胎儿胰腺组织中巢蛋白阳性细胞的分离和体外增殖以及诱导分化

目的 研究从胎儿胰腺组织中分离巢蛋白(Nestin)阳性细胞以及该细胞的体外扩增与向胰岛内分泌细胞分化的能力。方法 采用胶原酶消化法，从胎儿胰腺组织中分离获得胰岛样细胞簇(islet-like cell clusters，ICCs)，ICCs经手工挑拣后接种，待形成单层上皮样细胞后，进行传代培养和诱导分化。利用逆转录-聚合酶链反应(RT-PCR)、免疫荧光染色及放射免疫分析(RIA)等方法，检测该细胞中分子标志物的表达，并对其向胰岛内分泌细胞分化的能力进行鉴定。结果 (1)上述单层上皮样细胞具有很强的增殖能力，可至少连续传16代；(2)RT-PCR、免疫荧光染色分析显示，该细胞可表达干细胞的标志分子巢蛋白和ABCG2；(3)RT-PCR分析显示，在多种细胞因子和无血清的条件下，巢蛋白阳性细胞经诱导后可出现胰岛素、胰升糖素和胰十二指肠同源盒基因-1(PDX-1)mRNA的表达，而巢蛋白和Neurogenin3(Ngn3)mRNA表达消失。RIA分析也可检测到诱导后的细胞内有胰岛素产生。结论 从胎儿胰腺中分离得到的巢蛋白阳性细胞具有胰腺前体细胞的特性，在体外具有很强的增殖能力，并可诱导分化为胰岛内分泌细胞。该细胞有望为胰岛移植提供一种新的细胞来源。     

巢蛋白在人胚胎胰腺中的表达

目的 探讨人胚胎胰腺中干细胞特异性标志巢蛋白(Nestin)的表达及阳性细胞的分布。方法 用连续切片免疫组织化学染色方法检测16周胎龄人胚胎胰腺中巢蛋白、胰岛素、胰升糖素和导管上皮标志细胞角蛋白19(CK19)的表达及分布。结果 16周人胚胎胰腺中可检测到巢蛋白阳性细胞，分布于胰岛以及胰导管和腺泡结构中，并且以腺泡结构和小导管中的数目为多。部分胰岛素和CK19染色强阳性的细胞中巢蛋白染色为弱阳性，胰升糖素强阳性的部位则未见巢蛋白染色阳性的细胞。结论 人胚胎胰腺中存在巢蛋白表达的细胞；巢蛋白阳性细胞可能是胰腺组织中一个特殊的细胞亚群，代表了一群未成熟细胞。     

胰腺干细胞诱导分化为胰岛素分泌细胞的实验研究

目的研究大鼠胰腺组织干细胞向胰岛内分泌细胞分化及其在实验性糖尿病治疗中的应用。方法应用Nestin结合的免疫磁珠从大鼠胰腺导管细胞中分离和纯化干细胞,经体外扩增及诱导分化形成胰岛样结构,进行体外和体内的功能评价。结果大鼠胰腺干细胞经体外扩增和诱导分化后,表达胰岛素mRNA,并形成胰岛样结构。免疫荧光染色显示胰岛结构内含有大量胰岛素阳性细胞和少量胰高糖素阳性细胞。在葡萄糖刺激下干细胞分化出胰岛,有胰岛素释放,其释放量约为正常胰岛的39.4%。将干细胞分化来的胰岛移植到免疫缺陷的糖尿病小鼠后可以明显改善糖代谢的紊乱。结论胰腺组织干细胞可以在体外诱导分化成为具有治疗糖尿病功能的胰岛细胞。     

体外诱导大鼠骨髓间质干细胞分化为胰岛样细胞的研究

目的探索大鼠骨髓间质干细胞(MSCs)体外诱导分化为胰岛素分泌细胞的方法,为解决胰岛移植物来源匮乏问题提供新的思路。方法采用横向分化技术,将成年大鼠MSCs诱导成胰岛素分泌细胞;间接免疫荧光法鉴定诱导前后的细胞巢蛋白、胰岛素、胰高血糖素、生长抑素的表达;RTPCR法检测诱导前后的细胞胰岛素1、Pdx1、Pax6mRNA的表达;24h累积分泌量测定和胰岛素刺激实验评价诱导前后细胞的功能。结果诱导5h,巢蛋白阳性细胞为(44.6±7.3)%,诱导24h增至(61.8±8.4)%。此后,巢蛋白阳性细胞数开始下降,诱导第14天后,巢蛋白表达基本消失。而且,诱导后细胞可表达胰岛素、胰高血糖素、生长抑素等蛋白;表达胰岛素1及其多种转录因子基因mRNA;胰岛素刺激实验反应敏感,而诱导前MSCs不具备上述特点。结论体外大鼠MSCs可诱导成为胰岛素分泌细胞,为胰岛移植开辟新的研究思路。     

肝细胞生长因子及葡萄糖对人胰腺干细胞向胰岛素分泌细胞分化的影响

目的 探讨人胰腺干细胞体外扩增及其向胰岛素分泌细胞分化的影响因素.方法 应用剪碎消化法从孕4～6月龄引产的人胎儿胰腺中获得胰岛组织,选用差速贴壁法从胰岛中分离胰腺干细胞,经扩增后定向诱导成具有胰岛素分泌功能和胰岛样结构的胰岛样细胞团,观察肝细胞生长因子和葡萄糖对胰腺干细胞向胰岛素分泌细胞诱导分化以及胰岛样细胞团胰岛素分泌功能的影响.采用流式细胞仪和免疫组织化学法检测扩增前后nestin阳性细胞和ABCG2阳性细胞.行葡萄糖刺激的胰岛素释放试验,以酶联免疫吸附法（ELISA）检测每1×105个扩增细胞经定向诱导后形成的胰岛样细胞团释放的胰岛素量,比较肝细胞生长因子和葡萄糖对定向分化的影响.采用单因素方差分析或多样本均数两两比较进行统计学分析.结果 经过15代连续扩增,nestin阳性细胞和ABCG2阳性细胞分别扩增了27.9倍和37.8倍.扩增后的细胞经15 d定向诱导后形成具有胰岛素分泌功能和胰岛样结构的细胞团.葡萄糖刺激的胰岛素分泌试验显示,当诱导培养基中肝细胞生长因子浓度为0、5、10、15μg/L时,5.6 mmol/L葡萄糖刺激后,胰岛素分泌量分别为5.00、12.93、14.91和11.23μU;25.0 mmol/L葡萄糖刺激后,胰岛素分泌量分别为19.91、35.53、47.43和23.33 μU.诱导培养基中的葡萄糖浓度为0、2.8、5.6和11.2 mmol/L时,5.6 mmol/L葡萄糖刺激后,胰岛素分泌量分别为9.07、14.21、6.91和3.53 μU;25.0 mmol/L葡萄糖刺激后,胰岛素分泌量分别为26.64、48.97、37.63和34.20 μU.结论 人胰腺中nestin和ABCG2阳性胰腺干细胞具有很强的体外扩增能力.扩增后的胰腺干细胞仍然具有形成胰岛素分泌细胞的功能,经体外诱导后能形成胰岛样细胞团.在胰腺干细胞向胰岛素分泌细胞分化诱导过程中,适当浓度的肝细胞生长因子和葡萄糖有利于胰岛样细胞团的形成和胰岛素分泌.     

胎儿胰腺巢蛋白阳性细胞具有间充质表型特征

目的研究巢蛋白阳性细胞在胎儿胰腺中的分布以及其表面标志。方法采用免疫荧光染色法对巢蛋白阳性细胞在胎儿胰腺组织中的分布进行定位。分离和培养胎儿胰腺细胞,利用免疫荧光染色和流式细胞分析进行鉴定,并与骨髓间充质干细胞的表面标志比较。结果巢蛋白阳性细胞在胎儿胰腺间质中广泛分布;胎儿胰腺来源的巢蛋白阳性细胞的表面标志与骨髓间充质干细胞基本一致。结论从胎儿胰腺中分离的巢蛋白阳性细胞具有骨髓间充质干细胞表型特征。     

胰岛细胞增生症与胰腺内分泌部增生

胰岛细胞增生症一直被认为是一种引起婴儿高胰岛素血症的疾病。但在此病的定义和诊断标准方面存在争议。作者对几组胰内分泌部进行了研究,它们包括:①正常胰腺(从胎儿至成人,49例);②胰岛细胞增生症的胰腺(5例);③成人胰岛素瘤旁组织(8例)。用免疫组化及形态学测量方法重点观察了导管内分泌细胞增生、弥漫性内分泌细胞增生和间隔内胰岛等现象,测量了总内分泌区(TEA)和胰岛大小及胰岛内部内分泌细胞分布类型。     

体外高效诱导人胚胎干细胞定向分化为胰岛细胞的可行性研究

目的探讨体外诱导人胚胎干细胞(hESs)定向分化为胰岛细胞的可行性。方法体外分三阶段诱导hESCs定向分化为胰岛细胞。第一阶段:予以活化素-A、渥曼青霉素诱导分化形成定型内胚层;第二阶段:采用维甲酸(RA)、NOGGIN、碱性成纤维细胞生长因子(bFGF)诱导胰腺细胞定向分化;以EGF扩增胰腺祖细胞;第三阶段:采用尼克酰胺、唾液素4、bFGF及骨形成蛋白(BMP4)促进胰岛细胞成熟;观察诱导各阶段细胞形态变化,免疫荧光法鉴定胰十二指肠同源异型盒基因(PDX-1)、胰高糖素、胰岛素、C肽、葡萄糖转运子2(glut-2)表达。结果诱导14 d时hESs出现胰高糖素荧光表达;20 d时细胞出现PDX-1和C肽共表达;22 d形成的成熟胰岛细胞出现glut-2和胰岛素的阳性表达;胰岛素阳性细胞占17.1%,C肽阳性细胞占3.8%。结论体外可简单高效的诱导hESs定向分化为成熟胰岛细胞。     

小鼠胰腺干细胞的分离、培养与鉴定

目的从新生小鼠中分离培养并鉴定胰腺干细胞。方法利用组织块法分离新生小鼠胰腺源性干细胞,经RPMI1640+5%胎牛血清+双抗+1 mmol/L丙酮酸钠+1 mmol/L非必需氨基酸+10 ng/mL表皮生长因子+10 ng/mL成纤维生长因子+2 mmol/L谷氨酰胺的溶液培养,利用免疫组化分析胰腺源性干细胞的兔抗巢蛋白抗体(Nestin)、胰肠同源域因子(PDX-1)和葡葡糖转运子(GLUT-2)抗原表达;经10 mmol/L尼克酰胺诱导产生胰岛样细胞团并利用双硫腙(DTZ)染色分析其胰岛素分泌情况。结果分离培养的胰腺源性干细胞呈Nestin、PDX-1和GLUT-2阳性;诱导的胰岛样细胞团呈DTZ染色阳性。结论分离获得的新生小鼠胰腺源性干细胞在体外可分化为类胰岛,并具有胰岛素分泌功能。     

氧化应激对胰岛β细胞功能的影响

体外低糖培养胰岛β细胞,以葡萄糖/葡萄糖氧化酶(G/GO)为过氧化氢(H2O2)产生体系摸拟体内氧化应激水平,以流式细胞术检测细胞内H2O2,以RT-PCR。方法检测胰岛β细胞内胰腺十二指肠同源异型盒(PDX-1)的mRNA表达,检测胰岛素和C肽浓度。结果随着细胞内产生的H2O2逐渐升高,PDX-1的mRNA的表达逐步减少,胰岛素及C肽量也逐渐减少,而加入过氧化氢酶(CAT)清除H2O2后可部分逆转这一趋势。结论氧化应激可减少胰岛β细胞内的PDX-1 mRNA表达,从而减少胰岛β细胞胰岛素和C肽的分泌。     

Presence of endocrine and exocrine markers in EGFP-positive cells from the developing pancreas of a nestin/EGFP mouse

In order to purify and characterize nestin-positive cells in the developing pancreas a transgenic mouse was generated, in which the enhanced green fluorescent protein (EGFP) was driven by the nestin second intronic enhancer and upstream promoter. In keeping with previous studies on the distribution of nestin, EGFP was expressed in the developing embryo in neurones in the brain, eye, spinal cord, tail bud and glial cells in the small intestine. In the pancreas there was no detectable EGFP at embryonic day 11.5 (E11.5). EGFP expression appeared at E12.5 and increased in intensity through E14.5, E18.5 and post-natal day 1. Flow cytometry was used to quantify and purify the EGFP positive population in the E15.5 pancreas. The purified (96%) EGFP-expressing cells, which represent 20% of the total cell population, were shown by RT/PCR to express exocrine cell markers (amylase and P48) and endocrine cell markers (insulin 1, insulin 2, and Ngn3). They also expressed, at a lower level, PDX-1, Isl-1, and the islet hormones pancreatic polypeptide, glucagon and somatostatin as well as GLUT2, the stem cell marker ABCG2 and PECAM, a marker of endothelial cells. It was further shown by immunocytochemistry of the E15.5 pancreas that EGFP colocalised in separate subpopulations of cells that expressed nestin, insulin and amylase. These results support the conclusion that nestin expressing cells can give rise to both endocrine and exocrine cells. The ability to purify these putative progenitor cells may provide further insights into their properties and function.     

Immuolocalization of nestin in pancreatic tissue of mice at different ages

AIM： To localize nestin positive cells （NPC） in pancreatic tissue of mice of different ages. METHODS： Paraffin sections of 6-8 um of fixed pancreatic samples were mounted on poly-L-lysine coated slides and used for Immunolocalization using appropriate primary antibodies （Nestin, Insulin, Glucagon）, followed by addition of a fluorescently labeled secondary antibody. The antigen-antibody localization was captured using a confocal microscope （Leica SP 5 series）. RESULTS： In 3-6 d pups, the NPC were localized towards the periphery of the endocrine portion, as evident from immunolocalization of insulin and glucagon, while NPC were absent in the acinar portion. At 2 wk, NPC were localized in both the exocrine and endocrine portions. Interestingly, in 4-wk-old mice NPC were seen only in the endocrine portion, towards the periphery, and were colocalised with the glucagon positive cells. In the pancreas of 8- wk-old mice, the NPC were predominantly localized in the central region of the islet clusters, where immunostaining for insulin was at a maximum. CONCLUSION： We report for the first time the immunolocalization of NPC in the pancreas of mice of different ages （3 d to 8 wk） with reference to insulin and glucagon positive cells. The heterogeneous localization of the NPC observed may be of functional and developmental significance and suggest（s） that mice pancreatic tissue can be a potential source of progenitor cells. NPC from the pancreas can be isolated, proliferated and programmed to differentiate into insulin secreting cells under the appropriate microenvironment.     

Immunolocalization of nestin in pancreatic tissue of mice at different ages

AIM: To localize nestin positive cells (NPC) in pancreatic tissue of mice of different ages. METHODS: Paraffin sections of 6-8 μm of fixed pancreatic samples were mounted on poly-L-lysine coated slides and used for Immunolocalization using appropriate primary antibodies (Nestin, Insulin, Glucagon), followed by addition of a fluorescently labeled secondary antibody. The antigen-antibody localization was captured using a confocal microscope (Leica SP 5 series). RESULTS: In 3-6 d pups, the NPC were localized towards the periphery of the endocrine portion, as evident from immunolocalization of insulin and glucagon, while NPC were absent in the acinar portion. At 2 wk, NPC were localized in both the exocrine and endocrine portions. Interestingly, in 4-wk-old mice NPC were seen only in the endocrine portion, towards the periphery, and were colocalised with the glucagon positive cells. In the pancreas of 8- wk-old mice, the NPC were predominantly localized in the central region of the islet clusters, where immunostaining for insulin was at a maximum. CONCLUSION: We report for the first time the immunolocalization of NPC in the pancreas of mice of different ages (3 d to 8 wk) with reference to insulin and glucagon positive cells. The heterogeneous localization of the NPC observed may be of functional and developmental significance and suggest(s) that mice pancreatic tissue can be a potential source of progenitor cells. NPC from the pancreas can be isolated, proliferated and programmed to differentiate into insulin secreting cells under the appropriate microenvironment.     

Heterogenous expression of nestin in human pancreatic tissue precludes its use as an islet precursor marker

The discovery of a pancreatic adult stem cell would have significant implications for cell-based replacement therapies for type 1 diabetes mellitus. Nestin, a marker for neural precursor cells, has been suggested as a possible marker for islet progenitor cells. We have characterized the expression and localization of nestin in both the intact human pancreas and clinical human pancreatic islet grafts. Nestin was found to be expressed at different levels in the acinar component of human pancreatic biopsies depending on donor, as well as in ductal structures and islets to some degree. In islets, insulin-producing beta-cells rarely co-expressed the protein, and in the ducts a small percentage (1-2%) of cells co-expressed nestin and cytokeratin 19 (CK19) while most expressed only CK19 (90%) or nestin (5-10%) alone. Assessment of nestin expression in neonatal pancreatic sections revealed an increased number of islet-associated positive cells as compared with adult islets. Nestin immunoreactivity was also found in cells of the pancreatic vasculature and mesenchyme as evidenced by co-localization with smooth muscle actin and vimentin. Samples from post-islet isolation clinical islet grafts revealed a pronounced heterogeneity in the proportion of nestin-positive cells (<1-72%). Co-localization studies in these grafts showed that nestin is not co-expressed in endocrine cells and rarely (<5%) with cytokeratin-positive ductal cells. However, relatively high levels of co-expression were found with acinar cells and cells expressing the mesenchymal marker vimentin. In conclusion we have shown a diffuse and variable expression of nestin in human pancreas that may be due to a number of different processes, including post-mortem tissue remodeling and cellular differentiation. For this reason nestin may not be a suitable marker solely for the identification of endocrine precursor cells in the pancreas.