黄芪甲苷孵育的脂肪干细胞治疗糖尿病肾病
|
摘要
背景:研究表明,蛋白尿是糖尿病肾病进展的独立性危险因素,而蛋白尿的发生发展与足细胞的损伤密切相关。目的:观察移植黄芪甲苷孵育的人脂肪源性干细胞(astragaloside-incubated adipose stem cells,Ast-hADSCs)对糖尿病肾病模型大鼠肾损伤的修复作用。方法:人脂肪源性干细胞购自美国ATCC公司,雄性SD大鼠购自上海中医药大学动物实验中心,实验方案经上海中医药大学动物实验伦理委员会批准。将SD大鼠随机分为正常组、模型组、人脂肪源性干细胞组及Ast-hADSCs组,后3组应用左肾摘除联合链脲佐菌素注射法建立糖尿病肾病大鼠模型,2个干细胞干预组经尾静脉输注人脂肪源性干细胞或Ast-hADSCs悬液,2周1次,共6次,模型组予等量生理盐水。实验结束(23周)取材,检测肾功能、血脂、尿微量白蛋白及尿蛋白定量,苏木精-伊红及MASSON染色观察肾脏病理变化及肾小球面积,WT-1染色检测足细胞密度,激光共聚焦观察干细胞在肾脏的分布情况。结果与结论:①与模型组比较,2个干细胞干预组总胆固醇、尿微量白蛋白及尿蛋白定量均下降,但Ast-h ADSCs组下降更显著(P <0.05);②与模型组比较,2个干细胞干预组模型大鼠的三酰甘油、低密度脂蛋白、尿素氮、高密度脂蛋白水平无统计学意义(P> 0.05);③肾脏病理提示:2个干细胞干预组的肾脏损伤减轻,肾脏指数及肾小球截面积显著缩小,足细胞密度显著增加(P <0.01);Ast-hADSCs组模型大鼠肾脏病理改善明显(P <0.05);④激光共聚焦显示:干细胞干预两组模型大鼠肾组织内可见少量红色荧光表达。结果说明:经尾静脉移植人脂肪源性干细胞可提高糖尿病肾病模型大鼠足细胞的密度,减轻肾脏损伤,从而改善蛋白尿;经黄芪甲苷孵育48 h后,可在一定程度上提高人脂肪源性干细胞对糖尿病肾病模型大鼠受损肾组织的修复作用。
BACKGROUND: Proteinuria is an independent risk factor for the progression of diabetic nephropathy, and the occurrence and development of proteinuria is closely related to podocyte injury.OBJECTIVE: To observe the effect of astragaloside-incubated human adipose-derived stem cells(Ast-hADSCs) on renal injury in a rat model of diabetic nephropathy.METHODS: Human adipose-derived stem cells were purchased from ATCC, USA. Male Sprague-Dawley rats were purchased from the Animal Experimental Center of Shanghai University of Traditional Chinese Medicine. The study protocol was approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine. Rats were randomly divided into normal group, model group, hADSCs group and Ast-hADSC group. Animal models of diabetic nephropathy were established by left kidney extraction combined with streptozotocin injection in the latter three groups. hADSCs or Ast-hADSCs suspension was infused via the tail vein in the two intervention groups, every 2 weeks, for six sessions. Rats in the model group were given the same volume of normal saline. Levels of renal function indicators, blood lipids,urine microalbumin and urine protein were detected at 23 weeks. The pathological changes and glomerular area of the kidney were observed by hematoxylin-eosin staining and Masson staining. The podocyte density was detected by WT-1 staining. The distribution of stem cells in the kidney was observed by laser confocal microscopy.RESULTS AND CONCLUSION: Compared with the model group, there was a reduction in total cholesterol, urine microalbumin and urine protein levels decreased in the two intervention groups. Moreover, a greater reduction was found in the Ast-hADSC group than the hADSC group(P < 0.05). Triacylglycerol, low-density lipoprotein, urea nitrogen, high-density lipoprotein levels were similar in the model, hADSC and Ast-hADSC groups(P > 0.05). Renal pathological findings revealed a relief in renal damage and reduced renal index and glomerular cross-sectional area but significantly increased podocyte density in the hADSC and Ast-hADSC groups(P < 0.01). The renal pathological changes of the Ast-hADSC group were superior to those of the hADSC group(P < 0.05). Under the laser confocal microscope, a small amount of red fluorescence was expressed in the kidney. These results indicate that transplantation of human adipose-derived stem cells through the tail vein can increase the density of podocytes, reduce renal damage, and improve proteinuria in diabetic nephropathy rats.Incubation with astragaloside IV for 48 hours can promote the efficacy of human adipose-derived stem cells in the repair of damaged kidney tissue in diabetic nephropathy rats.
BACKGROUND: Proteinuria is an independent risk factor for the progression of diabetic nephropathy, and the occurrence and development of proteinuria is closely related to podocyte injury.OBJECTIVE: To observe the effect of astragaloside-incubated human adipose-derived stem cells(Ast-hADSCs) on renal injury in a rat model of diabetic nephropathy.METHODS: Human adipose-derived stem cells were purchased from ATCC, USA. Male Sprague-Dawley rats were purchased from the Animal Experimental Center of Shanghai University of Traditional Chinese Medicine. The study protocol was approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine. Rats were randomly divided into normal group, model group, hADSCs group and Ast-hADSC group. Animal models of diabetic nephropathy were established by left kidney extraction combined with streptozotocin injection in the latter three groups. hADSCs or Ast-hADSCs suspension was infused via the tail vein in the two intervention groups, every 2 weeks, for six sessions. Rats in the model group were given the same volume of normal saline. Levels of renal function indicators, blood lipids,urine microalbumin and urine protein were detected at 23 weeks. The pathological changes and glomerular area of the kidney were observed by hematoxylin-eosin staining and Masson staining. The podocyte density was detected by WT-1 staining. The distribution of stem cells in the kidney was observed by laser confocal microscopy.RESULTS AND CONCLUSION: Compared with the model group, there was a reduction in total cholesterol, urine microalbumin and urine protein levels decreased in the two intervention groups. Moreover, a greater reduction was found in the Ast-hADSC group than the hADSC group(P < 0.05). Triacylglycerol, low-density lipoprotein, urea nitrogen, high-density lipoprotein levels were similar in the model, hADSC and Ast-hADSC groups(P > 0.05). Renal pathological findings revealed a relief in renal damage and reduced renal index and glomerular cross-sectional area but significantly increased podocyte density in the hADSC and Ast-hADSC groups(P < 0.01). The renal pathological changes of the Ast-hADSC group were superior to those of the hADSC group(P < 0.05). Under the laser confocal microscope, a small amount of red fluorescence was expressed in the kidney. These results indicate that transplantation of human adipose-derived stem cells through the tail vein can increase the density of podocytes, reduce renal damage, and improve proteinuria in diabetic nephropathy rats.Incubation with astragaloside IV for 48 hours can promote the efficacy of human adipose-derived stem cells in the repair of damaged kidney tissue in diabetic nephropathy rats.
引文
[1]Collins AJ,Foley RN,Chavers B,et al.'United States Renal Data System 2011 Annual Data Report:Atlas of chronic kidney disease&end-stage renal disease in the United States.Am J Kidney Dis.2012;59(1 Suppl 1):A7,e1-420.
[2]Toth-Manikowski S,Atta MG.Diabetic Kidney Disease:Pathophysiology and Therapeutic Targets.J Diabetes Res.2015;2015:697010.
[3]Saran R,Li Y,Robinson B,et al.US Renal Data System 2014 Annual Data Report:Epidemiology of Kidney Disease in the United States.Am J Kidney Dis.2015;66(1 Suppl 1):Svii,S1-305.
[4]Checheri??IA,Turcu F,Dragomirescu RF,et al.Chronic complications in hemodialysis:correlations with primary renal disease.Rom JMorphol Embryol.2010;51(1):21-26.
[5]Stoumpos S,Jardine AG,Mark PB.Cardiovascular morbidity and mortality after kidney transplantation.Transpl Int.2015;28(1):10-21.
[6]杜俊文,吴韬,张坤,等.骨髓间充质干细胞移植改善糖尿病肾病大鼠血糖及尿总蛋白[J].中国组织工程研究,2016,20(6):855-860.
[7]何文涓,何晓升,袁志坚,等.黄芪甲苷对兔脂肪来源的间充质干细胞体外增殖和细胞周期的影响[J].中国生化药物杂志,011,32(6):466-468.
[8]王葳,姜燕,王巍巍,等.黄芪甲苷对脂肪源性干细胞体外生物学行为的影响[J].中国药理学通报,2013,29(2):220-224.
[9]王葳,姜燕,王巍巍,等.黄芪甲苷孵育脂肪源性干细胞对顺铂诱导肾小管上皮细胞凋亡的影响[J].中国组织工程研究,2014,18(28):4498-4503.
[10]姜燕,王葳,李泽争,等.黄芪甲苷孵育的脂肪源性干细胞对顺铂诱导的急性肾损伤小鼠的保护作用[J].中国中西医结合肾病杂志,2014,15(2):114-117.
[11]高俊丽,朱赟洁,王巍巍,等.左肾切除术联合链脲佐菌素腹腔注射诱导糖尿病肾病大鼠模型的建立[J].临床肾脏病杂志,2018,18(05):306-311.
[12]Coombe L,Kadri A,Martinez JF,et al.Current approaches in regenerative medicine for the treatment of diabetes:introducing CRISPR/CAS9 technology and the case for non-embryonic stem cell therapy.Am J Stem Cells.2018;7(5):104-113.
[13]Chandra V,G S,Phadnis S,et al.Generation of pancreatic hormone-expressing islet-like cell aggregates from murine adipose tissue-derived stem cells.Stem Cells.2009;27(8):1941-1953.
[14]Kajiyama H,Hamazaki TS,Tokuhara M,et al.Pdx1-transfected adipose tissue-derived stem cells differentiate into insulin-producing cells in vivo and reduce hyperglycemia in diabetic mice.Int J Dev Biol.2010;54(4):699-705.
[15]Kim D,Dressler GR.Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia.J Am Soc Nephrol.2005;16(12):3527-3534.
[16]Narayanan K,Schumacher KM,Tasnim F,et al.Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.Kidney Int.2013;83(4):593-603.
[17]Song B,Smink AM,Jones CV,et al.The directed differentiation of human iPS cells into kidney podocytes.PLoS One.2012;7(9):e46453.
[18]Lam AQ,Freedman BS,Morizane R,et al.Rapid and efficient differentiation of human pluripotent stem cells into intermediate mesoderm that forms tubules expressing kidney proximal tubular markers.J Am Soc Nephrol.2014;25(6):1211-1225.
[19]Ezquer ME,Ezquer FE,Arango-Rodriguez ML,et al.MSCtransplantation:a promising therapeutic strategy to manage the onset and progression of diabetic nephropathy.Biol Res.2012;45(3):289-296.
[20]Ezquer FE,Ezquer ME,Parrau DB,et al.Systemic administration of multipotent mesenchymal stromal cells reverts hyperglycemia and prevents nephropathy in type 1 diabetic mice.Biol Blood Marrow Transplant.2008;14(6):631-640.
[21]Wu S,Li L,Wang G,et al.Ultrasound-targeted stromal cell-derived factor-1-loaded microbubble destruction promotes mesenchymal stem cell homing to kidneys in diabetic nephropathy rats.Int J Nanomedicine.2014;9:5639-5651.
[22]Nagaishi K,Mizue Y,Chikenji T,et al.Mesenchymal stem cell therapy ameliorates diabetic nephropathy via the paracrine effect of renal trophic factors including exosomes.Sci Rep.2016;6:34842.
[23]Ezquer F,Giraud-Billoud M,Carpio D,et al.Proregenerative Microenvironment Triggered by Donor Mesenchymal Stem Cells Preserves Renal Function and Structure in Mice with Severe Diabetes Mellitus.Biomed Res Int.2015;2015:164703.
[24]Hamza AH,Al-Bishri WM,Damiati LA,et al.Mesenchymal stem cells:a future experimental exploration for recession of diabetic nephropathy.Renal Failure.2017;39(1):67-76.
[25]Lenoir N.Europe confronts the embryonic stem cell research challenge.Science.2000;287(5457):1425-1427.
[26]Takahashi K,Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell.2006;126(4):663-676.
[27]Ben-David U,Benvenisty N.The tumorigenicity of human embryonic and induced pluripotent stem cells.Nat Rev Cancer.2011;11(4):268-277.
[28]Zuk PA,Zhu M,Mizuno H,et al.Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.2001;7(2):211-228.
[29]Panés J,García-Olmo D,Van Assche G,et al.Expanded allogeneic adipose-derived mesenchymal stem cells(Cx601)for complex perianal fistulas in Crohn's disease:a phase 3 randomised,double-blind controlled trial.Lancet.2016;388(10051):1281-1290.
[30]Henry TD,Pepine CJ,Lambert CR,et al.The Athena trials:Autologous adipose-derived regenerative cells for refractory chronic myocardial ischemia with left ventricular dysfunction.Catheter Cardiovasc Interv.2017;89(2):169-177.
[31]Merceron C,Portron S,Masson M,et al.TThe effect of two-and three-dimensional cell culture on the chondrogenic potential of human adipose-derived mesenchymal stem cells after subcutaneous transplantation with an injectable hydrogel.Cell Transplant.2011;20(10):1575-1588.
[32]Aksu AE,Rubin JP,Dudas JR,et al.Role of gender and anatomical region on induction of osteogenic differentiation of human adipose-derived stem cells.Ann Plast Surg.2008;60(3):306-322.
[33]Wosnitza M,Hemmrich K,Groger A,et al.Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation.Differentiation.2007;75(1):12-23.
[34]Bourin P,Bunnell BA,Casteilla L,et al.Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells:a joint statement of the International Federation for Adipose Therapeutics and Science(IFATS)and the International Society for Cellular Therapy(ISCT).Cytotherapy.2013;15(6):641-648.
[35]Zack-Williams SD,Butler PE,Kalaskar DM.Current progress in use of adipose derived stem cells in peripheral nerve regeneration.World JStem Cells.2015;7(1):51-64.
[36]Baer PC,Geiger H.Adipose-derived mesenchymal stromal/stem cells:tissue localization,characterization,and heterogeneity.Stem Cells Int.2012;2012:812693.
[37]Lee RH,Kim B,Choi I,et al.Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.Cell Physiol Biochem.2004;14(4-6):311-324.
[38]Sheykhhasan M,Qomi RT,Ghiasi M.Fibrin Scaffolds Designing in order to Human Adipose-derived Mesenchymal Stem Cells Differentiation to Chondrocytes in the Presence of TGF-β3.Int J Stem Cells.2015;8(2):219-227.
[39]Mundra V,Gerling IC,Mahato RI.Mesenchymal Stem Cell-Based Therapy.Molecular Pharmaceutics.2012;10(1):77-89.
[40]Valina C,Pinkernell K,Song YH,et al.Intracoronary administration of autologous adipose tissue-derived stem cells improves left ventricular function,perfusion,and remodelling after acute myocardial infarction.Eur Heart J.2007;28(21):2667-2677.
[41]Zhang DZ,Gai LY,Liu HW,et al.Transplantation of autologous adipose-derived stem cells ameliorates cardiac function in rabbits with myocardial infarction.Chin Med J(Engl).2007;120(4):300-307.
[2]Toth-Manikowski S,Atta MG.Diabetic Kidney Disease:Pathophysiology and Therapeutic Targets.J Diabetes Res.2015;2015:697010.
[3]Saran R,Li Y,Robinson B,et al.US Renal Data System 2014 Annual Data Report:Epidemiology of Kidney Disease in the United States.Am J Kidney Dis.2015;66(1 Suppl 1):Svii,S1-305.
[4]Checheri??IA,Turcu F,Dragomirescu RF,et al.Chronic complications in hemodialysis:correlations with primary renal disease.Rom JMorphol Embryol.2010;51(1):21-26.
[5]Stoumpos S,Jardine AG,Mark PB.Cardiovascular morbidity and mortality after kidney transplantation.Transpl Int.2015;28(1):10-21.
[6]杜俊文,吴韬,张坤,等.骨髓间充质干细胞移植改善糖尿病肾病大鼠血糖及尿总蛋白[J].中国组织工程研究,2016,20(6):855-860.
[7]何文涓,何晓升,袁志坚,等.黄芪甲苷对兔脂肪来源的间充质干细胞体外增殖和细胞周期的影响[J].中国生化药物杂志,011,32(6):466-468.
[8]王葳,姜燕,王巍巍,等.黄芪甲苷对脂肪源性干细胞体外生物学行为的影响[J].中国药理学通报,2013,29(2):220-224.
[9]王葳,姜燕,王巍巍,等.黄芪甲苷孵育脂肪源性干细胞对顺铂诱导肾小管上皮细胞凋亡的影响[J].中国组织工程研究,2014,18(28):4498-4503.
[10]姜燕,王葳,李泽争,等.黄芪甲苷孵育的脂肪源性干细胞对顺铂诱导的急性肾损伤小鼠的保护作用[J].中国中西医结合肾病杂志,2014,15(2):114-117.
[11]高俊丽,朱赟洁,王巍巍,等.左肾切除术联合链脲佐菌素腹腔注射诱导糖尿病肾病大鼠模型的建立[J].临床肾脏病杂志,2018,18(05):306-311.
[12]Coombe L,Kadri A,Martinez JF,et al.Current approaches in regenerative medicine for the treatment of diabetes:introducing CRISPR/CAS9 technology and the case for non-embryonic stem cell therapy.Am J Stem Cells.2018;7(5):104-113.
[13]Chandra V,G S,Phadnis S,et al.Generation of pancreatic hormone-expressing islet-like cell aggregates from murine adipose tissue-derived stem cells.Stem Cells.2009;27(8):1941-1953.
[14]Kajiyama H,Hamazaki TS,Tokuhara M,et al.Pdx1-transfected adipose tissue-derived stem cells differentiate into insulin-producing cells in vivo and reduce hyperglycemia in diabetic mice.Int J Dev Biol.2010;54(4):699-705.
[15]Kim D,Dressler GR.Nephrogenic factors promote differentiation of mouse embryonic stem cells into renal epithelia.J Am Soc Nephrol.2005;16(12):3527-3534.
[16]Narayanan K,Schumacher KM,Tasnim F,et al.Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.Kidney Int.2013;83(4):593-603.
[17]Song B,Smink AM,Jones CV,et al.The directed differentiation of human iPS cells into kidney podocytes.PLoS One.2012;7(9):e46453.
[18]Lam AQ,Freedman BS,Morizane R,et al.Rapid and efficient differentiation of human pluripotent stem cells into intermediate mesoderm that forms tubules expressing kidney proximal tubular markers.J Am Soc Nephrol.2014;25(6):1211-1225.
[19]Ezquer ME,Ezquer FE,Arango-Rodriguez ML,et al.MSCtransplantation:a promising therapeutic strategy to manage the onset and progression of diabetic nephropathy.Biol Res.2012;45(3):289-296.
[20]Ezquer FE,Ezquer ME,Parrau DB,et al.Systemic administration of multipotent mesenchymal stromal cells reverts hyperglycemia and prevents nephropathy in type 1 diabetic mice.Biol Blood Marrow Transplant.2008;14(6):631-640.
[21]Wu S,Li L,Wang G,et al.Ultrasound-targeted stromal cell-derived factor-1-loaded microbubble destruction promotes mesenchymal stem cell homing to kidneys in diabetic nephropathy rats.Int J Nanomedicine.2014;9:5639-5651.
[22]Nagaishi K,Mizue Y,Chikenji T,et al.Mesenchymal stem cell therapy ameliorates diabetic nephropathy via the paracrine effect of renal trophic factors including exosomes.Sci Rep.2016;6:34842.
[23]Ezquer F,Giraud-Billoud M,Carpio D,et al.Proregenerative Microenvironment Triggered by Donor Mesenchymal Stem Cells Preserves Renal Function and Structure in Mice with Severe Diabetes Mellitus.Biomed Res Int.2015;2015:164703.
[24]Hamza AH,Al-Bishri WM,Damiati LA,et al.Mesenchymal stem cells:a future experimental exploration for recession of diabetic nephropathy.Renal Failure.2017;39(1):67-76.
[25]Lenoir N.Europe confronts the embryonic stem cell research challenge.Science.2000;287(5457):1425-1427.
[26]Takahashi K,Yamanaka S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell.2006;126(4):663-676.
[27]Ben-David U,Benvenisty N.The tumorigenicity of human embryonic and induced pluripotent stem cells.Nat Rev Cancer.2011;11(4):268-277.
[28]Zuk PA,Zhu M,Mizuno H,et al.Multilineage cells from human adipose tissue:implications for cell-based therapies.Tissue Eng.2001;7(2):211-228.
[29]Panés J,García-Olmo D,Van Assche G,et al.Expanded allogeneic adipose-derived mesenchymal stem cells(Cx601)for complex perianal fistulas in Crohn's disease:a phase 3 randomised,double-blind controlled trial.Lancet.2016;388(10051):1281-1290.
[30]Henry TD,Pepine CJ,Lambert CR,et al.The Athena trials:Autologous adipose-derived regenerative cells for refractory chronic myocardial ischemia with left ventricular dysfunction.Catheter Cardiovasc Interv.2017;89(2):169-177.
[31]Merceron C,Portron S,Masson M,et al.TThe effect of two-and three-dimensional cell culture on the chondrogenic potential of human adipose-derived mesenchymal stem cells after subcutaneous transplantation with an injectable hydrogel.Cell Transplant.2011;20(10):1575-1588.
[32]Aksu AE,Rubin JP,Dudas JR,et al.Role of gender and anatomical region on induction of osteogenic differentiation of human adipose-derived stem cells.Ann Plast Surg.2008;60(3):306-322.
[33]Wosnitza M,Hemmrich K,Groger A,et al.Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation.Differentiation.2007;75(1):12-23.
[34]Bourin P,Bunnell BA,Casteilla L,et al.Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells:a joint statement of the International Federation for Adipose Therapeutics and Science(IFATS)and the International Society for Cellular Therapy(ISCT).Cytotherapy.2013;15(6):641-648.
[35]Zack-Williams SD,Butler PE,Kalaskar DM.Current progress in use of adipose derived stem cells in peripheral nerve regeneration.World JStem Cells.2015;7(1):51-64.
[36]Baer PC,Geiger H.Adipose-derived mesenchymal stromal/stem cells:tissue localization,characterization,and heterogeneity.Stem Cells Int.2012;2012:812693.
[37]Lee RH,Kim B,Choi I,et al.Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.Cell Physiol Biochem.2004;14(4-6):311-324.
[38]Sheykhhasan M,Qomi RT,Ghiasi M.Fibrin Scaffolds Designing in order to Human Adipose-derived Mesenchymal Stem Cells Differentiation to Chondrocytes in the Presence of TGF-β3.Int J Stem Cells.2015;8(2):219-227.
[39]Mundra V,Gerling IC,Mahato RI.Mesenchymal Stem Cell-Based Therapy.Molecular Pharmaceutics.2012;10(1):77-89.
[40]Valina C,Pinkernell K,Song YH,et al.Intracoronary administration of autologous adipose tissue-derived stem cells improves left ventricular function,perfusion,and remodelling after acute myocardial infarction.Eur Heart J.2007;28(21):2667-2677.
[41]Zhang DZ,Gai LY,Liu HW,et al.Transplantation of autologous adipose-derived stem cells ameliorates cardiac function in rabbits with myocardial infarction.Chin Med J(Engl).2007;120(4):300-307.