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。     

Technique of pancreatic procurement for pancreatic islet isolation

AIM OF THE STUDY: The allograft of pancreatic islets represents a potential alternative to insulin therapy in patients suffering from the most severe forms of Type 1 diabetes. Here we report our experience of pancreatic procurement for isolation and islet allograft. MATERIALS AND METHODS: Pancreata were procured in brain-dead donors. The islets were isolated using techniques developed and validated in pigs and men. Injection of a given preparation was decided after quantitative and qualitative controls. Islets were transplanted in Type 1 diabetic patients already grafted with a kidney or suffering from severe and/or unstable diabetes, after percutaneous or surgical settlement of an intra-portal catheter. Patients received an "Edmonton-like" immunosuppressive protocol. Grafts were repeated once or twice until a total quantity of 10,000 transplanted islet-equivalents was obtained. RESULTS: Twenty-nine pancreata were procured and 14 preparations were grafted to 7 patients. Eleven graftings were done percutaneously and three were surgical. The initial function of the 14 transplants was confirmed by secretion of C-peptide and decrease of insulin doses. Insulin therapy was completely interrupted in the 5 patients having received at least two grafts. CONCLUSION: These preliminary clinical results confirmed that the isolation technique of human islets and the technique of pancreas procurement are mastered by our team. If the results of this assay (assessment one year after graft) confirm our hopes, we will be able to offer islet allografts to an increasing number of patients with severe Type 1 diabetes.     

胰腺癌干细胞研究进展

胰腺癌干细胞（PCSC）在胰腺癌的发生、发展过程中起着重要作用,且与胰腺癌的耐药、转移机制关系密切。PCSC的表面标志物、分离、鉴定、信号通路、微环境、耐药、转移和靶向治疗等方面的研究,将为临床胰腺癌的治疗提供新的方向。     

Evidence for stem cells in cultures of mouse prostate epithelial cells.

BACKGROUND: Primary cultures of mouse prostatic epithelial cells were studied to identify cells having stem cell characteristics. METHODS: Culture conditions supporting the growth of mouse prostatic epithelial cells were identified. Immunostaining identified cells expressing cytokeratin 5 (K5), cytokeratin 8 (K8), and bcl-2. Cells with clonogenic capacity were identified by replating dissociated cells from primary colonies. RESULTS: Only 2-4% of prostatic epithelial cells that were plated formed colonies. Most cells cultured for 2-4 days expressed bcl-2, K5, and K8. After 6 days in culture, colonies had two distinct phenotypes, star- or disc-shaped. In colonies of both phenotypes, only a few cells expressed K5 and bcl-2, while all cells expressed K8. Upon replating, two-thirds of star-shaped colonies gave rise to a single colony; of 29 disc-shaped colonies, none gave rise to new colonies. CONCLUSIONS: Colonies deriving from putative prostatic epithelial stem cells are identified. These cells express K5 and bcl-2 and are clonogenic.     

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.     

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。     

No-touch isolation techniques for pancreatic cancer

The rate of recurrence, including liver metastasis is high in pancreatic cancer, even when complete surgical resection is performed as a curative treatment. In patients with pancreatic cancer, the handling and grasping of the pancreas during surgery may increase the risk of liver metastasis, as squeezing may spread cancer cells via the portal vein. A no-touch isolation technique might prevent the spread of cancer cells via the hematogenous metastatic route in patients with pancreatic cancer. However, while no-touch isolation techniques are simple, feasible and, in theory, ideal procedures for the surgical treatment of pancreatic cancer, there have been no randomized controlled prospective studies to validate their advantages and their efficacy remains controversial. It is, therefore, worth investigating the use of no-touch isolation techniques in pancreatic cancer.     

肠上皮干细胞分离培养方法研究进展

肠上皮干细胞是位于肠黏膜陷窝内的具有自我更新和增殖分化为成熟肠上皮细胞功能的细胞。近年来,肠上皮干细胞的研究越来越受到重视,通过分离SP细胞、培养隐窝器官样组织和利用组织工程等方法研究肠上皮干细胞的分裂、分化过程,加深了对肠上皮干细胞的生物学特征的了解。但肠上皮干细胞的分离、培养和鉴别等研究方法尚未取得突破性进展。     

胰腺癌干细胞研究进展

肿瘤干细胞理论已逐步被人接受.胰腺癌干细胞表面分子标志初步确定为CD44+CD24+ESA+,将肿瘤干细胞理论运用于胰腺癌的研究,进一步研究胰腺癌于细胞特异性表面标志物及信号调控机制,可以提供胰腺癌药物作用干预的新靶点,希望能够寻找出一条治疗胰腺癌的新途径.     

Establishment of clonal colony-forming assay system for pancreatic stem/progenitor cells

Pluripotent stem cells found in a number of organs are usually in small cell populations. However, under adaptive stimulation, they enter the stage of growth and differentiation to compensate for the loss of differentiated cells. To analyze stem cell potential precisely, the exclusion of other differentiated cells and a clonal assay system are strongly required. In this study, we established a colony-forming assay system for pancreatic stem/progenitor cells in vitro. In this culture condition, they received signals for growth and differentiation, and formed clonal colonies including pancreatic endocrine-lineage cells, such as alpha and beta cells. By combining this culture system with flow cytometric cell sorting, pancreatic stem/progenitor cells will be enriched, and their potential can be analyzed precisely in single cell-based experiments.     

胰腺癌干细胞的研究进展

越来越多的证据表明肿瘤细胞中存在一小部分干细胞性质的肿瘤干细胞（cancer stem cell）．其具有自我更新及多向分化潜能。肿瘤干细胞是肿瘤细胞增殖、转移、复发和耐药的根源。胰腺癌是预后最差的肿瘤之一,肿瘤干细胞理论的提出将为胰腺癌的根治提供新的靶点和新的治疗方案。     

胰岛移植和胰岛干细胞研究现状

近年来,糖尿病患病率在全球呈现快速增长的趋势,我国人口患病率已接近5％,与发达国家类似。糖尿病已经成为患者和社会的巨大负担。目前,几乎所有1型糖尿病和部分胰岛素依赖的2型糖尿病患者都需要应用外源性胰岛素注射来控制血糖。严格控制血糖不能保证血糖稳定,亦不能避免远期并发症的发生,同时可能导致低血糖晕厥发作相对增多。因此,重建内源性胰岛素分泌系统是近年来研究者关注的热点。全胰腺移植始于20世纪60年代初期,     

大鼠神经干细胞与小鼠雪旺细胞混合培养的研究

外培目的观察小鼠雪旺细胞体养时对大鼠神经干细胞存活及分化的影响。方法获取Wistar鼠坐骨神经并采用组织块法分离和纯化雪旺细胞；体外分离新生乳鼠脑神经干细胞，将雪旺细胞和神经干细胞分别培养扩增后进行共培养。共分5组进行：实验组1（NSC悬浮＋SC悬浮＋DMEM／F12）；实验组2（NSC悬浮＋SC贴壁＋DMEM／F12）；试验对照组1（SC培养基＋NSC＋DMEM／F12）；试验对照组2（EGF／bFGF＋NSC＋DMEM／F12）；试验对照组3（NSC＋DMEM／F12）。倒置相差显微镜对各组培养细胞每天观察形态和计数，免疫组织化学检测混合培养细胞特异性标记物的表达：SC采用P0和S100，NS采用nestin标记，神经干细胞分化神经元分别采用GFAP、GalC、Tubulin-β染色。结果共培养组NF染色阳性神经元样细胞的百分率明显高干其他各组；实验组1克隆球直径明显高于其他各组，其平均直径为8μm；实验组神经元样细胞突起的长度比对照组的长，3周长度差为26．5-67．3μm。结论大鼠神经干细胞与小鼠雪旺细胞共培养使两者不仅能够共生，而且雪旺细胞能显著促进体神经干细胞分化为神经元样细胞；神经干细胞分化神经元突起增长并且有序排列成轴索样结构。     

Isolation and characterization of mouse mesenchymal stem cells

Objective

Mesenchymal stem cells (MSCs) have been studied in regenerative medicine because of their unique immunologic characteristics. However, before clinical application in humans, animal models are needed to confirm their safety and efficacy. To date, appropriate methods and sources to obtain mouse MSCs have not been identified. Therefore, we investigated MSCs isolated from 3 strains of mice and 3 sources for the development of MSCs in a mouse model.

Materials and Methods

Male BALB/c, C3H and C57BL/6 mice were used to isolate MSCs from various tissues including bone marrow (BM), compact bone, and adipose tissue. The MSCs were maintained in StemXVivo medium. Immunophenotypes of the MSCs were analyzed by FACS and their growth potential estimated by the number of colony-forming unit fibroblasts.

Results

All MSCs that were isolated from BM, compact bone, and adipose tissue showed plastic-adherent, fibroblastic-like morphologic characteristics regardless of the mouse strain or cell source. However, culture of BM MSCs was less successful than the other tissue types. The FACS phenotype analysis revealed that the MSCs were positive for CD29, CD44, CD105, and Sca-1, but negative for CD34, TER-119, CD45, and CD11b. According to the results of the characterization, the adipose tissue MSCs showed higher growth potential than did other MSCs.

Conclusion

The results of this study showed that culture of adipose tissue and compact bone-MSCs was easier than BM MSCs. Based on the results of immunophenotype and growth potential, C57BL/6 AT-MSCs might be a suitable source to establish a mouse model of MSCs.     

Cytotoxic activation of complement by mouse pancreatic islet cells

Human serum, or serum proteins excluded by Sephadex G-25, irreversibly inhibited the ability of mouse pancreatic islet cells to accumulate Rb+. The same treatment reduced the capacity of serum to subsequently inhibit Rb+ uptake by fresh islet cells or to lyse sensibilized sheep erythrocytes. Serum-treated islet cells exhibited electron microscopic signs of damage, including ruptures of the plasma membrane, swelling of mitochondria, and reduced electron density of the cytoplasmic ground substance. Serum induced a prompt insulin release, which was not inhibited by epinephrine. The serum effects were prevented by mild heating (50 degrees C or 56 degrees C, 30 min) but not by treating serum with 10 mM EGTA and 10 mM MgCl2, or with soybean trypsin inhibitor. Inhibition of Rb+ accumulation in response to human serum was also observed with dispersed mouse exocrine pancrease, liver, and spleen cells but not with whole islets. Homologous mouse serum had no effect on mouse liver or spleen cells but significantly decreased the Rb+ uptake by mouse islet cells. Autologous serum had no noticeable effect. It is suggested that mouse islet cells can activate complement via the alternative pathway and that triggering of this pathway is controlled by cellular discriminators of species, organ, and self.