| Objective:Diabetic nephropathy has become a serious problem in the progress of diabetes.The microvascular abnormalities and chronic hypoxia are closely related to the occurrence and development of diabetic nephropathy?DN?.The clinical treatment of DN is not effective so far.With the rise of stem cell therapy,it is expected to become the ideal treatment for DN.This study was to investigate the therapeutic and protective effects of human amniotic mesenchymal stem cells?hAMSCs?on DN and whether it can improve the renal hypoxia status of diabetic rats or improve and delay the progression of DN.Methods:HAMSCs were isolated,cultured and identified by the Key Laboratory of Cell Engineering of Zunyi Medical University and P3 generation cells were used.Induction of a rat model of type 1 diabetes by a single intraperitoneal injection of streptozocin?STZ?.Male SD rats?n=54?were randomly divided into 3 groups.NC group,the same amount of PBS were intraperitoneally injected,and the same amount of PBS was used to replace hAMSCs.DN group,no intervention was given after modeling,and an equal amount of PBS was injected instead of hAMSCs.MSC group,after the models were established,P3 hAMSCs were injected into the penile vein at 3 and 4W,respectively.Each group was divided into 3 time points,6 weeks group,8 weeks group,and 12 weeks group.Collecting urine using metabolic cages 24 hours before sacrifice of animals,blood and kidney were collected after anesthesia.24h urine protein,blood glucose and renal function were detcted after blood and urine were collected.Kidney tissues were embedded and frozen at-80°C.The renal pathological changes were observed by conventional HE and PAS staining.Immunohistochemical detection of HypoxyprobeTM-1M-1 positive expression to determine the area of hypoxia.Immunofluorescence was used to detect whether the anti-human marker antibody?MAB1281?was colonized in the kidney.Immunohistochemistry,immunofluorescence,Western Bloting and Real Time-PCR were used to detect the expression changes of renal hypoxia-related indicators?HIF-1?,PHD2,HO-1,SDF-1?.Results:?1?Blood glucose and 24-hour urine protein of DN group and MSC group were up-regulated compared with NC group at different time?P<0.05?;At the same time,the blood glucose and 24-hour urine protein of the MSC group decreased compared with the DN group,but the difference was only significant at 12W?P<0.05?.?2?The serum creatinine of DN group and MSC group were higher than NC group at different time;The blood urea level of MSC group 6 and 12W was lower than that of DN group?P<0.05?.?3?Histopathology showed an increase in renal weight index in DN group and MSC group,accompanied by glomerular hypertrophy,increased mesangial matrix,and thickening of renal tubular basement membrane.The islet structure damage,the islet cells disappeared atrophy,necrosis and inflammatory cell infiltration,and the kidney and pancreas damage in the MSC group was relatively reduced.?4?We observed the distribution of MAB1281 in the kidney tissues of the MSCs group at each time by immunofluorescence.DN and NC groups were not found.?5?Immunohistochemical staining of HypoxyprobeTM-1 showed that the NC group had positive staining at the junction of intramedullary and cortical,while the positive range of DN group expanded to cortex and darkened.The area of hypoxia in DN group was significantly higher than that in MSC group?P<0.05?.?6?Immunohistochemistry or fluorescence and Western blotting showed that HIF-1?,PHD2,HO-1 and SDF-1 were mainly expressed in renal tubular cells and some glomerular cells.The expressions of HIF-1?,PHD2,HO-1 and SDF-1 in DN group and MSC group were significantly higher than NC group?P<0.05?.The expression of HIF-1,HO-1 and SDF-1 in MSC group was significantly higher than that in DN group?P<0.05?,while the expression of PHD2 was down-regulated in DN group?P<0.05?.?7?The results of RT-PCR showed that the expressions of HIF-1?,PHD2,HO-1 and SDF-1 mRNA in DN group and MSC group were higher than NC group at different time.At the same time,the expression of HIF-1?,HO-1 and SDF-1 mRNA in the MSC group was up-regulated compared with the DN group.The expression differences were significant at 8W and 12W,6W and 8W,6W and 12W,respectively?P<0.05?.The expression of PHD2 mRNA in MSC group was down-regulated at each time compared with DN group,only 6W and 12W were significantly different?P<0.05?.Conclusion:The kidney of diabetic rats which were induce by STZ were accompanied by chronic hypoxia.Transplantation of hAMSCs could protect kidney function and improve hypoxia in diabetic rats.Transplantation of hAMSCs could alleviate the hypoxic state and improve renal pathology in the kidney of diabetic rats.After transplantation of hAMSCs.It may be related to the relief of diabetic nephropathy by improving the expression of the hypoxia-related factor HIF-PHD axis. |
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