Characterization of bovine pancreatic ductal cells isolated by a perfusion-digestion technique

Summary Bovine pancreatic ductal cells isolated by perfusing an enzyme solution into the lumen of the main duct were obtained as sheets of cells. Morphologic features of these cells were those of pancreatic ductal epithelial cells. These cells also contained alcian blue/periodic acid-Schiff positive material and bound lectins, and they stained for keratin in the same manner as intact ductal epithelium. In culture, the plating efficiency was high (13.6%) as determined by DNA content before and after 24 h plating, perhaps due to the gentle isolation technique and the isolation of sheets of cells rather than a single cell. Cell doubling time was 34.4 h in Eagle's minimal essential medium with 10% heat inactivated fetal bovine serum and antibodies, and over 95% of the cell incorporated [³H]thymidine during a 6 h labeling period after 4 d in primary culture. Isolated cells grew best in medium CMRL 1066 with 10% heat inactivated fetal bovine serum as determined by measuring DNA content. The paper is Publication 1100 from the Department of Pathology, University of Maryland School of Medicine. This study was supported in part by National Cancer Institute (Bethesda, MD) Contract NO1-CP-75947 and Grant CA-19197-06 through the National Pancreatic Cancer Project.     

Isolation and characterization of stromal progenitor cells from ascites of patients with epithelial ovarian adenocarcinoma

Background

At least one-third of epithelial ovarian cancers are associated with the development of ascites containing heterogeneous cell populations, including tumor cells, inflammatory cells, and stromal elements. The components of ascites and their effects on the tumor cell microenvironment remain poorly understood. This study aimed to isolate and characterize stromal progenitor cells from the ascites of patients with epithelial ovarian adenocarcinoma (EOA).

Methods

Seventeen ascitic fluid samples and 7 fresh tissue samples were collected from 16 patients with EOA. The ascites samples were then cultured in vitro in varying conditions. Flow cytometry and immunocytochemistry were used to isolate and characterize 2 cell populations with different morphologies (epithelial type and mesenchymal type) deriving from the ascites samples. The in vitro cell culture model was established using conditional culture medium.

Results

The doubling times of the epithelial type and mesenchymal type cells were 36 h and 48 h, respectively, indicating faster growth of the epithelial type cells compared to the mesenchymal type cells. Cultured in vitro, these ascitic cells displayed the potential for self-renewal and long-term proliferation, and expressed the typical cancer stem/progenitor cell markers CD44^high, CD24^low, and AC133⁺. These cells also demonstrated high BMP-2, BMP4, TGF-β, Rex-1, and AC133 early gene expression, and expressed EGFR, integrin α₂β₁, CD146, and Flt-4, which are highly associated with tumorigenesis and metastasis. The epithelial type cells demonstrated higher cytokeratin 18 and E-cadherin expression than the mesenchymal type cells. The mesenchymal type cells, in contrast, demonstrated higher AC133, CD73, CD105, CD117, EGFR, integrin α₂β₁, and CD146 surface marker expression than the epithelial type cells.

Conclusion

The established culture system provides an in vitro model for the selection of drugs that target cancer-associated stromal progenitor cells, and for the development of ovarian cancer treatments.     

Follicle-like structures formed by intestinal cell lines derived from the HT29-D4 adenocarcinoma cell line: morphological and functional characterization.

When cultured in high glucose containing medium, the human colon carcinoma cell line HT29-D4 and a clone derived by transfection with the MDR1 cDNA (MDR31) form multilayers of unorganized cells which are not polarized and are linked by desmosomes. Within these multilayers appear spontaneously large multicellular follicle-like-structures (FLS) where polarized cells linked by tight junctional complexes surround a lumen. Electron microscopy showed that some FLS display well developed brush borders with densely packed microvilli. Others have irregularly oriented microvilli of various lengths or are even completely devoid of apical differentiation. The lumen contains a variable amount of amorphous osmiophilic material. The apical surface of FLS forming cells express dipeptidylpeptidase IV, carcinoembryonic antigen, the mucin MUC1 and for the transfected cells the gp-170 protein. The organic anion fluorescein is transported from the cell to the lumen of FLS. Rhodamine 123, a substrate of the gp-170 ABC transporter is also concentrated in the lumen formed by MDR31 cells. Verapamil and cyclosporine A inhibited this last transport. Cyclic AMP stimulates the formation of these structures since treatment of post-confluent multilayers dramatically increased the number of FLS in HT29-D4 and MDR31 cell cultures within 24 h. The spontaneous formation of these morphologically and functionally polarized structures appeared at random and might respond to the coincidence of fluctuating parameters of the regulatory pathways (cAMP, Ca2+).     

A PAUF-neutralizing antibody targets both carcinoma and endothelial cells to impede pancreatic tumor progression and metastasis

Pancreatic adenocarcinoma up-regulated factor (PAUF) is expressed in pancreatic ductal adenocarcinoma (PDAC) and plays an important role in tumor progression and metastasis. Here we evaluate the anti-tumor efficacy of a human monoclonal antibody against PAUF, PMAb83, to provide a therapeutic intervention to treat the disease. PMAb83 reduced tumor growth and distant metastasis in orthotopically xenografted mice of human PDAC cells. PMAb83 treatment retarded proliferation along with weakened aggressiveness traits of the carcinoma cells. AKT/β-catenin signaling played a role in the carcinoma cell proliferation and the treated xenograft tumors exhibited reduced levels of β-catenin and cyclin D1. Moreover PMAb83 abrogated the PAUF-induced angiogenic responses of endothelial cells, reducing the density of CD31⁺ vessels in the treated tumors. In combination with gemcitabine, PMAb83 conferred enhanced survival of xenografted mice by about twofold compared to gemcitabine alone. Taken together, our findings show that PMAb83 treatment decreases the aggressiveness of carcinoma cells and suppresses tumor vascularization, which culminates in mitigated tumor growth and metastasis with improved survival in PDAC mouse models.     

Tissue culture of human epithelial cells from benign colonic tumors

Summary Human colonic epithelial cells from three classes of benign tumors have been reproducibly cultured free of fibroblasts for 8 wk using a supplemented Medium 199 (M 199S). The cultured colonic cells were identified as epithelial by the presence of junctional complexes (tight junctions, gap junctions, and desmosomes), a brush border on the apical surface, keratin fibrils, and by both a close-packed columnar or cuboidal morphology and the capability to transport water and ions to form hemicysts. Colony formation was initiated by groups of epithelial cells, not by single cells, and was inhibited by cocultivation with either lethally irradiated 3T3 cells or human diploid fibroblasts. Enhancement of epithelial colony formation was observed following culture on nonadherent, “floating” substrates compared with substrates attached directly to the bottom of the culture dish. Replication of epithelial cells in M 199S from the class of benign colonic tumors least prone to malignancy, the tubular, was significantly enhanced by epidermal growth factor (EGF). In contrast, EGF did not stimulate the growth of cells in M 199S from the other classes of benign tumors, the villotubular and the villous, which exhibit more malignant potential. These data imply that premalignant colonic epithelial cells lose responsiveness to growth modulation by EGF as they progress toward frank carcinoma. This study was supported by NCI Contract N01-CP43366 to M. L. and NCI Grant 1-R26-CA 28822 to E. F.     

阻断白色念珠菌粘附口腔膜上皮细胞的实验研究

白色念珠菌的粘附与其表面甘露糖结构有关,为探讨阻断粘附的方法,我们采用体外法测定其对口腔上皮细胞的粘附数量,显示：白色念珠菌粘附感染上皮细胞后,甘露糖可以在一定程度上减少粘附,绿慕安及刀豆素A可以有效地减少粘附。提示甘露糖及绿慕安可能有助于治疗白色念珠菌感染。     

脂多糖对人支气管上皮细胞STAT1、STAT3、STAT4、STAT6表达的影响

目的观察脂多糖对人支气管上皮细胞16HBESTAT1、STAT3、STAT4、STAT6表达的影响。方法采用普通RT—PCR检测16HBE细胞STAT1、STAT3、STAT4、STAT6的mRNA表达;Western印迹检测16HBE细胞STAT1、STAT4、STAT6的蛋白表达。分别采用不同浓度的脂多糖在不同的时间点处理16HBE细胞,采用Real—timePCR的方法检测16HBE细胞STAT1、STAT3、STAT4、STAT6的mRNA表达。结果1μg／m1的LPS处理16HBE细胞1h组、0．25μg／m1的LPS处理16HBE细胞4h组、1μg／ml的LPS处理16HBE细胞4h组STAT1、STAT4的mRNA表达较正常对照组显著增高（P〈0．01）;0．25μg／ml的LPS处理16HBE细胞2h组、1μg／ml的LPS处理16HBE细胞2h组、10μg／ml的LPS处理16HBE细胞2h组STAT1、STAT4的mRNA表达较正常对照组有所增高（P〈0．05）;1μg/ml的LPS处理16HBE细胞1h组STAT6的mRNA表达较正常对照组显著增高（P〈0．01）。所有LPS处理16HBE细胞组STAT3的mRNA表达均较正常对照组减低。结论人支气管上皮细胞表达STAT1、STAT3、STAT4、STAT6的mRNA和STAT1、STAT4、STAT6的蛋白,一定剂量的脂多糖在某些时间点分别刺激了人支气管上皮细胞STAT1、sTAT4、STAT6的mRNA表达。     

人胸腺上皮细胞培养上清液促进鼠胸腺细胞和细胞株增殖的研究(英文)

胸腺是细胞分化发育的场所,源于骨髓的前Ｔ细胞。在以胸腺上皮细胞为主体的基质细胞作用下,增殖、分化、成为成熟Ｔ细胞,迁移至外周。胸腺基质细胞通过细胞与细胞之间的直接接触和分泌细胞因子影响细胞分化发育的各个阶段。本文探讨了人胸腺上皮细胞培养上清液（ＴＥＣＳＮ）对鼠胸腺细胞、８５、Ｂｅ１３、ＨＤＭａｒ、Ｍｏｌｔ４、Ｒｅｈ以及Ｊｕｒｋａｔ细胞株增殖的影响。取人胸腺组织,用０．１５％的胰酶消化组织块后,将其接种到培养瓶中进行培养,收集上清液。将ＴＥＣＳＮ加到鼠胸腺细胞悬液中,２ｈ后加３ＨＴｄＲ,观察鼠胸腺细胞自发性掺入３ＨＴｄＲ有无改变。同时将ＴＥＣＳＮ＋ＣｏｎＡ加入鼠胸腺细胞悬液中培养４８ｈ,收获细胞前１８ｈ加入３ＨＴｄＲ,测定ＣＰＭ值。此外,ＴＥＣＳＮ加入８５细胞株及其它细胞株悬液中,观察ＴＥＣＳＮ对这些细胞株增殖的影响。上述各实验均设有对照组（Ｔａｂ．１）。结果表明：ＴＥＣＳＮ本身能增强鼠胸腺细胞３ＨＴｄＲ自发掺入,其３ＨＴｄＲＣＰＭ值明显大于对照组（Ｆｉｇ．１）。ＴＥＣＳＮ单独使用不影响胸腺细胞增殖,ＣｏｎＡ单独作用能促进鼠胸腺细胞增殖反应,而ＴＥＣＳＮ与ＣｏｎＡ共同使用,胸腺     

脂质体介导人β防御素2基因转染膀胱上皮的实验研究

目的 探讨脂质体介导人β防御-2（humaβ-defensin2,hBD2）基因体内、外转染膀胱上皮细胞的可行性。方法采用脂质体法体外转染人膀胱上皮细胞株T24及膀胱灌注大鼠体内转染,ELISA检测细胞上清及大鼠尿液中hBD：的表达;RT—PCR及Western印迹检测细胞及大鼠膀胱中hBD：的表达;组织免疫荧光检测hBD,在膀胱黏膜的表达。结果RT—PCR及Western印迹检测显示,脂质体介导pCAGG—hBD2体、内外转染膀胱上皮细胞后均可表达重组hBD2;转染细胞上清及大鼠尿液中hBD2浓度分别为（36．5±3．2）ng／10。细胞和（4．77±1．4）ng／ml;转基因hBD2主要表达于膀胱黏膜上皮层。结论脂质体介导hBD2基因体内、外转染膀胱上皮细胞后均能获得高效表达,为泌尿系感染的防御素基因治疗提供了实验依据。     

In vitro deregulation of markers characteristic of human prostate epithelial cells

We have screened primary cultures of human prostate for the expression of markers reported to be characteristic of specific cell lineages in vivo, in order to ascertain whether human prostate cells in vitro maintain and reflect their in vivo differentiated phenotypes and to evaluate the homogeneity of the populations of cells that can be derived from this tissue. Using single and dual stain immunofluorescent microscopy to analyse very early organoid and subsequently derived monolayer stage cultures, we have observed that expression of markers characteristic of human prostate epithelial cells in vivo is deregulated within 48h, indicating that dissociation of human prostate tissue and cultivation of prostate epithelial cells in culture can result in promiscuous expression of cell type specific markers of prostate epithelial cells. These observations have important implications for studies of cell lineage and differentiation of prostate cells in vitro.     

Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.     

Pancreatic cancer cells activate CCL5 expression in mesenchymal stromal cells through the insulin-like growth factor-I pathway

Mesenchymal stromal cells (MSCs) have a critical role in cancer progression and metastasis. Despite extensive studies of the physiological responses in cancer cells, the molecular mechanisms regulating gene expression in MSCs by cancer cells remain undefined. Here we demonstrate that CC chemokine ligand 5 (CCL5) expression was increased in MSCs co-cultured with pancreatic cancer cells (PCCs), and this activation was dependent on extracellular insulin-like growth factor (IGF-I). Moreover, CCL5 induction in MSCs was required for the activation of IGF-I pathway in PCCs. These results reveal a link between the IGF-I pathway in PCCs and CCL5 pathway in MSCs through the interaction of those cells.     

Reconstituted human gingival epithelium: Nonsubmerged in vitro model

Summary Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type I collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.     

人羊膜上皮细胞和胚胎干细胞

人胚胎干细胞有着巨大的医学应用前景,但人胚胎干细胞要求的生长条件很高,体外很难模拟其生长的体内环境,因此控制人胚胎干细胞的生长常不理想,而使用鼠胚胎成纤维细胞(MEF)作为滋养层则存在动物源性污染的问题。该文阐述人羊膜上皮细胞(HAEC)的特点及其作为滋养层培养胚胎干细胞的现状,并探讨基因组DNA甲基化修饰在胚胎干细胞分化过程中的作用,为建立更优化的培养系统提供依据。