| Objective:To acquire the purified rat bone marrow mesenchymal stem cells (BMSCs) through adherent culture for further study.Methods:BMSCs were isolated from rats marrow, adherent cultured and ampified in Mesenchymal Stem Cell Growth Medium containing low-glucose DMEM supplement with10%FBS. The morphology of BMSCs was observed under an optical microscope at different time. Cell growth curve was drawn according to the MTT assay results. Flow cytometry was used to determine the surface markers (CD34, CD44, CD45, CD29) expression. Differentiation into osteoblasts and adipocytes was induced by the conditioned culture mediums in vitro.The inductive results were tested by alizarin red staining and oil red O staining separately.Results:After the inoculation for48h, Some cells were firmLy adherent to the bottom of culture bottle. After4to5days of inoculation, Cell fusion can be observed. BMSCs were found at a good state, displaying uniform spindle shape and polarity arranged under inverted microscope. During cell passage, BMSCs were adherent to the bottom after a short suspension. Then after a short incubation period no more than one day, BMSCs began to display exponential growth phase. Five days later, BMSCs reached saturation density, stop growing and dispayed the plateau growth phase. Surface markers of the third passage cells were examined with flow cytometry. The cells were negative for CD34(14.93%) and CD45(4.48%) and positive for CD44(99.98%) and CD29(99.88%). Seven days after osteogenic introduction, The cells were polygonal and there were more granules in cytoplasm.13days later, the cytoplasm was filled with granules, The cells displyed colony-like growth and calcium deposition can be seen among cells.17days later, calcium nodule formed. At the twentith day, red mineralized nodules can be seen after alizarin red staining.72h after adipogenic introduction, The cells displayed enhanced three-dimensional appearing under inverted microscope and there were small lipid droplets in cells. About a week later, the number of lipid droplets increased and began to be fused. The cells become round or polygonal and displayed a large number of lipid deposition by oil red O staining.Conclusion:Adherent culture method can effectively isolated and purified rat bone marrow mesenchymal stem cells with multilineage differentiation capacity.The BMSCs can be used for further study. Objective:Bone marrow mesenchymal stem cells (BMSCs) live in the physiological environment of lower oxygen concentration compared with the common culture concentration (20%). Hypoxia can promote BMSCs proliferation and maintain the characteristics of differentiation potential. Hypoxic preconditioning means that the body or tissue get tolerance for more serious or even fatal ischemia or hypoxia after one or more short-term, non-lethal hypoxic stimulation. Therefore, this study aims to understand the biological characteristics of BMSCs after hypoxic preconditioning, and search for in vitro basis for BMSCs therapy in vivo with hypoxic preconditioning strategy.Methods:After passage and inoculation, BMSCs were cultured in the hypoxic incubator (1%O2,5%CO2). The effect of low oxygen concentration (1%) on BMSCs proliferation was analyzed according to the cell growth curve, which was drawn according to the MTT assay results. After preconditioned in hypoxic incubator(1%O2,5%CO2) for48h, the hypoxia preconditioned BMSCs (HpBMSCs) were sealed into the serum-free low glucose DMEM for12h,24h,36h,48h,60h,72h and84h. The HpBMSCs were collected and counted by the cell automatic counter after the placenta blue staining at each time point. The cell survival rates were calculated automatically. The BMSCs cultured under ruitine oxygen concentration (nBMSCs) were done by the same way. Flow cytometry was used to determine the surface markers (CD29, CD31, CD45, CD90) expression of BMSCs before and afer Hypoxic preconditioning. The HIF-1α protein expression in HpBMSCs was evaluated by Western-Blot technique. The VEGF mRNA expression was analyzed by RT-PCR technique. The VEGF protein expression in supernatant was determined by enzyme-linked immunosorbent assay(ELISA). Results:Compared with BMSCs cultured under normoxia(20%O2)> BMSCs cultured under hypoxia(1%O2) began to display exponential growth phase after a longer incubation period. Hypoxia(1%O2) was not the lethal condition for BMSCs. Cell survival rates of both groups reduced over time, while those of nBMSCs reduced faster. Surface markers expression of BMSCs after hypoxic preconditioning did not change greatly when compared with the results brfore hypoxic preconditioning. HpBMSCs did not differentiated into vascular endothelial cells. HIF-la protein expression was promoted after24h to48h hypoxic preconditioning, and there was no significant difference between the two timepoints. VEGF expression increased at both transcriptional and protein levels after hypoxic preconditioning.Conclusion:Culture under hypoxia(1%O2) does not produce a lethal effect on BMSCs and can be used for hypoxic preconditioning. Surface markers of BMSCs after hypoxic preconditioning did not change greatly and maitained the characteristics of sternness. Hypoxic preconditioning can promote the ability of BMSCs to survival under hypoxia/ischemic condition. The activation of the hypoxic response signal transduction pathway through HIF-1α may explain this. Hypoxic preconditioning may be a easy feasible strategy for cell therapy of ischemic disease. Background and Objective:Random flap is one kind of the most widely used skin flaps in plastic and reconstructive surgery. Its design was limited by the safe ratio of length to width, and partial distal necrosis of the ultra-long random flap.If the survival rate can be improved greatly, the random flap will be used more widly. It had been found that bone marrow mesenchymal stem cells (BMSCs) can improve the blood supply of the ischemic tissue,however, the low rate of transplanted cell survival was the critival problem.The aim of this study was to evaluate the effect of hypoxia preconditioned bone marrow mesenchymal stem cells (HpBMSCs) transplantation on the survival of ultra-long random skin flap in rats.Methods:Normoxic bone marrow mesenchymal stem cells (nBMSCs) were cultured under normoxia (20%O2) and HpBMSCs under hypoxia (1%O2) for48hours before transplantation. Thirty Sprague-Dawley rats were randomLy divided into control group, nBMSCs group and HpBMSCs group with each consisting of10rats. Survival area of ultra-long random skin flap on the dorsal of rats was measured seven days after flap surgery and labeled cell transplantation. Cell surviva/in vivo, microvessel density and vascular endothelial growth factor (VEGF) were evaluated by histological examination and enzyme-linked immunosorbent assay.Results:Compared with other two groups, flap survival area in HpBMSCs group was significantly larger (P<0.05). Microvessel density in HpBMSCs group (36.20±8.19) was higher than that in nBMSCs group (30.01±5.68) and control group (17.60±4.19)(P<0.05). VEGF in HpBMSCs group ((300.05±50.41) pg/g) was higher than those in nBMSCs group ((240.55±33.64) pg/g) and control group ((191.65±32.58) pg/g)(P <0.05). More fluorescence spots can be found in the flaps of the HpBMSCs group.Conclusion:HpBMSCs transplantation improves ultra-long random skin flap survival via promoting angiogenesis of more survival cells. Hypoxic preconditioning is a simple and effective strategy for the application of BMSCs transplantation.
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