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
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摘要
背景:目前,体外已成功将骨髓间充质干细胞定向诱导分化为能够分泌胰岛素的胰岛样细胞团,但是对细胞的形态变化规律、胰岛样细胞团中内分泌细胞的类型及其相互关系的研究报道较少。目的:观察骨髓间充质干细胞定向分化为胰岛样结构过程中细胞的形态变化、内分泌细胞的组成及分布规律。方法:取生长良好的第3代骨髓间充质干细胞,扩增培养至细胞集落汇合占据瓶底面积80%,加入胰腺组织裂解液连续诱导培养。以双硫腙染色方法对骨髓间充质干细胞定向分化形成的细胞团进行初步筛选,Mallory染色鉴别胰岛内分泌细胞的类型及其分布;采用免疫荧光细胞化学染色法检测胰岛内分泌细胞中胰岛素、C肽、胰高血糖素和生长抑素蛋白表达。结果与结论:①双硫腙染色显示,随着诱导时间延长,阳性细胞数量逐渐增多;②Mallory染色显示,胰岛样细胞团内α样细胞位于周边,β样细胞分布在中央及周边,成纤维样细胞分布在周围;③免疫荧光染色显示,胰岛样细胞团内有胰岛素、C肽、胰高血糖素和生长抑素阳性细胞;④结果表明,在特定的微环境条件下,骨髓间充质干细胞能分化为胰岛样结构,内含α、β、δ样细胞,周围有类成纤维细胞包绕。
BACKGROUND: Bone marrow mesenchymal stem cells have been induced into islet-like cell mass in vitro. However, little is reported on the morphological changes of cells, the types of endocrine cells in the islet-like cell mass and their relationships.OBJECTIVE: To investigate the morphological changes of cells in the differentiation of bone marrow mesenchymal stem cells into islet-like structure and to explore the composition and distribution of endocrine cells.METHODS: Passage 3 bone marrow mesenchymal stem cells growing well were cultured and expanded until the cell colonies occupies 80% of the bottom of the culture bottle, and the pancreatic tissue lysate was added for continuous induction. Dithizone staining was used to screen the islet-like cell mass directly differentiated from bone marrow mesenchymal stem cells. The types and distribution of endocrine cells were identified by Mallory staining. Expressions of insulin, C-peptide, glucagon and somatostatin protein were detected by immunofluorescence cytochemical staining.RESULTS AND CONCLUSION:(1) Dithizone staining showed that the number of positive cells was increased over the induction time.(2)Mallory staining showed the red α-like cells were located in the periphery of the islet-like cell mass, the yellow β-like cells located in the center and periphery, and the light blue fibroblasts were distributed around the cell mass.(3) Immunofluorescence staining showed insulin, C-peptide,glucagon and somatostatin positive cells in the islet-like cell mass. To conclude, under certain microenvironment, bone marrow mesenchymal stem cells can differentiate into islet-like structures which containing α, β, δ-like cells, and are surrounded by fibroblast-like cells.
BACKGROUND: Bone marrow mesenchymal stem cells have been induced into islet-like cell mass in vitro. However, little is reported on the morphological changes of cells, the types of endocrine cells in the islet-like cell mass and their relationships.OBJECTIVE: To investigate the morphological changes of cells in the differentiation of bone marrow mesenchymal stem cells into islet-like structure and to explore the composition and distribution of endocrine cells.METHODS: Passage 3 bone marrow mesenchymal stem cells growing well were cultured and expanded until the cell colonies occupies 80% of the bottom of the culture bottle, and the pancreatic tissue lysate was added for continuous induction. Dithizone staining was used to screen the islet-like cell mass directly differentiated from bone marrow mesenchymal stem cells. The types and distribution of endocrine cells were identified by Mallory staining. Expressions of insulin, C-peptide, glucagon and somatostatin protein were detected by immunofluorescence cytochemical staining.RESULTS AND CONCLUSION:(1) Dithizone staining showed that the number of positive cells was increased over the induction time.(2)Mallory staining showed the red α-like cells were located in the periphery of the islet-like cell mass, the yellow β-like cells located in the center and periphery, and the light blue fibroblasts were distributed around the cell mass.(3) Immunofluorescence staining showed insulin, C-peptide,glucagon and somatostatin positive cells in the islet-like cell mass. To conclude, under certain microenvironment, bone marrow mesenchymal stem cells can differentiate into islet-like structures which containing α, β, δ-like cells, and are surrounded by fibroblast-like cells.
引文
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[2]See EY, Toh SL, Goh JC. Multilineage potential of bone-marrow-derived mesenchymal stem cell cell sheets:implications for tissue engineering. Tissue Eng Part A. 2010;16(4):1421-1431.
[3]黄盛,郑伟,范志勇,等.不同微环境对骨髓间充质干细胞分化为产胰岛素细胞的影响[J].中华实验外科杂志, 2008, 25(3):368-370.
[4]Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells. 1978;4(1-2):7-25.
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[6]Ball SG, Shuttleworth AC, Kielty CM. Direct cell contact influences bone marrow mesenchymal stem cell fate. Int J Biochem Cell Biol. 2004;36(4):714-727.
[7]赵文婧,李琴,吴卓,等.心肌组织裂解液诱导骨髓间充质干细胞向心肌样结构的分化[J].中国组织工程研究与临床康复,2012,16(10):1729-1732.
[8]王慧丰,吴丽情,徐亦辰,等.不同诱导剂定向诱导BMSCs分化为胰岛样结构的比较[J].基因组学与应用生物学,2018, 37(4):1691-1696.
[9]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.
[10]Chandra V, Swetha G, Muthyala S, et al. Islet-like cell aggregates generated from human adipose tissue derived stem cells ameliorate experimental diabetes in mice. PLoS One. 2011;6(6):e20615.
[11]Wang H, Zhang W, Cai H, et al.α-Cell loss from islet impairs its insulin secretion in vitro and in vivo. Islets. 2011;3(2):58-65.
[12]Elliott AD, Ustione A, Piston DW. Somatostatin and insulin mediate glucose-inhibited glucagon secretion in the pancreaticα-cell by lowering cAMP. Am J Physiol Endocrinol Metab. 2015;308(2):E130-143.
[13]D’Ippolito G, Diabira S, Howard GA, et al. Marrow-isolated adult multilineage inducible(MIAMI)cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci. 2004;117(Pt 14):2971-2981.
[14]Prabhakaran MP, Venugopal JR, Ramakrishna S.Mesenchymal stem cell differentiation to neuronal cells on electrospun nanofibrous substrates for nerve tissue engineering. Biomaterials. 2009;30(28):4996-5003.
[15]刘晓芳,王韫芳,李亚里,等.干细胞治疗糖尿病的研究现状及展望[J].中国科学,2013,43(4):291-297.
[16]Rangappa S, Entwistle JW, Wechsler AS, et al.Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. J Thorac Cardiovasc Surg. 2003;126(1):124-132.
[17]艾国平,粟永萍,程天民.骨髓间充质干细胞的应用研究进展[J].中华创伤杂志,2002,18(9):572-574.
[18]Czubak P, Bojarska-Junak A, Tabarkiewicz J, et al. A modified method of insulin producing cells'generation from bone marrow-derived mesenchymal stem cells. J Diabetes Res.2014;2014:628591.
[19]于文浩,王亚丹,虎啸,等.DMSO联合高糖体外诱导兔骨髓间充质干细胞分化为胰岛样细胞的研究[J].中国细胞生物学学报, 2016,38(11):1325-1334.
[20]史光军,白国立,谭雪莹,等.PDX-1基因转染人脂肪间充质干细胞向胰岛样细胞分化及移植治疗1型糖尿病[J].中国组织工程研究, 2017,21(13):2062-2067.
[21]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. PLoS One. 2013;8(9):e76056.
[22]李琴,赵文婧,寇亚丽,等.心肌组织裂解液诱导骨髓间充质干细胞向心肌样细胞的分化[J].中国组织工程研究与临床康复, 2011,15(10):1726-1730.
[23]吴丽情,寇亚丽,赵文婧,等.体外定向诱导小鼠胰腺干细胞分化形成胰岛样结构[J].基础医学与临床, 2015, 35(12):1612-1616.
[24]Aamodt KI, Powers AC. Signals in the pancreatic islet microenvironment influenceβ-cell proliferation. Diabetes Obes Metab. 2017;19 Suppl 1:124-136.
[25]刘厚奇,蔡文琴.医学发育生物学[M]. 3版.北京:科学出版社,2012.
[26]Aboalola D, Han VKM. Different Effects of Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor-2 on Myogenic Differentiation of Human Mesenchymal Stem Cells. Stem Cells Int. 2017;2017:8286248.
[27]Xin Y, Jiang X, Wang Y, et al. Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia. PLoS One. 2016;11(1):e0145838.
[2]See EY, Toh SL, Goh JC. Multilineage potential of bone-marrow-derived mesenchymal stem cell cell sheets:implications for tissue engineering. Tissue Eng Part A. 2010;16(4):1421-1431.
[3]黄盛,郑伟,范志勇,等.不同微环境对骨髓间充质干细胞分化为产胰岛素细胞的影响[J].中华实验外科杂志, 2008, 25(3):368-370.
[4]Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells. 1978;4(1-2):7-25.
[5]Wilczek P,Zembala M,Cichon I,et al. The effect of the micromechanical stimulations on the human cardiac stem cells differentiations and morphology. Ann Transplant. 2009;14(1):37.
[6]Ball SG, Shuttleworth AC, Kielty CM. Direct cell contact influences bone marrow mesenchymal stem cell fate. Int J Biochem Cell Biol. 2004;36(4):714-727.
[7]赵文婧,李琴,吴卓,等.心肌组织裂解液诱导骨髓间充质干细胞向心肌样结构的分化[J].中国组织工程研究与临床康复,2012,16(10):1729-1732.
[8]王慧丰,吴丽情,徐亦辰,等.不同诱导剂定向诱导BMSCs分化为胰岛样结构的比较[J].基因组学与应用生物学,2018, 37(4):1691-1696.
[9]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.
[10]Chandra V, Swetha G, Muthyala S, et al. Islet-like cell aggregates generated from human adipose tissue derived stem cells ameliorate experimental diabetes in mice. PLoS One. 2011;6(6):e20615.
[11]Wang H, Zhang W, Cai H, et al.α-Cell loss from islet impairs its insulin secretion in vitro and in vivo. Islets. 2011;3(2):58-65.
[12]Elliott AD, Ustione A, Piston DW. Somatostatin and insulin mediate glucose-inhibited glucagon secretion in the pancreaticα-cell by lowering cAMP. Am J Physiol Endocrinol Metab. 2015;308(2):E130-143.
[13]D’Ippolito G, Diabira S, Howard GA, et al. Marrow-isolated adult multilineage inducible(MIAMI)cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential. J Cell Sci. 2004;117(Pt 14):2971-2981.
[14]Prabhakaran MP, Venugopal JR, Ramakrishna S.Mesenchymal stem cell differentiation to neuronal cells on electrospun nanofibrous substrates for nerve tissue engineering. Biomaterials. 2009;30(28):4996-5003.
[15]刘晓芳,王韫芳,李亚里,等.干细胞治疗糖尿病的研究现状及展望[J].中国科学,2013,43(4):291-297.
[16]Rangappa S, Entwistle JW, Wechsler AS, et al.Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype. J Thorac Cardiovasc Surg. 2003;126(1):124-132.
[17]艾国平,粟永萍,程天民.骨髓间充质干细胞的应用研究进展[J].中华创伤杂志,2002,18(9):572-574.
[18]Czubak P, Bojarska-Junak A, Tabarkiewicz J, et al. A modified method of insulin producing cells'generation from bone marrow-derived mesenchymal stem cells. J Diabetes Res.2014;2014:628591.
[19]于文浩,王亚丹,虎啸,等.DMSO联合高糖体外诱导兔骨髓间充质干细胞分化为胰岛样细胞的研究[J].中国细胞生物学学报, 2016,38(11):1325-1334.
[20]史光军,白国立,谭雪莹,等.PDX-1基因转染人脂肪间充质干细胞向胰岛样细胞分化及移植治疗1型糖尿病[J].中国组织工程研究, 2017,21(13):2062-2067.
[21]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. PLoS One. 2013;8(9):e76056.
[22]李琴,赵文婧,寇亚丽,等.心肌组织裂解液诱导骨髓间充质干细胞向心肌样细胞的分化[J].中国组织工程研究与临床康复, 2011,15(10):1726-1730.
[23]吴丽情,寇亚丽,赵文婧,等.体外定向诱导小鼠胰腺干细胞分化形成胰岛样结构[J].基础医学与临床, 2015, 35(12):1612-1616.
[24]Aamodt KI, Powers AC. Signals in the pancreatic islet microenvironment influenceβ-cell proliferation. Diabetes Obes Metab. 2017;19 Suppl 1:124-136.
[25]刘厚奇,蔡文琴.医学发育生物学[M]. 3版.北京:科学出版社,2012.
[26]Aboalola D, Han VKM. Different Effects of Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor-2 on Myogenic Differentiation of Human Mesenchymal Stem Cells. Stem Cells Int. 2017;2017:8286248.
[27]Xin Y, Jiang X, Wang Y, et al. Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia. PLoS One. 2016;11(1):e0145838.
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