巢蛋白和神经元素3在人胚胎胰腺中的表达和分布

目的探讨胰腺干细胞标志巢蛋白（nestin）和神经元素3（Ngn3）在人胚胎胰腺组织中的表达及其与内分泌细胞、导管细胞和外分泌细胞标志的共表达情况。方法以因病理因素行药物引产的12周-14周前人胚胎胰腺14例为材料,应用免疫荧光染色和逆转录聚合酶链反应（RT—PCR）方法检测nestin和Ngn3的表达情况,免疫荧光染色检测胰腺内分泌标志胰岛素、胰高血糖素和外分泌标志CK-PAN和CK-19。结果nestin和Ngn3在12～14周人胚胎胰腺组织均广泛表达。nestin和Ng．3阳性细胞中均无胰岛素及胰高血糖素表达;在胰岛中未见到nestin与Ngn3共表达,但在导管中可见共表达。RT—PCR结果显示12～14周人胚胎胰腺中均有nestin和Ngn3的表达。结论Ngn3和nestin在人胚胎胰腺未分化细胞中表达,而具有分泌功能的内分泌细胞无表达。     

成人胰腺干细胞分离及转分化为胰岛的研究

目的 通过对成人胰腺干细胞分离和转分化为胰岛过程的研究以便更进一步了解及改进胰腺干细胞分离、培养、鉴定方法。方法 成人胰腺组织以胶原酶消化，密度梯度离心法获得纯化的胰腺外分泌细胞、导管上皮细胞和胰岛。导管上皮细胞在体外共培养27d，观察细胞形态学变化及干细胞特异性转录基因PDX—1，CK—19蛋白等的表达。结果 上述方法可获得大量胰腺导管上皮细胞。体外培养第1天即可见PDX—1，CK—19阳性细胞，胰腺导管上皮细胞迅速分裂增殖并转变为有分化能力的干细胞继而转分化为三维结构的胰岛细胞。培养27d后，平均每克胰腺组织可生成760个胰岛。结论 用改进的方法可获得大量成人胰腺导管上皮细胞，并可在体外转分化为大量具有内分泌功能的胰岛，可能为克服胰岛移植的供体短缺提供一条新途径。     

新生大鼠胰腺nestin阳性细胞的体外分离、培养和分化的研究

目的探索新生大鼠胰腺nestin阳性细胞体外分离、培养并使之形成类胰岛样细胞团的方法。方法应用胶原酶消化新生大鼠胰腺,将消化的组织碎片培养形成类胰岛样细胞团后传代。免疫荧光细胞化学法检测贴壁细胞胰岛素、胰高血糖素及nestin的表达。RT-PCR检测培养1周的贴壁细胞nestin及细胞角蛋白(CK19)的表达。结果在pH 7．6条件下培养24-36 h有部分细胞贴壁生长,改用pH 7．4无血清RPMI 1640培养液培养18-24 d可形成类胰岛样细胞团,传代后可有单层细胞生长。培养24～36 h的贴壁细胞nestin呈阳性,但胰岛素、胰高血糖素阴性,形成的类胰岛样细胞团传代后24 h胰岛素、胰高血糖素呈阳性。培养1周的单层细胞经RT-PCR扩增获得nestin 片段,但未获得CK19相应的片段。结论类胰岛样细胞团传代培养后可表达胰岛素、胰高血糖素; 胰腺nestin阳性细胞具有胰岛干细胞的特点。     

成人胰腺干细胞转分化为胰岛的研究

目的：通过对成人胰腺干细胞转分化为胰岛过程的研究以便更深入了解及改进胰腺干细胞分离、培养、鉴定方法。方法：成人胰腺组织经胶原酶消化后,用密度梯度离心法将胰腺外分泌细胞、导管上皮细胞和胰岛分离、纯化,导管上皮细胞即具有转分化潜能的干细胞,在体外先后以CMRL1066和无血清DMEM／F12培养液共培养27d,在培养的不同时间点取样本于光镜和电镜下观察细胞形态学变化及干细胞特异性转录基因PDX－1,CK－19蛋白等单抗的免疫组化染色,并测定培养液中的淀粉酶和胰岛素含量。结果：上述方法可获得大量以往在胰岛分离时丢弃的胰腺导管上皮细胞。经体外一定条件的培养后,第1天即可见PDX－1,CK－19阳性细胞,胰腺导管上皮细胞迅速分裂增殖并转变为有分化能力的干细胞继而转分化为三维结构的胰岛细胞。培养27d后,平均每克胰腺组织可生成760个胰岛。结论：成人胰腺的导管上皮具有干细胞潜能并可在体外转分化为大量具有内分泌功能的胰岛,用此方法获得大量的胰岛可能为克服胰岛移植的供体短缺提供一条新的途径。     

新生鼠胰岛干细胞分离、培养和分化的实验研究

目的　探索新生鼠胰岛干细胞体外分离鉴定、培养以及体外分化的方法。方法　用胶原酶消化新生大鼠胰腺 ,将消化的组织碎片在pH7.4～ 7.6条件下经含血清RPMI 164 0及无血清RPMI164 0 (添加bFGF ,EGF ,N 2 )培养液中培养 ,观察形成类胰岛样细胞团的全过程。用胰岛素释放实验检测胰岛功能 ,免疫荧光法检测nestin的表达。结果　胰腺消化碎片培养 3 6h内 ,可见nestin阳性细胞贴壁 ,添加bFGF ,EGF ,N2后nestin阳性细胞快速增长 ,18～ 2 4d形成类胰岛样细胞团 ,并可表达胰岛素。结论　胰腺细胞中nestin阳性细胞具有胰岛干细胞特点 ,经体外培养可获得类胰岛样细胞团     

PDX-1促进大鼠骨髓间质干细胞体外分化为胰岛素分泌细胞

目的:研究胰腺十二指肠同源框-1(pancreatic duodenal homeobox-1,PDX-1)基因在骨髓间质干细胞(mesenchymal stem cells,MSCs)体外分化中的作用.方法:构建含有PDX-1基因的真核表达载体,转染介质Superfect介导重组载体转染MSCs,G418筛选阳性细胞后对转染组(PDX-1+MSCs)和未转染组(PDX-1-MSCs)均体外进行诱导分化,流式细胞仪检测胰岛素阳性细胞的比例,胰岛素ELISA试剂盒测定转染组诱导后细胞的胰岛素分泌功能.结果:限制性酶切分析和序列测定证实成功构建了含有PDX-1基因的真核表达载体,荧光显微镜观察证实Supefect高效介导重组载体转染MSCs;流式细胞仪发现PDX-1+MSCs分化为胰岛素分泌细胞的数量较PDX-1-MSCs明显增多(阳性率分别为28.23%±2.56%和7.08%±2.69%),25 mmol高浓度葡萄糖刺激的胰岛素分泌量(115.29±2.56 μ U/mL),5 mmol低浓度葡萄糖刺激的分泌量明显增高(56.61±4.82 μ U/mL).结论:PDX-1能增强大鼠MSCs体外分化为功能性胰岛素分泌细胞的能力,对开展胰岛移植治疗1型糖尿病有重要价值.     

大鼠胰腺导管上皮细胞体外分离与定向分化

目的分离和纯化大鼠的胰腺导管上皮细胞，在体外培养并诱导其向胰岛细胞定向分化。方法采用胶原酶逆行灌注法消化、密度梯度离心结合不同细胞贴壁差异性分离和纯化胰腺导管上皮细胞；以角蛋白-19（CK-19）免疫细胞化学染色进行鉴定；用RMPI1640＋含体积分数为10％的胎牛血清（FBS）培养基培养促进胰导管上皮细胞增殖，1周后，更换无血清培养基DMEM／F12并加入角朊细胞生长因子等进一步促进其增殖，细胞达80％汇合时传代，加入高糖及尼克酰胺促进胰导管上皮细胞向胰岛细胞定向分化；对胰岛样结构行双硫腙染色。结果CK-19染色结果证实所获细胞绝大多为导管上皮细胞。体外培养中导管上皮细胞24h开始贴壁，14-21d达80％融合并形成细胞克隆，第28d胰岛细胞样结构形成，且被双硫腙染成猩红色。结论采用密度梯度离心结合差异贴壁法可获得纯化的大鼠胰腺导管上皮细胞，在体外培养与诱导分化条件下可生成胰岛样结构。     

成人胰腺干细胞转分化为胰岛细胞过程中的形态学观察

目的：观察成人胰腺干细胞转化化为胰岛过程中的形态学变化，以便更深入了解有关机制。方法：人胰腺组织经胶原酶消化后，用梯度离心法将胰腺外分泌细胞和胰岛分离，混于外分泌组织中的导管上皮细胞即具有转分化潜能的干细胞。在体外经CMRL1066及不含血清的DMEM／F12加多种营养因子的培养液中培养27d，在培养的不同时间点取细胞作PDX－1、CK－19等单抗的免疫组化染色。光镜和电镜观察不同时间点的形态学变化。结论：上述方法可获得大量以往在胰岛分离时丢弃的胰腺导管上皮细胞。经体外一定条件的培养后，其形态学变化过程为：导管上皮细胞迅速分裂增殖转变为有分化能力的干细胞继而转分化为胰岛细胞。结论：成人胰腺的导管上皮具有干细胞潜能，并可在体外转分化为大量具有内分泌功能的胰岛。用此方法获得大量的胰岛可能为克服胰岛移植的供体短缺提供一条新的途径。     

糖尿病小鼠胰腺干细胞转分化为胰岛样细胞

目的 观察链脲佐菌素所致糖尿病小鼠胰腺干细胞是否能转分化为胰岛样细胞.方法 以链脲佐菌素建立糖尿病小鼠模型,分离培养其胰腺导管上皮细胞,经体外扩增及诱导培养后,以细胞免疫化学方法检测PDX1表达,行STZ染色和葡萄糖刺激的胰岛素释放试验鉴定其功能.结果 糖尿病小鼠胰腺干细胞经体外培养和诱导分化后,PDX1阳性,并形成胰岛样细胞团;胰岛样细胞对高糖刺激(15.0 mmol/L)的胰岛素释放较低糖(5.6 mmol/L)时增加了1.4倍(37.2±11.2比25.9±7.6,t =2.830,P＜0.05),DTZ染色阳性.结论 链脲佐菌素所致糖尿病小鼠胰腺干细胞在体外培养条件下可转分化为胰岛素分泌细胞.     

损伤胰腺组织提取液诱导大鼠骨髓间充质干细胞向胰岛样细胞分化的实验研究

目的：探讨胰岛损伤的胰腺组织提取液诱导大鼠骨髓间充质干细胞向胰岛素样细胞分化的作用.方法：通过大鼠腹腔注射链脲佐菌素（STZ）建立1型糖尿病模型,6 d后剥离胰腺提取胰腺组织提取液,与SD大鼠骨髓间充质干细胞共同培养18 d,体外诱导骨髓间充质干细胞向胰岛素分泌细胞分化,RT-PCR法检测诱导分化后细胞的胰岛细胞相关基因的表达,葡萄糖刺激胰岛素释放实验检测诱导后细胞在高糖作用下分泌胰岛素的功能.结果：经损伤胰腺组织提取液诱导后,间充质干细胞在形态上由梭形变成多边、不规则形,且逐渐聚集成岛状.免疫荧光检测诱导细胞的胰岛素表达呈阳性,RT-PCR检测诱导细胞表达胰岛素1（Ins1）、胰岛素2（Ins2）、葡萄糖转运子2（Glut-2）、神经源素3（Ngn3）、胰腺十二指肠同源盒1（Pdx1）、同源框蛋白（Isl-1）及脑神经源性分化因子（NeuroD）等胰岛相关基因,且具备高糖诱导促进胰岛素分泌的功能.结论：在损伤胰腺组织提取液诱导下,骨髓间充质干细胞分化为胰岛样细胞,并且具有表达胰岛相关基因和胰岛素分泌功能.     

Differentiation and maturation of porcine fetal islet cells in vitro and after transplantation

BACKGROUND: Porcine fetal pancreas is a potential source of beta cells for transplantation. The immaturity of the cells is a problem. We have defined the optimal conditions for in vitro propagation of this tissue before transplantation. METHODS: Porcine fetal pancreas tissue was obtained for tissue culture at various stages of development. Serum-containing and serum-free media and a variety of potential differentiation factors were tested. In vitro, the numbers of endocrine islet cells and their proliferation were quantified and functional maturity of the beta cells was assessed by perifusion. Growth and maturation of the cells was assessed 3 months after transplantation into nude mice. RESULTS: Highest beta cell mass was obtained from end-gestational, as compared with early fetal or neonatal, pancreas. Nicotinamide and sodium butyrate effectively increased the insulin content and the number of endocrine cells in culture. In combination, these factors led up to a 90-fold increase in the insulin content of islet-like cell clusters (ICC) as compared with untreated controls. However, a high level of cell death through apoptosis was observed in these maximally stimulated endocrine cells, and they did not survive as grafts when transplanted into nude mice. Instead, a serum-free culture medium containing 10 mM nicotinamide and 0.1 mM isobutylmethylxanthine was found to support both differentiation and proliferation of endocrine cells as loose ICCs. Insulin release from these ICCs was sensitive to glucose. When transplanted under the kidney capsule of normoglycemic nude mice, a high level of beta cell differentiation and function was evident only in the ICCs cultured in the serum-free medium, and in freshly isolated ICCs. When transplanted to hyperglycemic nude recipients, the cells cultured in serum-free medium for 3 weeks reversed hyperglycemia more consistently and rapidly than freshly isolated ICCs. CONCLUSIONS: Optimal maturation of porcine fetal pancreatic cells is obtained in serum-free medium supplemented with nicotinamide. Butyrate is a potent stimulus for beta cell differentiation but leads to increased apoptotic cell death.     

Generation of functional islet-like clusters after monolayer culture and intracapsular aggregation of adult human pancreatic islet tissue

BACKGROUND: Cellular replacement therapy represents a promising strategy for treating type I diabetes; however, such an approach is limited due to the inadequate availability of human donor tissue. Here we investigated the extent to which human islet tissue can be expanded in monolayer culture and brought back to islet function. METHODS: Adult human pancreatic cells were proliferated with a serum-free media in monolayer cultures through multiple passages. Expanded cells were dispersed and encapsulated in alginate-poly-l-lysine microcapsules wherein the cells spontaneously coalesced into islet-like clusters. Encapsulated cell clusters were subsequently transplanted into the peritoneal cavity of streptozotocin-induced diabetic severe combined immunodeficiency mice. RESULTS: The cultured monolayer cells secreted insulin in response to glucose stimulation and maintained endocrine gene expression. Encapsulated islet-like clusters displayed cellular architecture similar to freshly isolated and encapsulated adult human islets maintained in culture, exhibiting an immunoreactive core of insulin, glucagon, and somatostatin, as well as peripheral cytokeratin-19 staining. Encapsulated aggregates significantly reduced hyperglycemia in transplanted mice within 1 week and normoglycemia was achieved after 5 weeks. Human C-peptide was detected in transplanted mice concomitant with the reduction in hyperglycemia. Capsules recovered 8 weeks posttransplantation exhibited insulin immunoreactivity. CONCLUSIONS: Collectively, these data indicate that adult human pancreatic islet cells can be expanded by three serial passages while maintaining their endocrine properties and can yield functional islet-like cell clusters through intracapsular aggregation that reverse hyperglycemia in diabetic mice. This culture and aggregation process could serve as a platform for proliferation and differentiation studies of endocrine lineage cells.     

Effects of growth hormone and insulin-like growth factor I on endocrine function of human fetal islet-like cell clusters during long-term tissue culture

The effects of recombinant human growth hormone (GH, 1 micrograms/ml) and insulin-like growth factor I (IGF-I, 200 ng/ml) on the production of insulin and glucagon by human fetal islet-like cell clusters (ICCs) were studied in tissue culture. ICCs were derived after collagenase digestion and culture of pancreases from 16 fetuses (mean gestational age 15.6 wk). The ICCs were cultured with or without GH or IGF-I for 7 or 31 days. Basal rates of insulin and glucagon production were not altered by GH during the first 17 days of culture, but the release of both hormones was increasingly augmented by GH during the last 2 wk of culture (131% increase in insulin and 85% in glucagon compared with controls). ICCs cultured for 7 days in the presence of GH secreted more insulin when incubated for 120 min in 20 mM than in 2 mM glucose (2.1-fold response, P less than .05), whereas ICCs maintained in basal medium did not respond to glucose. GH had no effect on DNA and insulin content or insulin biosynthesis. Exogenous IGF-I caused a 28% suppression of insulin release (P less than .05) between days 4 and 10 of culture but induced a 49% increase in the mean secretion rate during the last week (days 25-31, P less than .01). Glucagon release was not affected by exogenous IGF-I. In contrast to GH, exogenous IGF-I induced a twofold increase in the DNA content of the 7-day--cultured ICCs. However, insulin biosynthesis and release were markedly suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of beta-cells in the native pancreas after pancreas allo-transplantation in the Spontaneously Diabetic Torii rat

BACKGROUND: We previously demonstrated the development of beta-cells in the native pancreas after syngeneic pancreas transplantation (PTx) in a model of type 2 diabetes, namely the Spontaneously Diabetic Torii (SDT; RT1 a) rat. In this study, we evaluated the effect of fully allogeneic PTx (allo-PTx) under immunosuppression on the native pancreases in the recipients. MATERIALS AND METHODS: Diabetic 25-week-old SDT rats were divided into two groups: untreated controls and PTx-treated recipients. Dark Agouti (RT1 a) pancreases were then transplanted into the SDT rats. FK506 was administered daily postoperatively. Each group was examined for 15 weeks. RESULTS: Control SDT rats showed a disappearance of the pancreatic and duodenal homeobox-1 (PDX-1) expression of the pancreases with the development of diabetes. In addition, the islets were gradually replaced by fibrosis, thus resulting in a marked decrease in the beta-cell mass at 40 weeks of age. On the other hand, in PTx recipients, islet-like cell clusters were found in the native pancreases. The beta-cell mass significantly increased in the native pancreases in the recipients at 10 and 15 weeks posttransplantation in comparison to the age-matched controls. Moreover, we observed the re-expression of PDX-1 in the islet-like cell clusters. Interestingly, insulin and glucagon double-positive stained cells in the mesenchyme and insulin single-positive cells in the ductal epithelium were also observed. CONCLUSIONS: Our results indicated that the benefits of avoiding glucose toxicity by allo-PTx under immunosuppression could therefore induce the PDX-1 expression in the native pancreases, thus potentially resulting in the development of beta-cells in type 2 diabetic recipients.     

Characterization of endocrine-rich monolayers of human fetal pancreas that display reduced immunogenicity

Fibroblast-free insulin-secreting monolayers of human fetal pancreas (14-20 wk of gestation) were formed by plating isletlike cell clusters (ICCs) obtained from partially digested pancreases on plates coated with bovine corneal matrix. Human fetal pancreatic cells, freshly digested with collagen, displayed a 17-fold response to human peripheral blood lymphocytes (HPBLs) in mixed-lymphocyte culture. After 14 days in culture, monolayers derived from ICCs exhibited a smaller, twofold response to HPBLs. By comparison, in monolayers produced from single-cell suspensions, fibroblast overgrowth remained a problem. The endocrine component of the monolayers was 65 +/- 13 and 43 +/- 8%, respectively, with the number of beta-cells being 51 and 9%. Cells from both monolayers displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+, and 0.6-1.3 microM 12-O-tetradecanoylphorbol-13-acetate but not to 20 mM glucose. Monolayers derived from ICCs synthesized DNA, proinsulin, and protein. This study showed that it is possible to establish an endocrine-rich monolayer of human fetal pancreas that has greatly reduced immunogenicity. The existence of residual activity to HPBLs suggests some additional form of immunosuppression is required to prevent rejection of this tissue when grafted into diabetic patients. Subculturing and cryopreservation may also be needed to achieve adequate numbers of beta-cells for clinical transplantation.     

Differentiation of fetal pig endocrine cells after allografting into the thymus gland

BACKGROUND: The thymus of large animals, such as the pig, is thought to be an appropriate site for transplanting adult islets, which contain numerous beta cells, for the purpose of reversing diabetes. Whether fetal islet-like cell clusters (ICCs), which contain few beta cells, will develop at this site, so that adequate amounts of insulin can be produced, is unknown. METHODS: Between 15,000 and 40,000 ICCs were injected into the thymus gland of six juvenile immunosuppressed pigs, and the animals were killed up to 30 days later. The graft was then examined histologically and comparisons made with untransplanted ICCs and those grafted into the omentum of immunosuppressed pigs. RESULTS: At transplantation, the percentage of cells in the ICCs containing insulin, glucagon, somatostatin, or pancreatic polypeptide was 9+/-1%, 13+/-2%, 9+/-1%, and 3+/-1% respectively. Within 9-30 days of transplantation into the thymus, the percentage of all endocrine cells increased, insulin to 41+/-3%, glucagon to 43+/-6%, somatostatin to 26+/-4%, and pancreatic polypeptide to 9+/-3%. There was co-localization of more than one hormone in some cells. Omental grafts contained a similar percentage of insulin and glucagon-containing cells, but significantly fewer somatostatin and pancreatic polypeptide-containing cells. CONCLUSIONS: Endocrine cells from the fetal pig pancreas will differentiate when transplanted into the thymus gland of the pig, making this a suitable site for grafting ICCs to test their ability to normalize blood glucose levels of diabetic recipients.     

Tissue culture of human fetal pancreas. Effects of human serum on development and endocrine function of isletlike cell clusters

The human fetal pancreas represents a source of insulin-producing beta-cells with a potential for transplantation to diabetic patients. It has previously been shown that such cells can be viably maintained in tissue culture media containing fetal calf serum (FCS) and that these explants continue to synthesize and release insulin. In this study the effects of human serum (HS) on the growth and function of human fetal pancreatic explants have been compared with those of FCS. For this purpose, pancreatic glands, obtained after prostaglandin-induced abortions, were briefly exposed to collagenase, and the digest was cultured in RPMI-1640 medium plus 10% pooled HS or FCS. The outgrowth of isletlike cell clusters (ICCs) was monitored. In 31 of 58 consecutively explanted glands, development of ICCs was observed. In the presence of FCS the outgrowth of ICC took place on top of a fibroblast monocellular cell layer; HS effected less growth of fibroblasts and increased the formation of ICCs about sevenfold compared with explants from the same glands maintained in FCS. However, in the explant cultures with HS, the cell number per ICC, expressed as DNA content, was reduced by 50%. In both FCS and HS the insulin content of the medium showed great variability and progressively declined from day 2 to day 5. The medium glucagon concentration also decreased but not to the same extent as that of insulin. Immunocytochemical-stained ICCs showed insulin- and glucagon-positive cells scattered among most nonstained, presumably nonendocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)