移植低氧预处理MSC对CKD大鼠肾功能的影响及机制

Effect and mechanism of transplantation of hypoxia-preconditioned MSC on renal function in CKD rats

目的探讨移植低氧预处理(HP)骨髓间充质干细胞(MSC)对慢性肾脏病(CKD)大鼠肾功能的影响及机制。方法取2只12周龄SD大鼠颈椎脱臼处死,剥离胫骨和股骨骨髓,分离并培养MSC,用流式细胞仪分析MSC表面抗原。将56只大鼠随机分为模型组40只、假手术(Sham)组12只、对照组4只。模型组用左肾切除及尾静脉注射阿霉素(ADR)建立CKD模型,并随机分为CKD组4只及ADR组、干细胞移植(MSC)组、低氧预处理干细胞移植(HP-MSC)组各12只。对照组于相同部位注射等体积生理盐水;Sham组做左侧肋脊角切口,随后缝合包扎,于相同部位注射等体积生理盐水。造模成功后将CKD组、对照组大鼠颈椎脱臼处死,取肾脏组织。将在常氧环境及低氧环境中培养的MSC分别移植至MSC组、HP-MSC组,Sham组、ADR组注射L-DMEM培养液。MSC移植第1、7天用全自动生化分析仪检测PRO、Hb、ALB、BUN和SCr水平,用HE染色法观察肾脏组织病理变化,用RT-PCR检测肾脏组织中基质细胞衍生因子1(SDF-1)mRNA表达。结果HP-MSC的细胞趋化因子受体4(CXCR)表达率高于常氧MSC(P<0.01)。移植第1、7天,与Sham组比较,ADR组PRO、BUN、SCr水平高,Hb、ALB水平低(P均<0.05)。移植第1天,MSC组、HP-MSC组、ADR组生化指标比较差异无统计学意义(P均>0.05);移植第7天,与ADR组比较,MSC组、HP-MSC组PRO、BUN、SCr水平低,Hb、ALB水平高(P均<0.05),且HP-MSC组PRO、ALB、BUN、SCr变化较MSC组明显(P均<0.05)。移植第7天,MSC组与HP-MSC组肾脏组织损伤减轻,MSC组肾小球尚可见局灶节段硬化,HP-MSC组肾小球袢扩张;与MSC组比较,HP-MSC组肾间质纤维化改善更明显,炎症反应更轻。与对照组比较,CKD组肾组织SDF-1 mRNA相对表达量高(P<0.05)。结论移植HP-MSC可改善CKD大鼠的肾功能,其机制可能与SDF-1-CXCR4信号轴有关。

Objective To investigate the effect and mechanism of transplantation of hypoxia-preconditioned(HP)bone marrow mesenchymal stem cells(MSC)on renal function in chronic kidney disease(CKD)rats.Methods Two 12-week-old SD rats were executed by breaking the neck,then the bone marrow of tibia and femur were stripped and MSC were isolated and cultured.Next,MSC surface antigen was analyzed by flow cytometry.After one-week acclimatization,56 male SD rats were randomly divided into three groups:Model group(n=40),Sham operation group(n=12)and Con‑trol group(n=4).In the Model group,CKD rat models were established by left nephrectomy and intravenous injection of adriamycin(ADR).After that,the model group was randomly divided into CKD group(n=4)and ADR group(n=12),stem cell transplantation(MSC)group(n=12)and hypoxia-preconditioned stem cell transplantation(HP-MSC)group(n=12).The rats in the Control group were injected with the same volume of normal saline at the same site;in the Sham opera‑tion group,the left costal ridge angle incision was sutured and bound,and the same volume of normal saline was injected into the same part.After the successful modeling,the rats in CKD group and Control group were killed by cervical disloca‑tion,and the renal tissues were taken.MSCs cultured in normoxic environment and hypoxic environment were transplanted to MSC group and HP-MSC group,respectively,while the Sham group and ADR group were injected with L-DMEM medi‑um.On the 1st day and 7th day after transplantation,the levels of PRO,Hb,ALB,BUN,and SCr were detected by auto‑matic biochemical analyzer,the pathological changes of renal tissues were observed by HE staining,and SDF-1 mRNA ex‑pression in the renal tissues was detected by RT-PCR.Results The expression rate of CXCR4 in the HP-MSC was higher than that in normoxia MSC(P<0.01).Compared with the Sham group,the levels of PRO,BUN and SCr in the ADR group were higher,and the levels of Hb and ALB were lower on the 1st day and the 7th day after transplantation(all P<0.05).On the 1st day after transplantation,there were no significant differences in biochemical indexes among MSC group,HP-MSC group and ADR group(all P>0.05).Compared with the ADR group,the levels of PRO,BUN and SCr in the MSC group and HP-MSC group were lower,and the levels of Hb and ALB were higher on the 7th day after transplanta‑tion(all P<0.05),and the changes of PRO,ALB,BUN,SCr in the HP-MSC group were significantly higher than those in the MSC group(all P<0.05).On the 7th day after transplantation,the damage of renal tissues was reduced in the MSC group and HP-MSC group,focal segmental sclerosis was still seen in glomerulus in the MSC group,and glomerular loop di‑lation was observed in the HP-MSC group.Compared with MSC group,the improvement of renal interstitial fibrosis and lighter inflammatory reaction in the HP-MSC group were more obvious.Compared with the Control group,the relative ex‑pression of SDF-1 mRNA in the CKD group was higher(P<0.05).Conclusion Transplantation of HP-MSC can improve the renal function of CKD rats and its mechanism may be related to SDF-1-CXCR4 axis.