Establishment and chemical transformation of human skin epithelial cells in vitro

Summary This paper describes the in vitro establishment and chemical treatment of human foreskin epithelial cells which transform the cells to an anchorage-independent state as demonstrated by growth in soft agar. The procedures described include (a) production of primary cultures of human epithelial cells, (b) cytotoxicity determination of putative chemical carcinogens, (c) chemical transformation protocol, and (d) evaluation of chemical transformation as indicated by anchorage-independent growth.     

Immunohistochemical identification of Ito cells and their myofibroblastic transformation in adult human liver

To identify Ito cells in normal and pathological adult human livers, immunohistochemical studies were performed by the avidin-biotin-peroxidase complex method using monoclonal antibodies for -smooth muscle actin (ASMA), desmin, and vimentin. Fifty one needle biopsies, 7 surgically resected specimens, and 5 autopsy specimens were studied. In the normal adult liver vascular smooth muscle cells and pericytes, together with perisinusoidal cells with thin cytoplasmic processes were positive for ASMA. These latter cells formed a loose and discontinuous layer along the sinusoidal walls. Immunoelectron microscopy showed that the ASMA-positive perisinusoidal cells were Ito cells containing fat droplets. The other sinusoidal lining cells were negative for ASMA. In chronic liver disease, ASMA-positive Ito cells showed an increase in number, size, and the intensity of immunostaining in areas of piecemeal necrosis), and formed a continuous cellular network. These cells were dendritic in shape with irregularly elongated cytoplasmic processes and contained an increased amount of microfilaments, in association with loss of the characteristic fat droplets. Thus, their ultrastructural features corresponded to those of myofibroblastic cells. Ito cells showed no staining for desmin in both normal and pathological livers. These results indicate that immunohistochemistry using an anti-ASMA antibody is a sensitive and reliable method for the identification of both normal and transformed Ito cells in adult human livers.     

In vitro transformation of adult rat liver cells by 3'-methyl-4-dimethylaminoazobenzene

Primary cultures of liver cells from normal adult rats were treated with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) at various concentrations for 6 days. 3'-Me-DAB treatment induced rapid proliferation of epithelial clear cells with chromosomal abnormalities and gamma-glutamyl transpeptidase (GGT) activity. In early culture, marker chromosomes were detected in 13 of 44 3'-Me-DAB-treated cultures but not in control cultures. GGT activity was not detected in the epithelial clear cells in either 3'-Me-DAB-treated or control cultures. In late culture, 21 cell lines established from 39 carcinogen-treated cultures consisted of 3 diploid cell lines, 5 pseudodiploid cell lines and 13 aneuploid cell lines. Eighteen of these 21 cell lines had marker chromosomes. Of the 2 cell lines established from 15 control cultures both were aneuploid, but a marker chromosome was detected in only one of these. GGT activity was detected in 11 of 21 cell lines established from the carcinogen-treated cultures but not in those from control cultures. Morphological features of the cell lines which varied from normal to cancerous included polymorphism, increased nuclear/cytoplasmic ratio and prominent nucleoli. No cell line established in this study developed tumors in host rats during a 1-year observation period.     

Mesenchymal-epithelial transformation of Ito cells in vitro

Cultured pure population of Ito cells isolated from adult rat liver expressed epithelial markers cytokeratin-8, α-fetoprotein, and γ-glutamyl transpeptidase after forming a dense monolayer. Mesenchymal-epithelial transformation of these cells is possible, which suggests them as candidates of hepatic stem cells. __________ Translated from Kletochnye Tekhnologii v Biologii i Medicine, No. 3, pp. 150–153, August, 2006     

EB病毒对人胚鼻咽上皮细胞的转化

为观察EBV和／或促癌物四癸酸佛波醇二酯（TPA）对其的转化作用，以人胚鼻咽上皮作体外原代组织培养，采用自B95－8细胞分离的EB病毒直接感染或结合TPA处理体外培养的人胚鼻咽上皮细胞，着重观察感染细胞在半固体培养基中的集落形成率；并采用PCR扩增法探讨EB病毒是否直接进入鼻咽上皮细胞。结果显示：单独EB病毒或灭活（56℃，30分钟）EB病毒加TPA感染时，病毒不能进入细胞导致表型改变；活性EB病毒结合TPA同时处理或先用EB病毒后用TPA处理时，EB病毒能直接进入细胞并导致细胞集落形成率明显增高（P＜0．05）。从而表明EB病毒体外能部分转化人胚鼻咽上皮细胞，其转化作用依赖于TPA的存在和病毒基因组的完整。     

人胎儿肝脏间充质干细胞的体外培养及生物学特性研究

目的建立一种在体外分离、培养人胎儿肝脏来源的间充质干细胞(MSC)的方法,并研究其生物学特性。方法采用双酶消化法分离获取人胎儿肝脏MSC并进行体外培养,待细胞达80%以上融合时传代,各传代细胞依次记为P1～P10代细胞,并于倒置相差显微镜下观察细胞形态。取P3、P7、P10代细胞,连续培养7d,观察分析细胞生长曲线。取P5代细胞,采用流式细胞仪检测MSC表面CD34、CD44、CD105、CD13、HLA-ABC、HLA-DR等抗原标志的表达。取P4代细胞,进行体外成骨细胞诱导培养后,采用碱性磷酸酶染色鉴定。冻存传代细胞,分别于2、6个月后复苏细胞,台盼蓝染色计数复苏后细胞存活率。结果原代细胞3d贴壁,6d后开始快速增长并形成集落,11d左右达80%～90%融合,多呈纤维样;传代培养后细胞维持纤维样形态。传代细胞生长曲线具有共同特征:潜伏期约24～48h,对数增殖期约3～4d,对数增殖期后第5～6天进入平台期。流式细胞仪检测显示,MSC表面CD34、HLA-DR呈阴性表达,CD44、CD105、CD13呈阳性表达,HLA-ABC呈弱阳性表达。碱性磷酸酶染色显示,诱导后细胞的细胞核染成均一的淡蓝色。冻存复苏后细胞存活率达90%以上,且与未冻存传代细胞相比具有相同的生长特性。结论本研究成功地从人胎儿肝脏培养出MSC,为MSC应用于科学研究和临床治疗提供了新的细胞来源。     

Growth inhibitory effects of IFN-beta on human liver cancer cells in vitro and in vivo.

We investigated the effects of interferon-beta (IFN-beta) on the growth of human liver cancer cells. The effects of IFN-beta with or without 5-fluorouracil (5-FU) on the proliferation of 13 liver cancer cell lines were investigated in vitro. Chronologic change in IFN-alpha receptor 2 (IFNAR-2) expression was monitored in hepatocellular carcinoma (HCC) cells (HAK-1B) cultured with IFN-beta. After HAK-1B cells were transplanted into nude mice, various doses of IFN-beta were administered, and the tumor volume, weight, histology, tumor blood vessel, and angiogenesis factor expression were examined. IFN-beta inhibited the growth of 11 cell lines with apoptosis in a dose-dependent and time-dependent manner. With IFN-beta, IFNAR-2 expression in HAK-1B cells was significantly downregulated from 6 to 12 h. IFN-beta induced a dose-dependent decrease in tumor volume and weight and a significant increase of apoptosis in the tumor. Both basic fibroblast growth factor (bFGF) and blood vessel number in the tumor decreased only in mice receiving the lowest dose (1000 IU) of IFN-beta. IFN-beta with 10 muM of 5-FU frequently induced synergistic antiproliferative effects. IFN-beta with or without 5-FU induces strong antitumor effects in HCC cells, and we conclude that IFN-beta is useful for the prevention and treatment of HCC.     

Plectin deficiency leads to both muscular dystrophy and pyloric atresia in epidermolysis bullosa simplex

Plectin is a cytoskeletal linker protein which has a long central rod and N‐ and C‐terminal globular domains. Mutations in the gene encoding plectin (PLEC) cause two distinct autosomal recessive subtypes of epidermolysis bullosa: EB simplex (EBS) with muscular dystrophy (EBS‐MD), and EBS with pyloric atresia (EBS‐PA). Previous studies have demonstrated that loss of full‐length plectin with residual expression of the rodless isoform leads to EBS‐MD, whereas complete loss or marked attenuation of expression of full‐length and rodless plectin underlies the more severe EBS‐PA phenotype. However, muscular dystrophy has never been identified in EBS‐PA, not even in the severe form of the disease. Here, we report the first case of EBS associated with both pyloric atresia and muscular dystrophy. Both of the premature termination codon‐causing mutations of the proband are located within exon 32, the last exon of PLEC. Immunofluorescence and immunoblot analysis of skin samples and cultured fibroblasts from the proband revealed truncated plectin protein expression in low amounts. This study demonstrates that plectin deficiency can indeed lead to both muscular dystrophy and pyloric atresia in an individual EBS patient. © 2010 Wiley‐Liss, Inc.     

Allelic 3p deletions in high-grade carcinomas after transformation in vitro of human uroepithelial cells.

Restriction fragment length polymorphism (RFLP) analysis for allelic losses on the chromosome arm 3p were performed on independent carcinomas produced in athymic nude mice after transformation in vitro of a pseudodiploid clonal SV40-immortalized human uroepithelial cell line (SV-HUC). We analyzed ten primary carcinomas with heterogeneous phenotypes for deletions on 3p by using three informative probes, D3S30, D3S2, and D3F15S2, which map to the 3p11-p14, 3p21.1, and 3p21 regions, respectively. Five of the ten primary cancers showed reduction to homozygosity with at least one of the probes, and all five cancers were high-grade and poorly differentiated. We also analyzed six carcinomas that arose after progression of low-grade cancers, either spontaneously or after exposure to a human bladder carcinogen, to higher grades (progressed carcinomas). Four of the six exhibited 3p allelic loss. No preferential loss of a specific 3p allele was observed in any of the carcinomas. In addition, whereas most of the carcinomas showed allelic loss for all three of the probes, indicating a large-scale deletion, several of the carcinomas exhibited losses for only one or two of the probes, thus making it possible, along with the cytogenetic data, to define the least common region of deletion to 3p13----p14.2. These results support the hypothesis that nonrandom loss of a gene or genes on 3p leads to the development of cancer. Furthermore, these findings associate deletion of a putative 3p13----p14.2 tumor suppressor gene region with the development of high-grade uroepithelial carcinomas.     

Expression of cytoskeletal proteins in hepatic stellate cells isolated from normal and cirrhotic rat liver

Hepatic stellate cells (HSC) are located in Disse spaces of normal rat liver. In their quiescent state they serve as a storage site for vitamin A. In fibrotic liver they become activated, proliferate and they undergo transdifferentiation into myofibroblast-like cells. Changes in the cell phenotype are accompanied by changes in the cellular cytoskeleton. We have studied the expression of alpha-smooth muscle actin and intermediate filament proteins vimentin, desmin and glial fibrillary acidic protein (GFAP) by immunocytochemistry in HSC cultured for 2 or 7 days after isolation. Normal or cirrhotic rat liver was perfused with solutions of pronase and collagenase and HSC were isolated by density gradient centrifugation of the resulting cell suspension. Liver cirrhosis was produced in rats by repeated carbon tetrachloride administration. Vimentin was detected in all cells from normal and cirrhotic liver. The concentration of desmin in the cells from cirrhotic liver was slightly higher than that in normal cells and it increased with time in culture. GFAP could be detected only in normal cells 2 days after their isolation. In contrast, alpha smooth muscle actin (alpha-SMA) was absent from normal cells at this time but its expression was pronouced later. In most cells from cirrhotic liver this antigen was already present on the second day of culture and its expression further increased.     

Initiation of apoptosis by actin cytoskeletal derangement in human airway epithelial cells

Changes in epithelial cell shape can lead to cell death and detachment. Actin filaments are cleaved during apoptosis, but whether disruption in the actin cytoskeletal network, as one manifestation of cell shape change, can itself induce apoptosis is not known. We tested this hypothesis in the airway epithelial cell line 1HAEo(-) and in primary airway epithelial cells by preventing actin filament elongation with cytochalasin D or by aggregating actin filaments with jasplakinolide. Disruption of actin filament integrity promptly induced apoptosis in adherent epithelial cells within 5 h. Jasplakinolide-induced apoptosis did not disrupt focal adhesions, whereas cytochalasin D-induced apoptosis decreased focal adhesion protein expression and occurred despite ligation of the fibronectin receptor. Death induction was abrogated by the caspase inhibitors z-VAD-fmk and Ac-DEVD-cho but not by blocking the Fas (CD95) receptor. Whereas cytochalasin D--induced apoptosis was associated with cleavage of pro-caspase-8, jasplakinolide-induced apoptosis was not. Both agents induced formation of a death-inducing signaling complex. These data demonstrate that disruption of actin filament integrity with either cytochalasin D or jasplakinolide induces apoptosis in airway epithelial cells but by different mechanisms, and suggest that actin may be an early modulator of apoptotic commitment.     

ANLN缺失后Hela细胞的力学特性与骨架蛋白变化分析

文题释义：Anillin蛋白(ANLN)：最早是以F-actin的绑定蛋白被发现的,后经研究表明其与细胞骨架的肌动蛋白纤维和肌球蛋白等主要组分相互作用,是一种高度保守的脚手架蛋白,在胞质分裂中稳定收缩环。在细胞分裂间期中维持正常的细胞-细胞连接和细胞形态。原子力显微镜：利用探针与测量样本之间的各种相互作用力,再通过激光束反射放大微悬臂形变的信号,能够以极高分辨率(纳米级)检测样本表面并成像,同时可对样本进行力学性能测试,获得样本表面特定点的力-距离曲线,再通过对曲线分析获得精确的力学特性,是近期运用最为广泛的工具之一。背景：目前关于ANLN蛋白在细胞分裂间期调节细胞形态、细胞-细胞间接完整性以及胞质分裂中稳定收缩环的作用已经明确,但其对细胞力学性能和骨架蛋白的影响还鲜有报道。目的：探讨ANLN缺失对分裂间期细胞力学特性与骨架蛋白的影响。方法：通过原子力显微镜分别测量正常Hela细胞与siRNA敲降ANLN的Hela细胞的表面弹性模量和破膜力;采用激光共聚焦显微镜观察正常Hela细胞与siRNA敲降ANLN的Hela细胞的肌球蛋白ⅡA以及肌动蛋白纤维分布特征。结果与结论：①敲降ANLN的Hela细胞的表面弹性模量明显高于未经处理的正常Hela细胞,与敲降ANLN的细胞相比,正常细胞的表面弹性模量更倾向于极性分布(两极间逐步降低),但两组细胞长轴边缘区域的破膜力并没有明显差别;②ANLN敲降组细胞在边缘位置有较低的肌球蛋白ⅡA分布;③ANLN敲降组近底层细胞-细胞连接区域的肌动蛋白纤维趋向更散乱,并且细胞内纤维束沿长轴排列不明显,中层有细胞间隙变大的倾向;④结果说明,敲降ANLN对细胞的边缘区域影响最大,ANLN的缺失会导致细胞边缘区域更频繁的重构,细胞需要聚集更多的骨架蛋白及其结合蛋白来稳定细胞状态,导致了更高的表面弹性模量。ORCID: 0000-0002-3501-4467(徐丽萌)中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Lipid transformation into glycogen in animal and human cells

On the basis of analysis of the literature and the author's data a hypothesis of transformation of lipids into glycogen in animal and human cells by means of microbodies and lysosomes as one of the universal adaptative reactions of the host is substantiated. In the opinion of the author, both microbodies and lysosomes may take part both in the process of oxidation of intracellular lipids up to intermediate metabolism products and in the process of glycogen synthesis from these products. As a result of enhanced oxidation and transformation of lipids into glycogen the energy of the cell improves and the possibility of development of fatty dystrophy decreases. The hypothesis is based on the structural association of microbodies, lysosomes, fatty and glycogen inclusions observed by the author in cells of various organs in different pathological processes, as well as on certain biochemical data.     

Characterization of cytoskeletal proteins in basal cells of human parotid salivary gland ducts

Summary From previous immunofluorescent, immunohistochemical and ultrastructural studies, myoepithelial cells have been reported to be absent from the striated and excretory ducts of human salivary gland. Yet recently, certain anti-cytokeratin monoclonal antibodies which specifically label the myoepithelium of salivary gland acini and intercalated ducts have also been found to stain basally situated cells in both striated and excretory ducts. In this study, we have used eight samples of normal human parotid gland (methacarn-fixed and frozen sections) to establish if basal cells of striated and excretory ducts have the cytoskeletal protein complement (actin and cytokeratins) of myoepithelial cells. Using a muscle-specific actin, HHF35, not only is the myoepithelium of acini and intercalated ducts stained in all cases, but stellate and spindle shaped cells are also detected all along the inter- and intralobular striated ducts in four of the eight examples. With double-labeled frozen sections and fluorescent microscopy, the actin-specific probe, phalloidin, and the myoepithelial-selective anti-cytokeratin 14 antibody, 312C8-1, confirm that the striated duct does have a population of basal cells with the cytoskeletal protein make-up of myoepithelium. The monoclonal antibody 8.12 (specific for cytokeratin 13 and 16) also stains some basal cells of striated and excretory ducts, as well as luminal cells of ducts at all levels, but does not label the myoepithelium of acini and intercalated ducts. Both the anti-cytokeratin antibodies and the actin-detecting mechanisms reveal that the basal cell population of striated and excretory ducts is more heterogeneous, and likely functionally more complex, than has been realized previously. Such findings are not in agreement with certain aspects of current theories of the histogenesis of salivary gland tumours.The authors appreciate the funding provided by the Moe Levin Family Foundation, Montreal, Canada