体外定向诱导小鼠胰腺干细胞分化形成胰岛样结构
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摘要
目的体外探讨胰腺干细胞形成胰岛样结构并对其进行相关检测,探寻胰腺干细胞分化为胰岛样结构可行性及初步鉴定的技术方法。方法从新生小鼠胰腺组织中通过原位传代扩增培养获得富足数量PSC,以尼克酰胺定向诱导PSC,观察细胞的形态变化、形成的细胞团双硫腙染色、免疫荧光组织化学染色及Mallory染色鉴定。结果PSC集落分布,细胞为单个大核、胞质折光透亮,巢蛋白(nestin)及碱性磷酸酶(AKP)染色阳性;诱导后聚集成团,最终形成具被膜包裹的细胞团;双硫腙染色阳性,胰岛素免疫荧光染色可见胞质被激发出绿色荧光的胰岛素阳性细胞;细胞团中央β样细胞及周围α样细胞,形成类似正常胰岛细胞组成的胰岛样结构。结论胰腺干细胞在适宜条件下可定向分化,形成胰岛样结构。
Objective To explore the method of induction of pancreatic stem cells into islet-like structures.Methods To collect and observe the morphological changes of pancreas stem cells after nicotinamide induction. The cell clusters were preformed with morphological observation,dithizone dyeing,Mallory dyeing and immunohistochemistry staining. Results The pancreas stem cells showed in colony distribution,with single big nuclear,refraction shining brilliantly,morphology was stable,nestin and AKP were both positive. After induction,the pancreatic stem cells gathered together and formed cell clusters,packed with intact membrane.The cells in the structures were positive for DTZ staining. With FITC tagging the insulin antibodies,the cells towards the center of the structures were insulin-producing cells of green fluorescence. While after Mallory staining,the structures were dressed into two different colours,in the centre of the cell cluster was faint yellow,periphery of cell cluster was red,the staining of the cell cluster was similar to the natural islets afterMallory staining. Conclusions Pancreatic stem cells could be induced into islet-like structures in vitro under appropriate conditions.
Objective To explore the method of induction of pancreatic stem cells into islet-like structures.Methods To collect and observe the morphological changes of pancreas stem cells after nicotinamide induction. The cell clusters were preformed with morphological observation,dithizone dyeing,Mallory dyeing and immunohistochemistry staining. Results The pancreas stem cells showed in colony distribution,with single big nuclear,refraction shining brilliantly,morphology was stable,nestin and AKP were both positive. After induction,the pancreatic stem cells gathered together and formed cell clusters,packed with intact membrane.The cells in the structures were positive for DTZ staining. With FITC tagging the insulin antibodies,the cells towards the center of the structures were insulin-producing cells of green fluorescence. While after Mallory staining,the structures were dressed into two different colours,in the centre of the cell cluster was faint yellow,periphery of cell cluster was red,the staining of the cell cluster was similar to the natural islets afterMallory staining. Conclusions Pancreatic stem cells could be induced into islet-like structures in vitro under appropriate conditions.
引文
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[2]Kunisada Y,Tsubooka-Yamazoe N,Shoji M,et al.Small molecules induce efficient differentiation into insulin-producing cells from human induced pluripotent stem cells[J].Stem Cell Res,2012,8:274-284.
[3]Lumelsky N,Blondel O,Laeng P,et al.Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets[J].Science,2001,292:1389-1394.
[4]Bonner-Weir S,Taneja M,Weir GC,et al.In vitro cultivation of human islets from expanded ductal tissue[J].Proc Natl Acad Sci U S A,2000,97:7999-8004.
[5]Schiesser JV,Wells JM.Generation of beta cells from human pluripotent stem cells:are we there yet?[J].Ann N Y Acad Sci,2014,1311:124-137.
[6]Kadam S,Muthyala S,Nair P,et al.Human placenta-derived mesenchymal stem cells and islet-like cell clusters generated from these cells as a novel source for stem cell therapy in diabetes[J].Rev Diabet Stud,2010,7:168-182.
[7]Lee SJ,Choi SE,Jung IR,et al.Protective effect of nicotinamide on high glucose/palmitate-induced glucolipotoxicity to INS-1 beta cells is attributed to its inhibitory activity to sirtuins[J].Arch Biochem Biophys,2013,535:187-196.
[8]Yang SJ,Choi JM,Kim L,et al.Nicotinamide improves glucose metabolism and affects the hepatic NAD-sirtuin pathway in a rodent model of obesity and type 2 diabetes[J].Nutr Biochem,2014,25:66-72.
[9]Cao LZ,Tang DQ,Horb ME,et al.High glucose is necessary for complete maturation of Pdx1-VP16-expressing hepatic cells into functional insulin-producing cells[J].Diabetes,2004,53:3168-3178.
[10]柳洁,张雪梅,李莉.Mallory氏胰岛细胞染色法的改良[J].现代生物医学进展,2007,7:1888-1889.
[11]Fukami A,Seino Y,Ozaki N,et al.Ectopic expression of GIP in pancreatic beta-cells maintains enhanced insulin secretion in mice with complete absence of proglucagon-derived peptides[J].Diabetes,2013,62:510-518.
[12]Marchetti P,Lupi R,Bugliani M,et al.A local glucagonlike peptide 1(GLP-1)system in human pancreatic islets[J].Diabetologia,2012,55:3262-3272.
[13]Wang H Zhang W,Cai H,et al.alpha-cell loss from islet impairs its insulin secretion in vitro and in vivo[J].Islets,2011,3:58-65.