the cells expressed CD29 but negative in CD34, 97.0% of cells expressed RT1 A but negative in RT1 B, indicating that when the primary cells which we extracted cultured to the third generation, they would be extremely pure, almost no hematopoietic stem cells or other hybrid cells. Put the third generation of BM MSCs culture under the lipid medium, using Oil Red O staining to see the red lipid droplet in the cells, under the bone formation medium, using Von Kossa staining to see the black salt deposit. These suggested that the cells we extrated were what we need. 2. Caco-2 were treated by TNF-a with different concentrations and different time. Through immune fluorescence, we could see that ZO-1 protein connected to a net, when the TNF-a concentration increased, the time extended, the net of ZO-1 would be destroy, the fluorescence intensity decreased. Through RT-PCR and Western blot, we detected that the expression levels of Occludin and ZO-1 reduced more and more obviously. But 100 ng/m L and 200 ng/m L TNF-a did not have the significant difference. Finally, we validated that the most suitable TNF-a condition was 100 ng/m L and 48 hours. 3. BM MSCs co-culture with injured Caco-2, RT-PCR showed that the levels of Occludin and ZO-1 m RNA on Caco-2 increased than injured Caco-2, the levels of CX3CR1 m RNA on BM MSCs increased too; Western blot showed that the levels of Occludin and ZO-1 protein on Caco-2 increased than injured Caco-2, the levels of CX3CR1 protein on BM MSCs increased too. The levels of m RNA were consistent with the levels of protein. This proved that BM MSCs has ability of repairing injured intestinal epithelial cells. 4. Anti-CX3CR1-BM MSCs co-culture with injured Caco-2, RT-PCR results showed that the levels of Occludin and ZO-1 m RNA on Caco-2 were less than the levels before blocking CX3CR1, but higher than the levels of injured Caco-2 group. Western blot results showed that the levels of Occludin and ZO-1 protein were lower than those before blocking CX3CR1, but higher than those of injured Caco-2 group. It revealed that the chemokine receptor CX3CR1 on BM MSCs could take part in repairing tight junctions between the injured intestinal epithelial cells.Conclusions: 1. The adherence screening method could be successfully extracted and cultured BM MSCs. 2. 100 ng/m L TNF-a was used to stimulate Caco-2 48 h to establish the stable injured intestinal epithelial model. 3. BM MSCs can through the CX3CR1 promoting the expressions of the intestinal epithelial tight junction protein and m RNA, protecting the injured intestinal mucosal epithelial cells. The effects were attenuated after blocking CX3CR1, confirmed that CX3CR1 participates in BM MSCs repairing tight junctions between the injured intestinal epithelial.