胰腺导管单克隆上皮样干细胞的多分化潜能
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摘要
体外诱导源于成年大鼠的胰腺导管单克隆上皮样干细胞分化形成胰岛、神经、脂肪及成骨细胞,探讨干细胞的多分化潜能。扩增培养源于成年大鼠的胰腺导管单克隆上皮样干细胞,采用不同的诱导液体外诱导其向胰岛、神经、脂肪及成骨细胞分化,并通过DTZ染色、糖刺激试验、免疫荧光反应、油红O染色、茜素红染色或Vonkossa染色的方法对分化细胞进行检测。结果显示,体外诱导培养干细胞分化形成类胰岛,DTZ染色阳性,糖刺激分泌胰岛素、C-肽;分化形成类神经细胞,表达神经元特异性烯醇化酶;分化形成类脂肪细胞,油红O染色阳性;分化形成类成骨细胞,其分泌物呈岛状矿化结节,茜素红和Vonkossa染色阳性。这表明,该源于成年大鼠的胰腺导管上皮样干细胞系具有多分化潜能。
Monoclonal epithelial stem cells derived from pancreatic duct of an adult rat were in vitro induced to differentiate into pancreatic islets, nerve cells, adipose cells and osteoblasts. And pluripotency of these stem cells was studied. Monoclonal epithelia stem cells derived from pancreatic duct of an adult rat were proliferated. With different inducement medium, epithelial stem cells were respectively induced to differentiate into pancreatic islets, nerve cells, adipose cells and osteoblasts. Characteristics of differentiated cells were respectively detected with DTZ staining, glucose stimulation experiment, immune-fluorescence reaction, oil red staining, alizarin red staining or Vonkossa staining. The results showed that in inducement culture conditions, monoclonal epithelial stem cells derived from pancreatic duct of an adult rat differentiated into functional pancreatic islet-like tissue that was positive for DTZ staining, secreted insulin and C-peptide using glucose stimulation. Monoclonal epithelial stem cells were induced to differentiate into nerve-like cells which expressed neuron specific enolase, and were induced to differentiated into adipose-like cells that displayed accumulation of lipid vacuoles as detected by oil red O. Also stem cells were induced to differentiate into osteoblast-like cells that excretion formed mineralized nodules. Alizarin red or Vonkossa staining was positive. This indicated that the monoclonal epithelial stem cells derived from pancreatic duct of an adult rat were pluripotent stem cells that could differentiate into cells from three germ layers.
Monoclonal epithelial stem cells derived from pancreatic duct of an adult rat were in vitro induced to differentiate into pancreatic islets, nerve cells, adipose cells and osteoblasts. And pluripotency of these stem cells was studied. Monoclonal epithelia stem cells derived from pancreatic duct of an adult rat were proliferated. With different inducement medium, epithelial stem cells were respectively induced to differentiate into pancreatic islets, nerve cells, adipose cells and osteoblasts. Characteristics of differentiated cells were respectively detected with DTZ staining, glucose stimulation experiment, immune-fluorescence reaction, oil red staining, alizarin red staining or Vonkossa staining. The results showed that in inducement culture conditions, monoclonal epithelial stem cells derived from pancreatic duct of an adult rat differentiated into functional pancreatic islet-like tissue that was positive for DTZ staining, secreted insulin and C-peptide using glucose stimulation. Monoclonal epithelial stem cells were induced to differentiate into nerve-like cells which expressed neuron specific enolase, and were induced to differentiated into adipose-like cells that displayed accumulation of lipid vacuoles as detected by oil red O. Also stem cells were induced to differentiate into osteoblast-like cells that excretion formed mineralized nodules. Alizarin red or Vonkossa staining was positive. This indicated that the monoclonal epithelial stem cells derived from pancreatic duct of an adult rat were pluripotent stem cells that could differentiate into cells from three germ layers.
引文
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2 Xiao M,An LL,Yang XY,Ge X,Qiao H,Zhao T,et al.Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats.Sci China C Life Sci 2008;51(9):779-88.
3 Lee J,Wen J,Park JY,Kim SA,Lee EJ,Song SY.Reversal of diabetes in rats using GLP-1-expressing adult pancreatic ductlike precursor cells transformed from acinar to ductal cells.Stem Cells Dev 2009;18(7):991-1002.
4 Chen XC,Liu H,Li H,Cheng Y,Yang L,Liu YF.In vitro expansion and differentiation of rat pancreatic duct derived stem cells into insulin secreting cells using a dynamic three dimensional cell culture system.Genet Mol Res 2016;15(2):gmr.15028808.
5 Tremblay JR,LeBon JM,Luo A.In vitro colony assays for characterizing Tri-potent progenitor cells isolated from the adult murine pancreas.J Vis Exp 2016;10(112):doi:10.3791/4016.
6 Han W,He X,Zhang M,Hu S,Sun F,Ren L,et al.Establishment of a porcine pancreatic stem cell line using T-REx.systeminducible Wnt3a expression.Cell Prolif 2015;48(3):301-10.
7 Shiroi A,Yoshikawa M,Yokota H,Fukui H,Ishizaka S,Tatsumi K,et al.Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.Stem Cells2002;20(4):284-92.
8 Peck AB,Cornelius JG,Schatz D,Ramiya VK.Generation of islets of langerhans from adult pancreatic stem cells.JHepatobiliary Pancreat Surg 2002;9(6):704-9.
9 Ramiya VK,Maraist M,Arfors KE,Schatz DA,Peck AB,Cornelius JG.Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.Nat Med 2000;6(3):278-82.
10 Cornelius JG,Tchernev V,Kao KJ.In vitro-generation of islets in long-term cultures of multipotent stem cells from adult mouse pancreas.Horm Metab Res 1997;29(6):271-7.
11 Jin L,Feng T,Shih HP,Zerda R,Luo A,Hsu J,et al.Colonyforming cells in the adult mouse pancreas are expandable in matrigel and form endocrine/acinar colonies in laminin hydrogel.Proc Natl Acad Sci USA 2013;110(10):3907-12.
12 Rovira M,Scott SG,Liss AS,Jensen J,Thayer SP,Leach SD,et al.Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas.Proc Natl Acad Sci USA 2010;107(1):75-80.
13 Huch M,Bonfanti P,Boj SF,Sato T,Loomans CJ,van de Wetering M,et al.Unlimited in vitro expansion of adult bipotent pancreas progenitors through the Lgr5/R spondin axis.EMBO J2013;32(20):2708-21.
14 Noguchi H,Oishi K,Ueda M,Yukawa H,Hayashi S,Kobayashi N,et al.Establishment of mouse pancreatic stem cell line.Cell Transplant 2009;18(5):563-71.
15 Bonner-Weir S,Taneja M,Weir GC,Tatarkiewicz K,Song KH,Sharma A,et al.In vitro cultivation of human islets from expanded ductal tissue.Proc Natl Acad Sci USA 2000;97(14):7999-8004.
16 Hoesli CA,Johnson JD,Piret JM.Purified human pancreatic duct cell culture conditions defined by serum-free high-content growth factor screening.PLoS One 2012;7(3):e33999.
17 Lee J,Sugiyama T,Liu Y,Wang J,Gu X,Lei J,et al.Expansion and conversion of human pancreatic ductal cells into insulinsecreting endocrine cells.Elife 2013;2:e00940.
18效梅,安立龙,葛欣,窦忠英.单克隆人胰腺干细胞分离培养体系的建立.分子细胞生物学报(Xiao Mei,An Lilong,Ge Xin,Dou Zhongying.Establishment of the isolation and culture system for monoclone human pancreatic stem cell.Journal of Molecular Cell Biology)2008;41(6):450-7.
19效梅,安立龙,窦忠英.单克隆人胰腺干细胞的形态和表达特征.解剖学报(Xiao Mei,An Lilong,Dou Zhongying.Morphology and protein expression features of the mono-clone human pancreatic stem cell.Acta Anatomica Sinica)2009;40(2):55-8.
20效梅,安立龙,窦忠英.单克隆人胰腺干细胞分化特性的研究.分子细胞生物学报(Xiao Mei,An Lilong,Dou Zhongying.Research on differentiation features of the monoclonal human pancreatic stem cell.Journal of Molecular Cell Biology)2008;41(6):457-64.
2 Xiao M,An LL,Yang XY,Ge X,Qiao H,Zhao T,et al.Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats.Sci China C Life Sci 2008;51(9):779-88.
3 Lee J,Wen J,Park JY,Kim SA,Lee EJ,Song SY.Reversal of diabetes in rats using GLP-1-expressing adult pancreatic ductlike precursor cells transformed from acinar to ductal cells.Stem Cells Dev 2009;18(7):991-1002.
4 Chen XC,Liu H,Li H,Cheng Y,Yang L,Liu YF.In vitro expansion and differentiation of rat pancreatic duct derived stem cells into insulin secreting cells using a dynamic three dimensional cell culture system.Genet Mol Res 2016;15(2):gmr.15028808.
5 Tremblay JR,LeBon JM,Luo A.In vitro colony assays for characterizing Tri-potent progenitor cells isolated from the adult murine pancreas.J Vis Exp 2016;10(112):doi:10.3791/4016.
6 Han W,He X,Zhang M,Hu S,Sun F,Ren L,et al.Establishment of a porcine pancreatic stem cell line using T-REx.systeminducible Wnt3a expression.Cell Prolif 2015;48(3):301-10.
7 Shiroi A,Yoshikawa M,Yokota H,Fukui H,Ishizaka S,Tatsumi K,et al.Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone.Stem Cells2002;20(4):284-92.
8 Peck AB,Cornelius JG,Schatz D,Ramiya VK.Generation of islets of langerhans from adult pancreatic stem cells.JHepatobiliary Pancreat Surg 2002;9(6):704-9.
9 Ramiya VK,Maraist M,Arfors KE,Schatz DA,Peck AB,Cornelius JG.Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells.Nat Med 2000;6(3):278-82.
10 Cornelius JG,Tchernev V,Kao KJ.In vitro-generation of islets in long-term cultures of multipotent stem cells from adult mouse pancreas.Horm Metab Res 1997;29(6):271-7.
11 Jin L,Feng T,Shih HP,Zerda R,Luo A,Hsu J,et al.Colonyforming cells in the adult mouse pancreas are expandable in matrigel and form endocrine/acinar colonies in laminin hydrogel.Proc Natl Acad Sci USA 2013;110(10):3907-12.
12 Rovira M,Scott SG,Liss AS,Jensen J,Thayer SP,Leach SD,et al.Isolation and characterization of centroacinar/terminal ductal progenitor cells in adult mouse pancreas.Proc Natl Acad Sci USA 2010;107(1):75-80.
13 Huch M,Bonfanti P,Boj SF,Sato T,Loomans CJ,van de Wetering M,et al.Unlimited in vitro expansion of adult bipotent pancreas progenitors through the Lgr5/R spondin axis.EMBO J2013;32(20):2708-21.
14 Noguchi H,Oishi K,Ueda M,Yukawa H,Hayashi S,Kobayashi N,et al.Establishment of mouse pancreatic stem cell line.Cell Transplant 2009;18(5):563-71.
15 Bonner-Weir S,Taneja M,Weir GC,Tatarkiewicz K,Song KH,Sharma A,et al.In vitro cultivation of human islets from expanded ductal tissue.Proc Natl Acad Sci USA 2000;97(14):7999-8004.
16 Hoesli CA,Johnson JD,Piret JM.Purified human pancreatic duct cell culture conditions defined by serum-free high-content growth factor screening.PLoS One 2012;7(3):e33999.
17 Lee J,Sugiyama T,Liu Y,Wang J,Gu X,Lei J,et al.Expansion and conversion of human pancreatic ductal cells into insulinsecreting endocrine cells.Elife 2013;2:e00940.
18效梅,安立龙,葛欣,窦忠英.单克隆人胰腺干细胞分离培养体系的建立.分子细胞生物学报(Xiao Mei,An Lilong,Ge Xin,Dou Zhongying.Establishment of the isolation and culture system for monoclone human pancreatic stem cell.Journal of Molecular Cell Biology)2008;41(6):450-7.
19效梅,安立龙,窦忠英.单克隆人胰腺干细胞的形态和表达特征.解剖学报(Xiao Mei,An Lilong,Dou Zhongying.Morphology and protein expression features of the mono-clone human pancreatic stem cell.Acta Anatomica Sinica)2009;40(2):55-8.
20效梅,安立龙,窦忠英.单克隆人胰腺干细胞分化特性的研究.分子细胞生物学报(Xiao Mei,An Lilong,Dou Zhongying.Research on differentiation features of the monoclonal human pancreatic stem cell.Journal of Molecular Cell Biology)2008;41(6):457-64.