The Effect Of Hypoxic Preconditioning Of Bone Marrow Derived Mesenchymal Stem Cells On Rat Acute Kidney Injury

Part one The isolation, culturing and identification of rat BMSCs and establishment of an optimal hypoxic preconditioning model of BMSCs with CoCl2Objective:To isolate, culture and identify rat bone marrow derived mesenchymal stem cells (BMSCs) in vitro, and establish an optimal hypoxic preconditioning model with CoCl2.Methods:BMSCs were cultured by whole bone marrow adherence method in vitro (3-4generations), and inverted microscope was used to observe morphology. The characteristics of BMSCs were identified by inducing adipogenic and osteogenic differentiation and surface markers (CD29、D9C0、CD45) with flow cytometry (FCM). BMSCs were incubated in cell culture mediums with different concentrations of CoCl2(final concentration of CoCl2were0,50,100,150,200,300,400,600μM) and incubated for different times, including0,6,12,24,48,72h. MTT assay and FCM were applied to to detect the growth inhibition rates and the the apoptosis rates of BMSCs,to establish the best model of CoCl2induced hypoxic preconditioning.Results:l.Most of primary cells were of shuttle and irregular shape,these cells grew in parallel or vortex with a similar fibrobalstoid spindle-shaped morphology after several passages.2.Cultured BMSCs had the capacities for adipogenic and osteogenic differentiation and highly expressed CD29and CD90, and negative for CD45.3.The concentration of CoCl2in cell culture medium was200μM and the incubation time was24h,which was the optimal model of CoCl2induced hypoxic preconditioning.Conclusion:1.BMSCs can be isolated and expanded in vitro by using whole bone marrow adherent cultivation.2.The optimal hypoxic preconditioning model of BMSCs was incubated with CoCl2at a final concentration of200μM/L for24h. Part two Effects and possible mechanism of hypoxic preconditioning on the migration of rat BMSCs in vitroObjective:To confirm the enhancement effect of migration ability of BMSCs in vitro and its underlying mechanism.Methods:BMSCs were isolated and cultured by whole bone marrow adherence method in vitro (3-4generations). After silencing HIF-1α by siRNA technique and blocking CXCR4by its antagonist AMD3100,BMSCs were divided into four groups, including normal culture group, hypoxia culture group, hypoxia HIF-1α siRNA group and hypoxia AMD3100group. The migration ability of BMSCs with hypoxic preconditioning was analyzed by scratch wound healing assay and transwell migration assay. The protein and mRNA expressions of HIF-1α and CXCR4of BMSCs were detected by western blotting and real-time PCR.Results:1.Compared with control group (scratch wound healing assay:0.200±0.011; Transwell migration assay:8.5±1.7), the migration ability of BMSCs in hypoxic preconditioning group (scratch wound healing assay:0.396±0.018;Transwell migration assay:21.0±4.5) was higher(P<0.05), after silencing HIF-1α or blocking CXCR4by AMD3100, the migration ability of BMSCs in hypoxic preconditioning group was decreased and had no difference with control group (P>0.05).2.The protein and mRNA levels of HIF-1α and CXCR4of BMSCs in hypoxic preconditioning group were significantly higher than in control group(P<0.05), both of them showed lower expression after silencing HIF-1α compared with hypoxic preconditioning group(P<0.05).Conclusion:Hypoxic preconditioning can enhance the migration ability of BMSCs in vitro by the activation of HIF-1α and up-regulatbn of CXCR4. Part three The benefit and mechanism of hypoxia-preconditioned BMSCs transplantation for the cell therapy of rat ischemia-reperfusion injuryObjective:To study the influence of hypoxia-preconditioning on BMSCs therapy of rat ischemia-reperfusion injury, and the effect on BMSCs homing.Methods:SD rats were cross-clasped for40minutes followed by reperfusion to establish the ischemia-reperfusion(I/R) rat model,and animals were divided into3groups:carotidly injection of cell culture medium group(control group); carotidly injection of normoxia cultured BMSCs group(normoxia+BMSCs group) and hypoxia-preconditioning BMSCs group(Co+BMSCs group).The renal function was estimated by measurement of serum creatinine(Scr) and blood urea nitrogen(BUN) concentrations,and histological changes were observed by hematoxylin and eosin(HE) staining after injection at24h、48h、72h and1week. BMSCs were labeled with Fe3O4-PLL,after injection of the labeled cells,3.0T MRI system was used to follow the distribution of transplanted cells by measuring the changes of signal intensity of renal cortex.Results:1.BMSCs can be successfully and efficiently labeled with SPION at the concentration of30μg/ml.2.The BUN and Scr levels of both two BMSCs groups were significantly lower than the contro gtoup(P<0.05)after injection at24h and lweek, the Co+BMSCs group was even lower than the normoxia+BMSCs group (P<0.05);the renal tubulointerstitial damage of Co+BMSCs group was significantly lower than that of the normoxia+BMSCs group and the control group at24h after injection(P<0.05),and the renal tubular epithelial cells of the Co+BMSCs group recovered fastest at48h afere injection and until1week,there were even no chronic changes in the Co+BMSCs group.3.The labeled Co+BMSCs group showed higher loss of signal intensity in the renal cortex on T2*-weigheted MR images compared with normoxia+BMSCs group at2h afere injection (P<0.05),and till72h,the Co+BMSCs group still showed significantly lower of signal intensity while the normoxia+BMSCs group showed almost no loss of signal intensity.Conclusion:Hypoxic preconditioned BMSCs transplantation can enchance the recovery of rat IR injure through the mobilization of BMSCs homing to the kiney.