Proliferation And Migration Of Bone Marrow Mesenchymal Stem Cells By Co-culture With TM4Sertoli Cells

Bone marrow mesenchymal stem cells (BM-MSCs) are gained easily and are capable of differentiating into a variety of cell types such as chondrocytes, osteoblasts, fat cells, muscle cells and liver cells, BM-MSCs are considered as a promising option in the field of regenerative medicine and tissue engineering. Sertoli cells (SCs) are well known as the "nurse cells" because they can secrete many growth cytokines. The purpose of thesis focuses on the BM-MSCs proliferation and migration by co-culture with TM4sertoli cells. The main results are as follows:(1) An in vitro transwell system was set up after identified the BM-MSCs by flow cytometry. The optimal cell proportion between TM4cells and BM-MSCs was10:1and optimal co-cultured time was48h. Furthermore, cell cycle analysis showed that co-culture with the TM4cells accelerated the progress of BM-MSCs from the G1to the S phase. In addition, the apoptosis of BM-MSCs changed little when co-cultured with TM4cells.(2) The effect of epithelial growth factor (EGF), interleukin-6(IL-6), stem cell factor (SCF) and insulin-like growth factor-1(IGF-1) on the proliferation of BM-MSCs were researched in the paper. The results clarified that the proliferation of epithelial growth factor EGF-treated BM-MSCs was remarkably higher than other groups, increasing the number of cells by1.58times compared with the control. Meanwhile, the cell density was increased by1.29times in the IGF-1-treated group and1.27times in SCF-treated group, respectively. However, For the IL-6-treated group, it did not show a statistical significance in comparison of control group. Apparently, EGF does a great contribution to the proliferation of BM-MSCs. In addition, BM-MSCs in co-culture had markedly increased expression of p53, Mdm2and phospho-Akt, as well as the cell cycle signals phospho-CDC2and cyclin Dl. The results were then quantified by Quantity One software. When LY294002(5μM), the PI3K/AKT-specific inhibitor, was added to the co-cultured group for one hour, a decrease in cell proliferation of approximately37.5%was observed. At the same time, the expression of phospho-Akt was also inhibited; however, the total Akt expression remained unchanged based on the statistical data by Quantity One software. It implies that the PI3K/AKT pathway could be involved in the enhanced BM-MSCs proliferation by co-culture with TM4cells.(3) The migration cell numbers of the co-cultured group increased by about6.1times compared with the control group. And the transforming growth factors β3(TGFβ3), stromal cell-derived factor-la (SDF-la) and MAPK pathway might be involved in the enhanced BM-MSCs migration by co-culture with TM4cells. And there was no synergy effect between the TGFβ3and SDF-la on the migration of BM-MSCs, but the inhibition effect.In conclusion, the proliferaton and migration of BM-MSCs could be enhanced by co-culture with TM4sertoli cells, at least EGF, TGFβ3and PI3K/AKT pathway involved in it.