人脐带间充质干细胞联合免疫干预治疗1型糖尿病小鼠的实验研究
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摘要
背景:1型糖尿病是一种以T细胞介导的胰岛β细胞损伤为特征的自身免疫性疾病,实验拟针对其免疫机制进行干预,并用干细胞修复受损的胰岛β细胞,探索治疗1型糖尿病的新方法。目的:观察人脐带间充质干细胞联合免疫干预治疗1型糖尿病小鼠的疗效。方法:取靶向敲除调节性T细胞的BALB/c Foxp3-DTR-EGFP阳性小鼠50只,随机选择6只作为正常对照组,剩余小鼠通过腹腔注射链脲佐菌素及白喉毒素制备1型糖尿病模型,造模成功后,随机分为4组:模型组给予等量生理盐水;免疫干预组皮下注射地塞米松(10μg)+胰岛素(10μg)混合液;人脐带间充质干细胞组经尾静脉移植人脐带间充质干细胞1×10~6/只;联合治疗组采用以上两种治疗方法。治疗后4周,观察各组小鼠血糖、C肽及体质量变化,胰腺组织病理及胰岛素染色阳性面积。结果与结论:①与正常对照组对比,模型组血糖升高(P <0.05), C肽及体质量均下降(P <0.05),胰岛严重萎缩,胰岛细胞减少,胰岛素染色阳性面积减小;②与模型组对比,免疫干预组血糖有所下降但差异不显著(P>0.05),C肽及体质量改变不显著(P> 0.05),胰岛细胞增多,胰岛素阳性面积增大;③与模型组对比,人脐带间充质干细胞组及联合治疗组血糖降低,但差异不显著(P> 0.05),C肽及体质量均升高(P <0.05),胰岛细胞增多,胰岛素染色阳性面积增大;④结果表明,人脐带间充质干细胞、免疫干预均能改善1型糖尿病小鼠的胰岛功能,联合治疗效果更佳。
BACKGROUND: Type 1 diabetes mellitus is a T cell-mediated autoimmune disease resulting in pancreatic islet cell damage. In this study,immunotherapy was used to deal with type 1 diabetes mellitus and stem cell transplantation was used to repair damaged islet β cells,attempting to explore a new treatment for type 1 diabetes mellitus.OBJECTIVE: To observe the efficacy of human umbilical cord mesenchymal stem cells combined with immunotherapy for the treatment of type 1 diabetic mice.METHODS: Fifty BALB/c Foxp3-DTR-EGFP positive mice with targeted knockout of regulatory T cells were selected, six of which were randomly selected as normal control group and the remaining of which were intraperitoneally injected with streptozotocin and diphtheria toxin to prepare an animal model of type 1 diabetes mellitus. After successful modeling, randomization was performed in model mice and there were four groups: model group(normal saline), immunotherapy group(subcutaneous injection of dexamethasone(10 μg) and insulin(10 μg)mixture), cell transplantation group(injection of human umbilical cord mesenchymal stem cells(1×106 cells per mouse) through the tail vein,and combined treatment group(the combination of immunotherapy and cell transplantation as described above). At 4 weeks after treatment,changes in blood glucose, C-peptide, body mass, pancreatic histopathology and insulin-positive area were observed in each group.RESULTS AND CONCLUSION:(1) Compared with the normal control group, the blood glucose level of the model group increased(P < 0.05),the C peptide level and body mass decreased(P < 0.05), and the islet was severely atrophied, with decreased number of islet cells and reduced insulin-positive area.(2) Compared with the model group, the blood glucose level of the immunotherapy group decreased(P > 0.05),the C-peptide level and body mass did not change significantly(P > 0.05), the islet cells increased in number, and the insulin-positive area increased.(3) Compared with the model group, the blood glucose level of the cell transplantation group and the combined treatment group decreased(P > 0.05), the C peptide level and body mass increased(P < 0.05), the islet cells increased in number, and the insulin-positive area increased. These findings reveal that either human umbilical cord mesenchymal stem cells or immunotherapy can improve the islet function of type 1 diabetic mice, and the combination treatment has better outcomes.
BACKGROUND: Type 1 diabetes mellitus is a T cell-mediated autoimmune disease resulting in pancreatic islet cell damage. In this study,immunotherapy was used to deal with type 1 diabetes mellitus and stem cell transplantation was used to repair damaged islet β cells,attempting to explore a new treatment for type 1 diabetes mellitus.OBJECTIVE: To observe the efficacy of human umbilical cord mesenchymal stem cells combined with immunotherapy for the treatment of type 1 diabetic mice.METHODS: Fifty BALB/c Foxp3-DTR-EGFP positive mice with targeted knockout of regulatory T cells were selected, six of which were randomly selected as normal control group and the remaining of which were intraperitoneally injected with streptozotocin and diphtheria toxin to prepare an animal model of type 1 diabetes mellitus. After successful modeling, randomization was performed in model mice and there were four groups: model group(normal saline), immunotherapy group(subcutaneous injection of dexamethasone(10 μg) and insulin(10 μg)mixture), cell transplantation group(injection of human umbilical cord mesenchymal stem cells(1×106 cells per mouse) through the tail vein,and combined treatment group(the combination of immunotherapy and cell transplantation as described above). At 4 weeks after treatment,changes in blood glucose, C-peptide, body mass, pancreatic histopathology and insulin-positive area were observed in each group.RESULTS AND CONCLUSION:(1) Compared with the normal control group, the blood glucose level of the model group increased(P < 0.05),the C peptide level and body mass decreased(P < 0.05), and the islet was severely atrophied, with decreased number of islet cells and reduced insulin-positive area.(2) Compared with the model group, the blood glucose level of the immunotherapy group decreased(P > 0.05),the C-peptide level and body mass did not change significantly(P > 0.05), the islet cells increased in number, and the insulin-positive area increased.(3) Compared with the model group, the blood glucose level of the cell transplantation group and the combined treatment group decreased(P > 0.05), the C peptide level and body mass increased(P < 0.05), the islet cells increased in number, and the insulin-positive area increased. These findings reveal that either human umbilical cord mesenchymal stem cells or immunotherapy can improve the islet function of type 1 diabetic mice, and the combination treatment has better outcomes.
引文
[1]Battaglia M,Atkinson MA.The Streetlight Effect in Type 1 Diabetes.Diabetes.2015;64(4):1081-1090.
[2]Gülden E,Palm N,Herold KC.MAIT Cells:A Link between Gut Integrity and Type 1 Diabetes.Cell Metab.2017;26(6):813-815.
[3]Atkinson MA,von Herrath M,Powers AC,et al.Current concepts on the pathogenesis of type 1 diabetes-considerations for attempts to prevent and reverse the disease.Diabetes Care.2015;38(6):979-988.
[4]Wang HS,Shyu JF,Shen WS,et al.Transplantation of insulinproducing cells derived from umbilical cord stromal mesenchymal stem cells to treat NOD mice.Cell Transplant.2011;20(3):455-466.
[5]Qu H,Liu X,Ni Y,et al.Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells.J Transl Med.2014;12:135.
[6]申义,王意忠,时瀚,等.人脐带间充质干细胞分化胰岛样细胞过程中胰岛素和巢蛋白的表达[J].中国组织工程研究,2016,20(50):7500-7506.
[7]Kuchroo P,Dave V,Vijayan A,et al.Paracrine factors secreted by umbilical cord-derived mesenchymal stem cells induce angiogenesis in vitro by a VEGF-independent pathway.Stem Cells Dev.2015;24(4):437-450.
[8]Shen C,Lie P,Miao T,et al.Conditioned medium from umbilical cord mesenchymal stem cells induces migration and angiogenesis.Mol Med Rep.2015;12(1):20-30.
[9]Li T,Xia M,Gao Y,et al.Human umbilical cord mesenchymal stem cells:an overview of their potential in cell-based therapy.Expert Opin Biol Ther.2015;15(9):1293-1306.
[10]Ding D,Chang Y,Shyu W,et al.Human umbilical cord mesenchymal stem cells:a new era for stem cell therapy.Cell Transplant.2015;24(3):339-347.
[11]Lu LL,Liu YJ,Yang SG,et al.Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesissupportive function and other potentials.Haematologica.2006;91(8):1017-1026.
[12]Wilckens T,De Rijk R.Glucocorticoids and immune function:unknown dimensions and new frontiers.Immunol Today.1997;18(9):418-424.
[13]Londono P,Komura A,Hara N,et al.Brief dexamethasone treatment during acute infection prevents virus-induced autoimmune diabetes.Clin Immunol.2010;135(3):401-411.
[14]Harrison LC,Wentworth JM,Zhang Y,et al.Antigen-based vaccination and prevention of type 1 diabetes.Curr Diab Rep.2013;13(5):616-623.
[15]Harrison LC,Honeyman MC,Steele CE,et al.Pancreatic beta-cell function and immune responses to insulin after administration of intranasal insulin to humans at risk for type 1 diabetes.Diabetes Care.2004;27(10):2348-2355.
[16]于文龙,高宏,余霄龙,等.脐带间充质干细胞移植治疗初发1型糖尿病[J].中国组织工程研究与临床康复,2011,15(23):4363-4366.
[17]谭晓俊,王镇,李晓莹,等.脐带间充质干细胞治疗1型糖尿病远期疗效观察三例报告[J].中华内分泌代谢杂志,2016,32(12):1030-1032.
[18]Geng S,Zhang H,Zhou X,et al.Diabetes tolerogenic vaccines targeting antigen-specific inflammation.Hum Vaccin Immunother.2015;11(2):522-530.
[19]Campbell-Thompson ML,Atkinson MA,Butler AE,et al.The diagnosis of insulitis in human type 1 diabetes.Diabetologia.2013;56(11):2541-2543.
[20]Krogvold L,Skog O,Sundstrom G,et al.Function of Isolated Pancreatic Islets From Patients at Onset of Type 1 Diabetes:Insulin Secretion Can Be Restored After Some Days in a Nondiabetogenic Environment In Vitro:Results From the DiViD Study.Diabetes.2015;64(7):2506-2512.
[21]Marhfour I,Lopez XM,Lefkaditis D,et al.Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes.Diabetologia.2012;55(9):2417-2420.
[22]Imai Y,Dobrian AD,Morris MA,et al.Lipids and immunoinflammatory pathways of beta cell destruction.Diabetologia.2016;59(4):673-678.
[23]Grzesik WJ,Nadler JL,Machida Y,et al.Expression pattern of12-lipoxygenase in human islets with type 1 diabetes and type 2diabetes.J Clin Endocrinol Metab.2015;100(3):E387-E395.
[24]Willcox A,Richardson SJ,Bone AJ,et al.Analysis of islet inflammation in human type 1 diabetes.Clin Exp Immunol.2009;155(2):173-181.
[25]Visperas A,Vignali DA.Are Regulatory T Cells Defective in Type 1Diabetes and Can We Fix Them?.J Immunol,2016;197(10):3762-3770.
[26]D'Alessio FR,Tsushima K,Aggarwal NR,et al.CD4+CD25+Foxp3+Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury.J Clin Invest.2009;119(10):2898-2913.
[27]Liesz A,Suri-Payer E,Veltkamp C,et al.Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke.Nat Med.2009;15(2):192-199.
[28]Chen X,Zhou B,Li M,et al.CD4+CD25+FoxP3+regulatory T cells suppress Mycobacterium tuberculosis immunity in patients with active disease.Clin Immunol.2007;123(1):50-59.
[29]Qiao YC,Shen J,Hong XZ,et al.Changes of regulatory T cells,transforming growth factor-beta and interleukin-10 in patients with type 1 diabetes mellitus:A systematic review and meta-analysis.Clin Immunol.2016;170:61-69.
[30]Ghonaim MM,El-Edel RH,Kamal ES,et al.T-Regulatory Cell Subsets in Children with Type 1 Diabetes Mellitus:Relation to Control of the Disease.Endocr Metab Immune Disord Drug Targets.2017;17(3):238-245.
[31]Lampeter EF,Signore A,Gale EA,et al.Lessons from the NODmouse for the pathogenesis and immunotherapy of human type 1(insulin-dependent)diabetes mellitus.Diabetologia.1989;32(10):703-708.
[32]Lenzen S.Animal models of human type 1 diabetes for evaluating combination therapies and successful translation to the patient with type 1 diabetes.Diabetes Metab Res Rev.2017;33(7).doi:10.1002/dmrr.2915.
[33]Geng S,Zhang H,Zhou X,et al.Diabetes tolerogenic vaccines targeting antigen-specific inflammation.Hum Vaccin Immunother.2015;11(2):522-530.
[34]Zipris D.Innate immunity in type 1 diabetes.Diabetes Metab Res Rev.2011;27(8):824-829.
[35]Hu J,Wang Y,Wang F,et al.Effect and mechanisms of human Wharton s jelly-derived mesenchymal stem cells on type 1diabetes in NOD model.Endocrine.2015;48(1):124-134.
[36]Wang D,Chen K,Du W T,et al.CD14+monocytes promote the immunosuppressive effect of human umbilical cord matrix stem cells.Exp Cell Res.2010;316(15):2414-2423.
[37]Ribeiro A,Laranjeira P,Mendes S,et al.Mesenchymal stem cells from umbilical cord matrix,adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B,natural killer and T cells.Stem Cell Res Ther.2013;4(5):125.
[38]Tang RJ,Shen SN,Zhao XY,et al.Mesenchymal stem cells-regulated Treg cells suppress colitis-associated colorectal cancer.Stem Cell Res Ther.2015;6:71.
[39]Wang D,Huang S,Yuan X,et al.The regulation of the Treg/Th17balance by mesenchymal stem cells in human systemic lupus erythematosus.Cell Mol Immunol.2017;14(5):423-431.
[40]Montanucci P,Alunno A,Basta G,et al.Restoration of t cell substes of patients with type 1 diabetes mellitus by microencapsulated human umbilical cord Wharton jelly-derived mesenchymal stem cells:An in vitro study.Clin Immunol.2016;163:34-41.
[41]赵子粼,叶常青,罗建春,等.脐带间充质干细胞移植对心力衰竭大鼠外周血CD_4~+CD_(25)~+Foxp_3~+Treg细胞表达的影响[J].山东医药,2015,55(41):13-15.
[42]El-Hossary N,Hassanein H,El Ghareeb AW,et al.Intravenous vs intraperitoneal transplantation of umbilical cord mesenchymal stem cells from Wharton s jelly in the treatment of streptozotocininduced diabetic rats.Diabetes Res Clin Pract.2016;21:102-111.
[43]Sun X,Hao H,Han Q,et al.Human umbilical cord-derived mesenchymal stem cells ameliorate insulin resistance by suppressing NLRP3 inflammasome-mediated inflammation in type2 diabetes rats.Stem Cell Res Ther.2017;8(1):241.
[2]Gülden E,Palm N,Herold KC.MAIT Cells:A Link between Gut Integrity and Type 1 Diabetes.Cell Metab.2017;26(6):813-815.
[3]Atkinson MA,von Herrath M,Powers AC,et al.Current concepts on the pathogenesis of type 1 diabetes-considerations for attempts to prevent and reverse the disease.Diabetes Care.2015;38(6):979-988.
[4]Wang HS,Shyu JF,Shen WS,et al.Transplantation of insulinproducing cells derived from umbilical cord stromal mesenchymal stem cells to treat NOD mice.Cell Transplant.2011;20(3):455-466.
[5]Qu H,Liu X,Ni Y,et al.Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells.J Transl Med.2014;12:135.
[6]申义,王意忠,时瀚,等.人脐带间充质干细胞分化胰岛样细胞过程中胰岛素和巢蛋白的表达[J].中国组织工程研究,2016,20(50):7500-7506.
[7]Kuchroo P,Dave V,Vijayan A,et al.Paracrine factors secreted by umbilical cord-derived mesenchymal stem cells induce angiogenesis in vitro by a VEGF-independent pathway.Stem Cells Dev.2015;24(4):437-450.
[8]Shen C,Lie P,Miao T,et al.Conditioned medium from umbilical cord mesenchymal stem cells induces migration and angiogenesis.Mol Med Rep.2015;12(1):20-30.
[9]Li T,Xia M,Gao Y,et al.Human umbilical cord mesenchymal stem cells:an overview of their potential in cell-based therapy.Expert Opin Biol Ther.2015;15(9):1293-1306.
[10]Ding D,Chang Y,Shyu W,et al.Human umbilical cord mesenchymal stem cells:a new era for stem cell therapy.Cell Transplant.2015;24(3):339-347.
[11]Lu LL,Liu YJ,Yang SG,et al.Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesissupportive function and other potentials.Haematologica.2006;91(8):1017-1026.
[12]Wilckens T,De Rijk R.Glucocorticoids and immune function:unknown dimensions and new frontiers.Immunol Today.1997;18(9):418-424.
[13]Londono P,Komura A,Hara N,et al.Brief dexamethasone treatment during acute infection prevents virus-induced autoimmune diabetes.Clin Immunol.2010;135(3):401-411.
[14]Harrison LC,Wentworth JM,Zhang Y,et al.Antigen-based vaccination and prevention of type 1 diabetes.Curr Diab Rep.2013;13(5):616-623.
[15]Harrison LC,Honeyman MC,Steele CE,et al.Pancreatic beta-cell function and immune responses to insulin after administration of intranasal insulin to humans at risk for type 1 diabetes.Diabetes Care.2004;27(10):2348-2355.
[16]于文龙,高宏,余霄龙,等.脐带间充质干细胞移植治疗初发1型糖尿病[J].中国组织工程研究与临床康复,2011,15(23):4363-4366.
[17]谭晓俊,王镇,李晓莹,等.脐带间充质干细胞治疗1型糖尿病远期疗效观察三例报告[J].中华内分泌代谢杂志,2016,32(12):1030-1032.
[18]Geng S,Zhang H,Zhou X,et al.Diabetes tolerogenic vaccines targeting antigen-specific inflammation.Hum Vaccin Immunother.2015;11(2):522-530.
[19]Campbell-Thompson ML,Atkinson MA,Butler AE,et al.The diagnosis of insulitis in human type 1 diabetes.Diabetologia.2013;56(11):2541-2543.
[20]Krogvold L,Skog O,Sundstrom G,et al.Function of Isolated Pancreatic Islets From Patients at Onset of Type 1 Diabetes:Insulin Secretion Can Be Restored After Some Days in a Nondiabetogenic Environment In Vitro:Results From the DiViD Study.Diabetes.2015;64(7):2506-2512.
[21]Marhfour I,Lopez XM,Lefkaditis D,et al.Expression of endoplasmic reticulum stress markers in the islets of patients with type 1 diabetes.Diabetologia.2012;55(9):2417-2420.
[22]Imai Y,Dobrian AD,Morris MA,et al.Lipids and immunoinflammatory pathways of beta cell destruction.Diabetologia.2016;59(4):673-678.
[23]Grzesik WJ,Nadler JL,Machida Y,et al.Expression pattern of12-lipoxygenase in human islets with type 1 diabetes and type 2diabetes.J Clin Endocrinol Metab.2015;100(3):E387-E395.
[24]Willcox A,Richardson SJ,Bone AJ,et al.Analysis of islet inflammation in human type 1 diabetes.Clin Exp Immunol.2009;155(2):173-181.
[25]Visperas A,Vignali DA.Are Regulatory T Cells Defective in Type 1Diabetes and Can We Fix Them?.J Immunol,2016;197(10):3762-3770.
[26]D'Alessio FR,Tsushima K,Aggarwal NR,et al.CD4+CD25+Foxp3+Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury.J Clin Invest.2009;119(10):2898-2913.
[27]Liesz A,Suri-Payer E,Veltkamp C,et al.Regulatory T cells are key cerebroprotective immunomodulators in acute experimental stroke.Nat Med.2009;15(2):192-199.
[28]Chen X,Zhou B,Li M,et al.CD4+CD25+FoxP3+regulatory T cells suppress Mycobacterium tuberculosis immunity in patients with active disease.Clin Immunol.2007;123(1):50-59.
[29]Qiao YC,Shen J,Hong XZ,et al.Changes of regulatory T cells,transforming growth factor-beta and interleukin-10 in patients with type 1 diabetes mellitus:A systematic review and meta-analysis.Clin Immunol.2016;170:61-69.
[30]Ghonaim MM,El-Edel RH,Kamal ES,et al.T-Regulatory Cell Subsets in Children with Type 1 Diabetes Mellitus:Relation to Control of the Disease.Endocr Metab Immune Disord Drug Targets.2017;17(3):238-245.
[31]Lampeter EF,Signore A,Gale EA,et al.Lessons from the NODmouse for the pathogenesis and immunotherapy of human type 1(insulin-dependent)diabetes mellitus.Diabetologia.1989;32(10):703-708.
[32]Lenzen S.Animal models of human type 1 diabetes for evaluating combination therapies and successful translation to the patient with type 1 diabetes.Diabetes Metab Res Rev.2017;33(7).doi:10.1002/dmrr.2915.
[33]Geng S,Zhang H,Zhou X,et al.Diabetes tolerogenic vaccines targeting antigen-specific inflammation.Hum Vaccin Immunother.2015;11(2):522-530.
[34]Zipris D.Innate immunity in type 1 diabetes.Diabetes Metab Res Rev.2011;27(8):824-829.
[35]Hu J,Wang Y,Wang F,et al.Effect and mechanisms of human Wharton s jelly-derived mesenchymal stem cells on type 1diabetes in NOD model.Endocrine.2015;48(1):124-134.
[36]Wang D,Chen K,Du W T,et al.CD14+monocytes promote the immunosuppressive effect of human umbilical cord matrix stem cells.Exp Cell Res.2010;316(15):2414-2423.
[37]Ribeiro A,Laranjeira P,Mendes S,et al.Mesenchymal stem cells from umbilical cord matrix,adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B,natural killer and T cells.Stem Cell Res Ther.2013;4(5):125.
[38]Tang RJ,Shen SN,Zhao XY,et al.Mesenchymal stem cells-regulated Treg cells suppress colitis-associated colorectal cancer.Stem Cell Res Ther.2015;6:71.
[39]Wang D,Huang S,Yuan X,et al.The regulation of the Treg/Th17balance by mesenchymal stem cells in human systemic lupus erythematosus.Cell Mol Immunol.2017;14(5):423-431.
[40]Montanucci P,Alunno A,Basta G,et al.Restoration of t cell substes of patients with type 1 diabetes mellitus by microencapsulated human umbilical cord Wharton jelly-derived mesenchymal stem cells:An in vitro study.Clin Immunol.2016;163:34-41.
[41]赵子粼,叶常青,罗建春,等.脐带间充质干细胞移植对心力衰竭大鼠外周血CD_4~+CD_(25)~+Foxp_3~+Treg细胞表达的影响[J].山东医药,2015,55(41):13-15.
[42]El-Hossary N,Hassanein H,El Ghareeb AW,et al.Intravenous vs intraperitoneal transplantation of umbilical cord mesenchymal stem cells from Wharton s jelly in the treatment of streptozotocininduced diabetic rats.Diabetes Res Clin Pract.2016;21:102-111.
[43]Sun X,Hao H,Han Q,et al.Human umbilical cord-derived mesenchymal stem cells ameliorate insulin resistance by suppressing NLRP3 inflammasome-mediated inflammation in type2 diabetes rats.Stem Cell Res Ther.2017;8(1):241.