| Objective: To investigate the repairing effect of hypoxia-preconditioning on bonemarrow derived-mesenchymal stem cells transplantation in 5/6 nephrectomy rats.Methods:1. The bone marrow-derived mesenchymal stem cells were separated by densitygradient centrifugation assay from Sprague Dawley rat, the observation of cellmorphology under inverted microscope, and flow cytometry to detect the surfacemarkers, the 3-4 generation of cells was used in the experiment. Adopting mixed gashypoxia chamber(1%O2+5%CO2+94%N2) to hypoxic preconditioning BMSC establishthe experimental model. The experiment was divided into 3 groups: the normal culturedMSC(control group), hypoxia preconditioning 24 h group(hypoxia 24 h group),hypoxia preconditioned 48 h group(hypoxia 48 h group), the cell apoptosis were detectdby flow cytometry Annexin V-FITC/PI after hypoxic preconditioning. The m RNA andprotein expressions of HIF-la, CXCR4 and VEGF of BMSC were detected by reversetranscription polymerase chain reaction and western blotting;and compare the differentof each group antiapoptosis ability and cytokines expression.2. The model of 5/6 nephrectomy was used to establish animal model of chronic renalfailure, the animasls were randomly divided into four groups: Sham group(sham4operation group), model group(5/6 nephrectomy group), BMSC group(normoxia BMSC transplantation group), hp-BMSC group(hypoxiapreconditioning 24 h BMSC transplantation group). Rats of every group, respectively,were injected by PBS, PBS, BMSC and hp-BMSC cell suspension though the dorsalpenile venous after a week of 5/6 nephrectomy. After injection of 1weeks, 3weeks, 6weeks, rats were killed, the serum and renal tissue were collected toexamine serum urea nitrogen(BUN) and serum cretinine(SCr), and to observe the renalpathological changes. At the same time, using the Dio green fluorescent dye labeledBMSC in vitro, compare the quantity difference in renal homing of normoxic cultureBMSC and hypoxic preconditioning BMSC after transplantation.Results:1. Primary cells were spindle shaped, oval, adherent colony growth, after 3generation, the cell size and shape are uniform, spindle or radial growth. A display byflow cytometry showed that early and late cells both no apoptosis in controlgroup, hypoxia preconditioned 24h(hypoxia 24h) group in early and late theapoptosis rates were not significantly different, with the extension ofcell hypoxia, hypoxia preconditioned 48h(hypoxia 48h) group, early apoptosis, lateapoptosis and total quantity apoptosis ware significantly increased(P<0.05).RT-PCR and WB show that, compared with control group and hypoxia 48 h group, theexpression of HIF-1α 、 CXCR4 and VEGF in hypoxia 24 h group ware significantlyincreased(P<0.05), while the control group and hypoxia 48 h group compared,there isno significant difference between the expression of HIF-1α、CXCR4 and VEGF.2. At each time point, the BUN and Scr levels of model group rats were higher thanthose in Sham groups(P<0.05), the BMSC group and hp-BMSC group was lower thanthat in Model group(P<0.05), compared with BMSC group, the BUN and Scr levels in6 weeks decreased more significantly in hp-BMSC group(P<0.05); Fluorescencelocalization showed in hp-BMSC group the survival of BMSC more than BMSC groups.After cell transplantation, two groups of rats renal pathological damage was milder thanthos in Model groups, but hp-BMSC group treatment effect is more obvious than BMSCgroup.Conclusion:Application of hypoxic preconditioning BMSC culture can improve theBMSC antiapoptosis ability, promote BMSC homing to the damaged kidney, prolongthe cells in kidney colonization time. Compared with normoxic cultureBMSC transplantation, hypoxic preconditioning BMSC transplantation can moreeffective improve the rat renal function of chronic renal failure and repair the impairedrenal tissue. |
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