| Objectives:1.To isolate,purify and culture BMSCs and tracking them by immunofluorescet labeling.To compare the effect of normal culture and hypoxic preconditioning on the expression of CXCR4/CXCR7 on the surface of BMSCs.2.To investigate the expression of SDF-lm RNA in the injured kidney tissues by establishing the ADR-induced chronic kidney disease model in rats.3.To measure the effect of the treatment of transplantation of normal culture and hypoxia-treated BMSCs in the ADR rat model(the control group was also established).To draw the comparison of the location of BMSCs in the kidney tissues and analyze the effect of SDF-1/CXCR4 on the targeted homing of BMSCs to the impaired kidney.Methods:1.Kill two male SD rats and collect bone marrow from the bilateral femur and tibia to obtain the BMSCs.The expression of cell surface antigen of BMSCs was examined by flow cytometry.Labelling the BMSCs(4PDL)by transfection using the adenoviruses carrying green immunofluorescent protein(GFP).The BMSCs(4PDL)were divided into two groups,the C1 group continued normal culture and the C2 group was treated with low oxygen environment which contains 2%O2?93%N2?5%CO2 for 48h.And then detect the expression of CXCR4/CXCR7 by flow cytometry respctively after fixed by Penicillin-Streptomycin.2.After 50 male SD rats were excised from the left kidney 4 weeks ago,the rats were treated with adriamycin 2.5mg/Kg once a week for 2 weeks.After the establishment of chronic kidney disease model of SD rats,4 model rats and 4 normal rats were sacrificed for pathological examination.And the expression of SDF-1 mRNA was detected by PCR.3.The model rats were divided into three groups(12 rats in each group):adriamycin chronic kidney disease model group(ADR group),transplanted group with normal cultured BMSCs(T1 group),transplanted group with low oxygen treated BMSCs(T2 group).What's more,another 12 SD rats with the same batch were chosen as normal control group(N group).After general anesthesia,the right renal artery was separated as the injection approaches.The T1 group were injected into C1 group cell suspension 2 ml and the T2 group were injected into C2 group cell suspension 2 ml.For comparison,ADR group and N group were injected into 2ml of L-DMEM culture medium.Blood samples were collected at the first day and the 7th day after transplantation.The changes of urinary protein,hemoglobin,blood urea nitrogen,serum creatinine and serum albumin were measured by the blood of the rats'inner canthal vein.4 rats chosen from each group were sacrificed and the renal tissues were stained with HE for histological examination.And the distribution of BMSCs were observed using confocal fluorescence microscopy.Results:1.The culture and identification of BMMSs and the expression of CXCR4/CXCR7:The BMSCs showed typical adherent growth characteristics,fibroblast-like morphology and spindle-shaped appearance after 3-4 times of passages.CD29 and CD44 were highly expressed on the cell surface while CD11b,CD4 were of low expression.The BMSCs'expression rate of green fluorescence was 75%after transfected with adenovirus carrying green fluorescent protein.And the luminous effect was stable,the cell morphology was consistent with normal cells'.The expression of CXCR4 was susceptible low oxygen environment in comparison to CXCR7.The flow cytometry showed that the expression of CXCR4 unregulated significantly after hypoxia treatment while the expression of CXCR7 did not have the statistical difference.2.The rats that were induced chronic kidney disease showed significantly increase of the daily urine protein,the serum creatinine,and the urea nitrogen and the rats were actually in the condition of hypoproteinemia and anemia.The histological examination showed glomerular sclerosis,renal interstutual fibrosis and intrarenal vascularsclerosis.The expression of SDF-1 mRNA in renal tissue of rats with ADR chronic kidney disease was increased comparing with control group.3.The success rate of renal artery puncture operation was 100%.The renal function of ADR group rats continued to decline.The levels of urinary protein,SCr and BUN in the T1 and T2 groups were significantly lower than those in the ADR group,and the levels of hemoglobin and serum albumin were significantly increased(P<0.05).When compared T1 group with T2 group,the results showed the levels of daily urinary protein,SCr,BUN and serum albumin in T2 group were significantly lower than those in T1 group,and the hemoglobin content did not change significantly(P>0.05).The histological examination showed severe inflammatory response,glomerular sclerosis,renal interstutual fibrosis and intrarenal vascularsclerosis in ADR group whie the T1 group showed mild glomerular steatosis,and insignificant atrophy of renal tubular and the T2 group had less inflammatory cells and slight renal interstitial fibrosis and most of the glomerular capillary loop dilated obviously.Fluorescence confocal microscopy showed that T1 and T2 group rats showed green fluorescence on day 1,and more obvious at day 7 and there was a significantly discrepancy between the two groups which means the contain of green fluorescence in T2 group was higher than T1.The green fluorescence of T1 group was mainly distributed in renal tubular epithelial cells and some mesangial areas.However,in group T2,green fluorescence was widely distributed in glomeruli,renal tubular epithelial cells,glomerular mesangial area and tubulointerstitial capillaries.Conclusions:1.Rat BMSCs were easily extracted,isolated and purified.And the cells of high purity were easily obtained by several times' subculture.The hypoxic environment can induce the significantly increased expression of CXCR4 on the cell surface,but had no significant effect on CXCR7.2.Unilateral nephrectomy and tail vein injection of doxorubicin induced chronic kidney disease model is simple,effective,and reproducible.ADR-induced high expression of SDF-1 in renal tissue of chronic kidney disease provided foundational supports for SDMS-1/CXCR4 axis-induced BMSCs' homing to damaged kidney.3.Transplantation of BMSCs by renal artery has a significant therapeutic effect on rat model of chronic kidney disease.It can reduce the amount of urinary protein,serum creatinine and blood urea nitrogen in a short term,decrease the hemoglobin and decrease serum albumin,and thus improve renal function.The pathological examination showed obvious repair to damaged tissue especially to the renal tubular.The homing cells to the injured renal tissue with high expression of SDF-1 tended to be the cells with high expression of CXCR4.The therapeutic effect of those cells was better than the cells with low expression of CXCR4.In conclusion,the SDF-1/CXCR4 axis plays an important role in the homing and locating to the injured renal tissue in rats and thus improves the curative effect. |
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