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。     

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.     

同种异体胰岛及胰腺干细胞来源的胰岛样结构序贯移植治疗糖尿病

目的 观察同种异体大鼠胰岛及胰腺干细胞来源的胰岛样结构序贯移植在糖尿病治疗中的作用.方法 分离胰腺组织获得胰岛及胰腺导管上皮细胞,将具有干细胞潜能的胰腺导管上皮细胞在体外培养27d.将新鲜分离的胰岛(200±50)个及诱导分化2周的胰腺干细胞来源的胰岛样结构(2×106)个序贯移植到糖尿病大鼠的肾被膜下观察大鼠的血糖及生存情况.结果 将胰岛及胰腺干细胞来源的胰岛样结构序贯移植到同一糖尿病大鼠3周后血糖仍在5 mmol/L水平,对照组血糖无明显下降.结论 胰腺干细胞可诱导分化为分泌胰岛素的胰岛样结构,胰岛及胰腺干细胞来源的胰岛样结构序贯移植对大鼠糖尿病有治疗作用.     

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。     

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     

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。     

Nestin阳性细胞的研究进展

胰岛干细胞移植有望能治疗Ⅰ型和部分Ⅱ型糖尿病。从胰腺组织分离得到的Nestin阳性细胞,可以转分化为胰腺的内、外分泌细胞和肝细胞,因而被认为是胰岛干细胞;但是也有研究表明Nestin阳性细胞不能转分化为胰岛细胞,主要与未成熟胰岛细胞向胰岛细胞过渡过程中的血管生成有关,并向未成熟胰岛细胞传递信号,促进胰岛细胞的成型(formation)并维持胰岛细胞的形态功能。本文就Nestin阳性细胞的研究进展进行综述。     

Nestin阳性细胞的研究进展

胰岛干细胞移植有望能治疗Ⅰ型和部分Ⅱ型糖尿病。从胰腺组织分离得到的Nestin阳性细胞，可以转分化为胰腺的内、外分泌细胞和肝细胞，因而被认为是胰岛干细胞；但是也有研究表明Nestin阳性细胞不能转分化为胰岛细胞，主要与未成熟胰岛细胞向胰岛细胞过渡过程中的血管生成有关，并向未成熟胰岛细胞传递信号，促进胰岛细胞的成型(formation)并维持胰岛细胞的形态功能。本文就Nestin阳性细胞的研究进展进行综述。     

CK-19与胰腺干细胞的研究现状

细胞角蛋白19(CK19)在胰腺干细胞有阳性表达,是胰腺干细胞的分子标志物之一,其在胰腺干细胞的鉴定及转分化方面有特别意义。     

两种酶对猪胰岛消化分离效果的对比

目的　研究单纯酶 (胶原酶Ⅳ )与复合酶 (由胶原酶Ⅰ、Ⅳ、弹性蛋白酶、纤维素酶组成 )对成年猪胰岛的分离效果。方法　采用经胰管灌注消化酶的方法分离胰岛 ,用双硫腙染色做胰岛计数 ,台盼蓝染色判定胰岛活化水平 ,胰岛培养第 2d、第 4d、第 6d测定胰岛素含量 ,培养第 6d做胰岛活性评价 ,电镜观察胰岛的形态结构。结果　经复合酶消化法制备的胰岛数量为 (4 915±l0 42 )个胰岛 /克胰腺 ;而经单纯酶消化的胰岛数量为 (30 12± 989)个胰岛 /克胰腺。两组比较差异显著 (P <0 .0 5 )。结论　复合酶消化法是一种优于单纯酶消化法的成年猪胰岛分离方法。     

Characterization of endocrine progenitor cells and critical factors for their differentiation in human adult pancreatic cell culture

We have reproduced a previously described method for the in vitro generation of endocrine cells in adult human pancreatic tissue culture. The aim of this study was to characterize the nature of pancreatic progenitor cells and to identify the factors necessary for their differentiation in this model. During monolayer expansion, two types of cells proliferated sequentially; first cytokeratin 19 (CK19)-positive ductal epithelial cells and then nestin-positive fibroblastoid cells. After the bromodeoxyuridine-labeled cells were traced in differentiated islet buds, some of the proliferating ductal cells had differentiated into endocrine cells, whereas nestin-positive cells could not give rise to endocrine tissue. Serum-free culture was found to be an absolute requirement for the endocrine differentiation to occur. Also, overlay of the cells with Matrigel was essential, whereas nicotinamide had a potentiating effect. The in vitro-generated islet buds released insulin in response to glucose nearly as efficiently as native islets. When transplanted under the kidney capsule of nude mice, only one of five grafts demonstrated further growth with foci of both endocrine and exocrine differentiation. Our results support the previous notion that pancreatic progenitor cells represent a subpopulation of ductal epithelial cells. No evidence was found for the development of endocrine cells from nestin-positive stem cells.     

Multipotent progenitor cells isolated from adult human pancreatic tissue

The supply of islet cells is a limiting factor for the widespread application of islet transplantation of type-1 diabetes. Islets constitute 1% to 2% of pancreatic tissue, leaving approximately 98% as discard after islet isolation and purification. In this report we present our data on the isolation of multipotent progenitor cells from discarded adult human pancreatic tissue. The collected cells from discarded nonislet fractions, after enzymatic digestion and gradient purification of islets, were dissociated for suspension culture in a serum-free medium. The cell clusters grown to a size of 100 to 150 mum contained cells staining for stage-specific embryonic antigens, but not insulin or C-peptide. To direct cell differentiation toward islets, clusters were recultured in a pancreatic differentiation medium. Insulin and C-peptide-positive cells by immunocytochemistry appeared within a week, reaching over 10% of the cell population. Glucagon and somatostatin-positive cells were also detected. The cell clusters were found to secrete insulin in response to glucose stimulation. Cells from the same clusters also had the capacity for differentiation into neural cells, as documented by staining for neural and glial cell markers when cultured as monolayers in media containing neurotrophic factors. These data suggest that multipotent pancreatic progenitor cells exist within the human pancreatic tissue that is typically discarded during islet isolation procedures. These adult progenitor cells can be successfully differentiated into insulin-producing cells, and thus they have the potential for treatment of type-1 diabetes mellitus.     

源于体外培养胰岛中表达Ngn3的细胞是胰腺内分泌前体细胞的新证据

目的 评估表达Ngn3的细胞对成体胰岛维护和更新的作用及其意义。方法 用6—8周龄的C57BL／6小鼠分离胰岛。胆总管内插管成功后,切除胰腺组织并用浓度为2．5mg／ml胶原蛋白酶V灌注消化。手工拣取胰岛后,置于60mm培养皿内,每个培养皿内含10—12个胰岛,用RPMI-1640(12．5mmol／L HEPES,5．2mmol／L葡萄糖和2％胎牛血清)培养液培养。用A6抗体,胰岛素抗体,胰高血糖素抗体,Nestin抗体,Ngn3抗体和5′—溴—2—脱氧尿嘧啶(5-bromo-2′-deoxy-uridine,Brdu)抗体进行胰岛细胞免疫荧光染色。结果 在增殖的胰岛细胞中,只有不到15％的细胞表达Ngn3,其中约有30％以上的细胞同时表达A6。在培养的胰岛细胞中存在表达Ngn3的细胞,这一结果与其他在胚胎发育和成体胰岛中的研究结果一致。本研究发现培养的胰岛细胞中存在同时表达A6和Ngn3的细胞。A6被认为是可分化为胆管上皮细胞的肝脏前体细胞的标记物。胰岛内表达A6的细胞可能来源于成体胰管,并迁移至胰岛内。细胞表达A6的同时表达Ngn3,表明这些细胞在胰岛内可分化为内分泌细胞。结论 培养的胰岛细胞中发现共同表达Ngn3和A6的细胞,表明胰岛内的4种内分泌细胞可能来源于成体胰岛中的同一内分泌细胞系。A6和Ngn3是对胰岛内成体干细胞研究很有帮助的标记物。这可以让我们进一步了解IPC的增殖和分化,并且有可能通过胰岛内成体干细胞来治疗糖尿病。     

Human pancreatic ductal cells: large-scale isolation and expansion

The in vitro differentiation of pancreatic stem cells has recently been shown to represent a new source of beta cells for cell therapy in diabetes. Human ductal cell differentiation, in vitro, has been documented in three-dimensional (3D) culture and recently substantiated. Although encouraging, the optimization of the ductal cell source, expansion and differentiation ex vivo are mandatory for clinical relevance. We compared three sources of human ductal cells (hDC) (method A1-2, B, and C). The classical main duct isolation of hDC by explant (A1), or enzymatic digestion (A2), was compared with two indirect methods: from 3D cultured human islet/duct-enriched fractions (B) and dedifferentiated exocrine fractions (C). Method A: few viable hDC were obtained from the main duct. Method B: embedding islet/duct rich fraction in 3D collagen gels expands the cytokeratin 19 (CK19)-positive ductal component in the form of ductal cysts, as we described previously; monolayers derived from digested cysts were 80% ductal (CK19). Method C: initially adherent amylase-positive exocrine clusters contained 12% (CK19) to 22% (CK7) ductal cells. One-week exocrine cultures were amylase negative and 46% (CK19) to 63% (CK7) ductal. Cell viability varied: <20% (A1), 81+/-12% (B), 91+/-2% (C). Extrapolating total yields we obtained (+/-SEM): 10.5+/-4.6 x 10(3) (A1), 36+/-18 x 10(3) (A2), 292+/-50 x 10(6) (B), 1696+/-526 x 10(6) (C) viable hDC per pancreas. A secondary monolayer expansion of cyst-derived hDC (method B) was achieved with NuSerum (4.2-fold on plastic, 2.6-fold on 804G matrix; p < 0.05 vs. control cells on plastic). First passage exocrine-derived ductal cells also responded to matrix and to growth factors, albeit not significantly. In conclusion, this study demonstrated that an abundant hDC supply can be obtained from islet/duct or exocrine fractions followed by monolayer expansion with NuSerum. If their differentiation capacity is confirmed, in particular exocrine-derived ductal cells may represent a promising abundant source of islets for allogenic and autologous diabetes cell therapy.     

Differentiation of affinity-purified human pancreatic duct cells to beta-cells

To test whether pancreatic duct cells are in vitro progenitors, they were purified from dispersed islet-depleted human pancreatic tissue using CA19-9 antibody. The purified fraction was almost entirely CK19+ with no insulin+ cells, whereas the unpurified cells (crude duct) were 56% CK19+ and 0.4% insulin+ of total cells (0.7% of CK19+ cells). These cells were expanded as monolayers, aggregated under serum-free conditions, and transplanted into normoglycemic NOD/SCID mice. In crude duct grafts, insulin+ cells increased to 6.1% of CK19+ cells. Purified duct cells had slow expansion and poor aggregation, as well as engraftment. The addition of 0.1% cultured stromal cells improved these parameters. These stromal cells contained no CK19+ cells and no insulin by either quantitative RT-PCR or immunohistochemistry; stromal cell aggregates and grafts contained no insulin+ cells. Aggregation of purified duct plus stromal preparations induced insulin+ cells (0.1% of CK19+ cells), with further increase to 1.1% in grafts. Insulin mRNA mirrored these changes. In these grafts, all insulin+ cells were in duct-like structures, while in crude duct grafts, 85% were. Some insulin+ cells coexpressed duct markers (CK19 and CA19-9) and heat shock protein (HSP)27, a marker of nonislet cells, suggesting the transition from duct. Thus, purified duct cells from adult human pancreas can differentiate to insulin-producing cells.