骨髓间充质干细胞与胰岛细胞共培养对胰岛功能的影响
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摘要
背景:骨髓间充质干细胞能分泌多种生长因子并支持造血,但能否保护和修复胰岛细胞功能尚不明确。目的:探讨大鼠骨髓间充质干细胞与其胰岛细胞共培养对胰岛功能的影响。方法:从八九周龄的雄性SD大鼠中提取股骨骨髓,采用密度梯度离心法和贴壁培养法相结合提取骨髓间充质干细胞;另取八九周龄的雄性SD大鼠,从胆总管插管注入1 g/L胶原酶P 10 mL,剪下胰腺,采用Histopaque不连续密度梯度离心法分离与纯化胰岛细胞,在显微镜下用1 mL的pipet移液器手工挑取筛选薄膜完整的胰岛细胞。将第3代骨髓间充质干细胞与胰岛细胞进行共培养,共分为4组:对照组(单纯大鼠胰岛细胞培养)、直接共培养组(接种有骨髓间充质干细胞的培养板内加入胰岛细胞进行共培养)、间接共培养组1(利用transwell培养皿进行间接共培养)、间接共培养组2(利用骨髓间充质干细胞培养液培养胰岛细胞)。培养过程中观察胰岛形态及生长状况,在培养后第3,7,14天用ELISA法检测胰岛素分泌量。实验方案经延边大学附属医院医学伦理委员会批准。结果与结论:(1)4组之间采用方差分析,骨髓间充质干细胞与胰岛细胞共培养能明显提高胰岛素的分泌量,而且共培养第7天胰岛素分泌量达高峰;(2)在培养后第7天时,间接共培养组1的胰岛素分泌量明显高于直接共培养组,差异有显著性意义(P<0.05);(3)结果表明,骨髓间充质干细胞与胰岛细胞利用transwell培养皿进行间接共培养可以明显提高胰岛素的分泌量,而且共培养7 d效果较好。
BACKGROUND: Bone marrow mesenchymal stem cells can secrete various growth factors and support hematopoiesis, but whether they can protect and repair the function of islet cells remains unclear.OBJECTIVE: To investigate the effect of rat bone marrow mesenchymal stem cells co-cultured with islet cells on rat islet function. METHODS: Bone marrow was extracted from male Sprague-Dawley rats aged 8-9 weeks. Bone marrow mesenchymal stem cells were isolated from bone marrow using density gradient centrifugation combined with adherent culture. Collagenase P(1 g/L, 10 mL) was injected into the common bile duct of another male Sprague-Dawley rats aged 8-9 weeks, and the pancreas was cut off to isolate and purify islet cells using Histopaque discontinuous density gradient. Islet cells with intact cell membrane were selected by hand using 1-mL pipet pipette under microscope, and co-cultured with third-generation bone marrow mesenchymal stem cells. The experiment was divided into control group(single islet cell culture), direct co-culture group(islet cells co-cultured with bone marrow mesenchymal stem cells in culture plates), indirect culture group 1(indirect co-culture with Transwell dish), and indirect culture group 2(islet cell culture with bone marrow mesenchymal stem cell medium). The morphology and growth of islets were observed during culture. Insulin secretion was measured by ELISA on the 3~(rd), 7~(th), and 14~(th) days after culture. The study protocol was approved by the Ethics Committee of Yanbian University Affiliated Hospital. RESULTS AND CONCLUSION: The results of variance analysis showed that insulin secretion was significantly increased when islet cells were co-cultured with bone marrow mesenchymal stem cells, and reached a peak on the 7~(th) day of co-culture. Insulin secretion was significantly higher in the indirect co-culture group 1 than the direct co-culture group(P < 0.05). To conclude, the indirect co-culture method of Transwell dish can significantly improve insulin secretion, and the rational co-culture time is 7 days.
BACKGROUND: Bone marrow mesenchymal stem cells can secrete various growth factors and support hematopoiesis, but whether they can protect and repair the function of islet cells remains unclear.OBJECTIVE: To investigate the effect of rat bone marrow mesenchymal stem cells co-cultured with islet cells on rat islet function. METHODS: Bone marrow was extracted from male Sprague-Dawley rats aged 8-9 weeks. Bone marrow mesenchymal stem cells were isolated from bone marrow using density gradient centrifugation combined with adherent culture. Collagenase P(1 g/L, 10 mL) was injected into the common bile duct of another male Sprague-Dawley rats aged 8-9 weeks, and the pancreas was cut off to isolate and purify islet cells using Histopaque discontinuous density gradient. Islet cells with intact cell membrane were selected by hand using 1-mL pipet pipette under microscope, and co-cultured with third-generation bone marrow mesenchymal stem cells. The experiment was divided into control group(single islet cell culture), direct co-culture group(islet cells co-cultured with bone marrow mesenchymal stem cells in culture plates), indirect culture group 1(indirect co-culture with Transwell dish), and indirect culture group 2(islet cell culture with bone marrow mesenchymal stem cell medium). The morphology and growth of islets were observed during culture. Insulin secretion was measured by ELISA on the 3~(rd), 7~(th), and 14~(th) days after culture. The study protocol was approved by the Ethics Committee of Yanbian University Affiliated Hospital. RESULTS AND CONCLUSION: The results of variance analysis showed that insulin secretion was significantly increased when islet cells were co-cultured with bone marrow mesenchymal stem cells, and reached a peak on the 7~(th) day of co-culture. Insulin secretion was significantly higher in the indirect co-culture group 1 than the direct co-culture group(P < 0.05). To conclude, the indirect co-culture method of Transwell dish can significantly improve insulin secretion, and the rational co-culture time is 7 days.
引文
[1]梁梦璐,胡永华.1型糖尿病病因流行病学研究进展[J].中华疾病控制杂志,2013,17(4):349-353.
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[3]Kim JY,Lee JI,Jeong JH,et al.Functional evaluation of chondrocyte sheeting immunodelusive immunoisolated bioartificial pancreas.Transplant Proc.2010;42(3):903-906.
[4]Jin SM,Kim KW.Is islet transplantation a realistic approach to curing diabetes.Korean J Intern Med.2017;32(1):62-66.
[5]Dunn TB,Wilhelm JJ,Bellin MD,et al.Autologous islet transplantation:challenges and lessons.Curr Opin Organ Transplant.2017;22(4):364-371.
[6]张德国,谭雪莹,唐楠,等.人脂肪间充质干细胞尾静脉移植治疗大鼠急性肝衰竭[J].中国组织工程研究,2019,23(9):1416-1421.
[7]Rawal S,Williams SJ,Ramachandran K,et al.Integration of mesenchymal stem cells into islet cell spheroids improves long-term viability,but not islet function.Islets.2017;9(5):87-98.
[8]刘红静,王慧丰,韦雅淑.胰腺组织裂解液诱导骨髓间充质干细胞分化为胰岛样结构的鉴定[J].中国组织工程研究,2019,23(13):2088-2093.
[9]Gabr MM,Zakaria MM,Refaie AF,et al.Insulin-producing cells from adult human bone marrow mesenchymal stem cells control streptozotocin-induced diabetes in nude mice.Cell Transplant.2013;22(1):133-145.
[10]Xie H,Wang Y,Zhang H,et al.Role of injured pancreatic extract promotes bone marrow-derived mesenchymal stem cells efficiently differentiate into insulin-producing cells.PLoSOne.2013;8(9):e76056.
[11]Kim JW,Vang S,Luo JZQ,et al.Human Islet Co-Cultured with Bone Marrow Mesenchymal Stem Cells in 3D Scaffolding May Augment Pancreatic Beta Cell Function.Journal of biomaterials and tissue engineering.2017;7(3):203-209.
[12]Unsal IO,Ginis Z,Pinarli FA,et al.Comparison of therapeutic characteristics of islet cell transplantation simultaneous with pancreatic mesenchymal stem cell transplantation in rats with Type 1 diabetes mellitus.Stem Cell Rev.2015;11(3):526-532.
[13]Wen D,Peng Y,Liu D,et al.Mesenchymal stem cell and derived exosome as small RNA carrier and Immunomodulator to improve islet transplantation.J Control Release.2016;238:166-175.
[14]Corradi-Perini C,Santos TM,Camara NOS,et al.Co-transplantation of Xenogeneic Bone Marrow-derived Mesenchymal Stem Cells Alleviates Rejection of Pancreatic Islets in Non-obese Diabetic Mice.Transplant Proc.2017;49(4):902-905.
[15]徐昕博.糖尿病治疗的研究现状及进展[J].养生保健指南,2019(7):213-214.
[16]Moravej H,Salehi A,Shamsedini A,et al.Complementary and Alternative Medicine to Treat Diabetes Mellitus Type 1 in Southern Iran.Iran J Med Sci.2016;41(1):71-72.
[17]Heller T,Kloos C,Lehmann T,et al.Mortality and its Causes in a German Cohort with Diabetes Mellitus Type 1 after 20Years of Follow-Up:The JEVIN Trial.Exp Clin Endocrinol Diabetes.2018;126(6):387-393.
[18]Kramer G,Kuniss N,Kloos C,et al.Principles and frequency of self-adjustment of insulin dose in people with diabetes mellitus type 1 and correlation with markers of metabolic control.Diabetes Res Clin Pract.2016;116:299-305.
[19]Bro?ováJ,?echurováD,LacigováS.Metabolic syndrome in patients with diabetes mellitus type 1,prevalence,impact on morbidity and mortality.Vnitr Lek.2016;62(11 Suppl 4):S8-14.
[20]Pathak S,Regmi S,Nguyen TT,et al.Polymeric microsphere-facilitated site-specific delivery of quercetin prevents senescence of pancreatic islets in vivo and improves transplantation outcomes in mouse model of diabetes.Acta Biomater.2018;75:287-299.
[21]Tchokonte-Nana V,Manda JK.Early islets and mesenchyme from an injured adult pancreas improve syngeneic engraftments and islet graft function in diabetic rats.Acta Histochem.2018;120(4):356-362.
[22]He Y,Zhang D,Zeng Y,et al.Bone Marrow-Derived Mesenchymal Stem Cells Protect Islet Grafts Against Endoplasmic Reticulum Stress-Induced Apoptosis During the Early Stage After Transplantation.Stem Cells.2018;36(7):1045-1061.
[23]Carlsson PO,Espes D,Sedigh A,et al.Transplantation of macroencapsulated human islets within the bioartificial pancreasβAir to patients with type 1 diabetes mellitus.Am JTransplant.2018;18(7):1735-1744.
[24]Cao XK,Li R,Sun W,et al.Co-combination of islets with bone marrow mesenchymal stem cells promotes angiogenesis.Biomed Pharmacother.2016;78:156-164.
[25]Wang H,Strange C,Nietert PJ,et al.Autologous Mesenchymal Stem Cell and Islet Cotransplantation:Safety and Efficacy.Stem Cells Transl Med.2018;7(1):11-19.
[26]He Y,Zhang D,Zeng Y,et al.Bone Marrow-Derived Mesenchymal Stem Cells Protect Islet Grafts Against Endoplasmic Reticulum Stress-Induced Apoptosis During the Early Stage After Transplantation.Stem Cells.2018;36(7):1045-1061.
[27]Li HT,Jiang FX,Shi P,et al.In vitro reprogramming of rat bmMSCs into pancreatic endocrine-like cells.In Vitro Cell Dev Biol Anim.2017;53(2):157-166.
[28]Cantarelli E,Citro A,Pellegrini S,et al.Transplant Site Influences the Immune Response After Islet Transplantation:Bone Marrow Versus Liver.Transplantation.2017;101(5):1046-1055.
[29]李宏图,林学文,李彩虹,等.负向调节因子重编程诱导大鼠骨髓间充质干细胞向胰岛内分泌细胞分化的研究[J].中国医科大学学报,2016,45(1):1-6.
[30]Westenfelder C,Gooch A,Hu Z,et al.Durable Control of Autoimmune Diabetes in Mice Achieved by Intraperitoneal Transplantation of"Neo-Islets,"Three-Dimensional Aggregates of Allogeneic Islet and"Mesenchymal Stem Cells"Stem Cells Transl Med.2017;6(7):1631-1643.
[2]Kharroubi AT,Darwish HM.Diabetes mellitus:The epidemic of the century.World J Diabetes.2015;6(6):850-867.
[3]Kim JY,Lee JI,Jeong JH,et al.Functional evaluation of chondrocyte sheeting immunodelusive immunoisolated bioartificial pancreas.Transplant Proc.2010;42(3):903-906.
[4]Jin SM,Kim KW.Is islet transplantation a realistic approach to curing diabetes.Korean J Intern Med.2017;32(1):62-66.
[5]Dunn TB,Wilhelm JJ,Bellin MD,et al.Autologous islet transplantation:challenges and lessons.Curr Opin Organ Transplant.2017;22(4):364-371.
[6]张德国,谭雪莹,唐楠,等.人脂肪间充质干细胞尾静脉移植治疗大鼠急性肝衰竭[J].中国组织工程研究,2019,23(9):1416-1421.
[7]Rawal S,Williams SJ,Ramachandran K,et al.Integration of mesenchymal stem cells into islet cell spheroids improves long-term viability,but not islet function.Islets.2017;9(5):87-98.
[8]刘红静,王慧丰,韦雅淑.胰腺组织裂解液诱导骨髓间充质干细胞分化为胰岛样结构的鉴定[J].中国组织工程研究,2019,23(13):2088-2093.
[9]Gabr MM,Zakaria MM,Refaie AF,et al.Insulin-producing cells from adult human bone marrow mesenchymal stem cells control streptozotocin-induced diabetes in nude mice.Cell Transplant.2013;22(1):133-145.
[10]Xie H,Wang Y,Zhang H,et al.Role of injured pancreatic extract promotes bone marrow-derived mesenchymal stem cells efficiently differentiate into insulin-producing cells.PLoSOne.2013;8(9):e76056.
[11]Kim JW,Vang S,Luo JZQ,et al.Human Islet Co-Cultured with Bone Marrow Mesenchymal Stem Cells in 3D Scaffolding May Augment Pancreatic Beta Cell Function.Journal of biomaterials and tissue engineering.2017;7(3):203-209.
[12]Unsal IO,Ginis Z,Pinarli FA,et al.Comparison of therapeutic characteristics of islet cell transplantation simultaneous with pancreatic mesenchymal stem cell transplantation in rats with Type 1 diabetes mellitus.Stem Cell Rev.2015;11(3):526-532.
[13]Wen D,Peng Y,Liu D,et al.Mesenchymal stem cell and derived exosome as small RNA carrier and Immunomodulator to improve islet transplantation.J Control Release.2016;238:166-175.
[14]Corradi-Perini C,Santos TM,Camara NOS,et al.Co-transplantation of Xenogeneic Bone Marrow-derived Mesenchymal Stem Cells Alleviates Rejection of Pancreatic Islets in Non-obese Diabetic Mice.Transplant Proc.2017;49(4):902-905.
[15]徐昕博.糖尿病治疗的研究现状及进展[J].养生保健指南,2019(7):213-214.
[16]Moravej H,Salehi A,Shamsedini A,et al.Complementary and Alternative Medicine to Treat Diabetes Mellitus Type 1 in Southern Iran.Iran J Med Sci.2016;41(1):71-72.
[17]Heller T,Kloos C,Lehmann T,et al.Mortality and its Causes in a German Cohort with Diabetes Mellitus Type 1 after 20Years of Follow-Up:The JEVIN Trial.Exp Clin Endocrinol Diabetes.2018;126(6):387-393.
[18]Kramer G,Kuniss N,Kloos C,et al.Principles and frequency of self-adjustment of insulin dose in people with diabetes mellitus type 1 and correlation with markers of metabolic control.Diabetes Res Clin Pract.2016;116:299-305.
[19]Bro?ováJ,?echurováD,LacigováS.Metabolic syndrome in patients with diabetes mellitus type 1,prevalence,impact on morbidity and mortality.Vnitr Lek.2016;62(11 Suppl 4):S8-14.
[20]Pathak S,Regmi S,Nguyen TT,et al.Polymeric microsphere-facilitated site-specific delivery of quercetin prevents senescence of pancreatic islets in vivo and improves transplantation outcomes in mouse model of diabetes.Acta Biomater.2018;75:287-299.
[21]Tchokonte-Nana V,Manda JK.Early islets and mesenchyme from an injured adult pancreas improve syngeneic engraftments and islet graft function in diabetic rats.Acta Histochem.2018;120(4):356-362.
[22]He Y,Zhang D,Zeng Y,et al.Bone Marrow-Derived Mesenchymal Stem Cells Protect Islet Grafts Against Endoplasmic Reticulum Stress-Induced Apoptosis During the Early Stage After Transplantation.Stem Cells.2018;36(7):1045-1061.
[23]Carlsson PO,Espes D,Sedigh A,et al.Transplantation of macroencapsulated human islets within the bioartificial pancreasβAir to patients with type 1 diabetes mellitus.Am JTransplant.2018;18(7):1735-1744.
[24]Cao XK,Li R,Sun W,et al.Co-combination of islets with bone marrow mesenchymal stem cells promotes angiogenesis.Biomed Pharmacother.2016;78:156-164.
[25]Wang H,Strange C,Nietert PJ,et al.Autologous Mesenchymal Stem Cell and Islet Cotransplantation:Safety and Efficacy.Stem Cells Transl Med.2018;7(1):11-19.
[26]He Y,Zhang D,Zeng Y,et al.Bone Marrow-Derived Mesenchymal Stem Cells Protect Islet Grafts Against Endoplasmic Reticulum Stress-Induced Apoptosis During the Early Stage After Transplantation.Stem Cells.2018;36(7):1045-1061.
[27]Li HT,Jiang FX,Shi P,et al.In vitro reprogramming of rat bmMSCs into pancreatic endocrine-like cells.In Vitro Cell Dev Biol Anim.2017;53(2):157-166.
[28]Cantarelli E,Citro A,Pellegrini S,et al.Transplant Site Influences the Immune Response After Islet Transplantation:Bone Marrow Versus Liver.Transplantation.2017;101(5):1046-1055.
[29]李宏图,林学文,李彩虹,等.负向调节因子重编程诱导大鼠骨髓间充质干细胞向胰岛内分泌细胞分化的研究[J].中国医科大学学报,2016,45(1):1-6.
[30]Westenfelder C,Gooch A,Hu Z,et al.Durable Control of Autoimmune Diabetes in Mice Achieved by Intraperitoneal Transplantation of"Neo-Islets,"Three-Dimensional Aggregates of Allogeneic Islet and"Mesenchymal Stem Cells"Stem Cells Transl Med.2017;6(7):1631-1643.