Effects Of MiR-155 On Immunologic Factors And Its Mechanism Of Mesenchymal Stem Cells Under Hypoxic Environment

Objective: To investigate the effects of proliferation, cycle and miR-155 on immunologic factors and its mechanism of human umbilical cord mesenchymal stem cells under hypoxia.Method: After stripping off arteries and veins, the remaining parts of umbilical cord were cut into small sections and cultured with DMEM/F12 containing 10% fetal bovine serum. Adhere cells were obtained and the morphology of cells was observed under inverted phase contrast microscope. The growth curves of them were drawn by CCK-8 and the cell cycle and surface antigens were detected by Flow Cytometry. The micro-RNA sequences targeting miR-155 mimic and mimic NC genes were designed and transfected into hUC-MSCs by lipofectamineTM 2000. Lipopolysaccharide is used to stimulate the immunofaction of hUC-MSCs under hypoxic environment. The experiment was set-up into six groups, 1the blank control group was hUC-MSCs cells that was not treated, 2the control group treated by LPS, 3miR-155 mimic-NC + LPS, 4miR-155 mimic + LPS, 5hypoxia + miR-155 mimic-NC + LPS, 6hypoxia + miR-155 mimic + LPS. Transfectional efficiency of miR-155 and immune-related genes（IL-6, IL-8, iNOS, SDF-1a, TGF-β, HIF-1α） were detected by q RT-PCR. The related protein（iNOS and HIF-1α） and supernatant cytokines（IL-6, IL-8, TGF-β, SDF-1α） were analyzed by Western Blot and ELISA, Respectively.Result: Seven to ten days after primary culture, adhere cells came out from fragments. The hUC-MSCs harvested were homogenous population and exhibited a spindle-shaped fibroblastic morphology. The cell growth curve showed that hUC-MSCs had a strong ability of proliferation. The cells were positive for CD29、CD73、CD90、CD105, while negative for CD31、CD14、CD34、CD45、CD11b、HLA-DR. More than 80% cells of hUC-MSCs were found at G0/G1 phase. CoCl₂ induced-chemical hypoxia may promote the proliferation of hUC-MSCs and depend on the role of times and concentrations of CoCl₂. Mi R-155 was transfected into hUC-MSCs effectively（P<0.05）. In miR-155 high-expressed groups, the expressions of IL-8 and IL-6 were up-regulated and iNOS was markedly suppressed. Hypoxia up-regulated expressions of HIF-1α and promoted the regulation of miR-155. There was statistical significance（P<0.05）. However, miR-155 had no significant effect on the expression of SDF-1α, TGF-β, and had not statistical significance（P>0.05）.Conclusion: hUC-MSCs could be cultured and proliferated in vitro and could be used for tissue engineering. CoCl₂ induced-hypoxia environment may promote the proliferation of hUC-MSCs and the optimal concentration is 200μmol/L CoCl₂. However, higher concentration（≥250μmol/L CoCl₂） inhibits the proliferation. Hypoxia environment may promote miR-155 to inhibit immunosuppression of MSCs, through up-regulating the expression of HIF-1α, which down-regulated iNOS protein.