Differentiated Mouse Epithelial Cell Line with Hepatocyte Characteristics

An epithelial cell line, 3105, with an unusual growth pattern has been derived from the liver of an (NZBxNZW)F₁ mouse. When confluent, it forms a monolayer of closely packed cells interspersed with holes that do not fill in during cultivation. By electron microscopy, the line has tight and intermediate junctions as well as desmosomes typical of epithelial cells. It produces several enzymes normally present in liver including hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, glucose-6 phosphatase, and alkaline phosphatase; has cytochromes P-450 and b₅; and spontaneously releases xenotropic but not ecotropic endogenous mouse type C viruses. Inoculation of the cell line into athymic nude mice gives rise to benign cysts in 2–3 months. This mouse epithelial line with hepatocyte characteristics should be helpful to investigators as a cell model of normal liver cell differentiation.     

Neuronal and Glial Properties Coexist in a Novel Mouse CNS Immortalized Cell Line

A mes-c-myc A1 (A1) cell line was generated by retroviral infection of cultured embryonic mesencephalic cells and selected by neomycin resistance. A1 cells cease to divide and undergo morphological differentiation after serum withdrawal or addition of c-AMP. Proliferating or morphologically differentiated A1 cells are all positive for vimentin and nestin, a marker of neural precursor, and show neuronal markers such as microtubule-associated protein 1, neuron-specific enolase and peripherin, and the glial marker glial fibrillary acidic protein. Neuronal and glial markers coexist in single cells. Furthermore, A1 cells show presence of glutamic acid decarboxylase 67 mRNA and its embryonic form EP10 and accumulate the neurotransmitter GABA. Electrophysiological studies demonstrate that morphologically differentiated A1 cells display voltage-gated sodium and potassium channels in response to depolarizing stimuli. A1 cells thus represent a novel, bipotent neural cell line useful for studying CNS differentiation and plasticity, as well as the molecular mechanisms underlying development of GABAergic neurotransmission.     

稳定整合pTet-on载体小鼠胚胎干细胞株的建立

目的建立稳定整合Tet-on基因的ES-D3细胞系,用于人胰岛素基因(pTRE2-human-Ins)在ES-D3细胞中诱导表达调控的研究。方法通过电穿孔转染的方法,将pTet-on质粒导入ES-D3细胞中,利用G418的药物选择特性,对转染的ES-D3细胞进行压力筛选,用PCR和Southern blot方法进行DNA整合鉴定,并用瞬时转染荧光素酶报告基因(pTRE-Luc)对筛选的Tet-on阳性克隆株的功能进行鉴定。结果经400μg/mL的G418压力筛选后,获得了11个细胞克隆株,特异性核苷酸引物检测细胞基因组DNA,有9个ES-D3细胞株可以扩增出相应的核苷酸片段,部分Tet-on阳性ES-D3株Southern blot鉴定结果表明Tet-on基因片段已整合入ES-D3细胞基因组DNA,荧光素酶报告基因功能鉴定获得1株诱导表达倍率为21.31的ES-D3细胞株,且Tet-on基因阳性ES-D3细胞的形态和生长速度与正常ES-D3细胞没有差异。结论通过电穿孔转染的方法成功地获得1株诱导表达倍率为21.31的高表达ES-D3细胞株,为进行目的基因(pTRE2-human-Ins)在ES-D3细胞中转染和诱导表达打下了基础。     

金雀异黄素对小鼠红白血病细胞系MEL的抑制作用

选择小鼠红白血病细胞系MEL作为金雀异黄素 (genistein)的作用对象 ,以探讨其对MEL的抑制作用及其可能的作用机理 .MTT和生长曲线表明 ,金雀异黄素对MEL细胞具有很强的生长抑制作用 (半数最大抑制效应浓度IC50 为 7 12mg L) .流式细胞术显示 ,金雀异黄素能够使MEL细胞发生G2 M期阻滞 ,但是诱导MEL凋亡作用并不明显 .DNA凝胶电泳发现金雀异黄素能够造成MEL细胞DNA的损伤 .联苯胺染色显示金雀异黄素对MEL细胞具有很强的诱导分化作用 ,且这种作用是时间和剂量依赖性的 .RT PCR显示 ,金雀异黄素能够抑制MEL细胞中原癌基因c myc的表达 .生物流变学检测发现 :金雀异黄素作用后 ,MEL细胞表面电荷密度减少、渗透脆性增大、细胞最大变形能力下降     

稳定表达重组小鼠FAPα蛋白细胞株的构建

采用RT-PCR方法从BALB/c胚鼠成纤维细胞克隆FAPα基因,将其连接至表达栽体pTd-FL-N,转化Stbl3感受态.筛选重组质粒,经酶切、PCR检测及测序鉴定,证实表达质粒构建正确.将表达载体转染HEK293细胞,经G418筛选单克隆阳性细胞,采用Western blot技术证实稳定表达FAPα细胞株构建成功.通过流式细胞术确定FAPa蛋白可定位表达于细胞膜上.     

表皮生长因子对鼠胚成纤维细胞细胞周期的调节作用

本文观察了表皮生长因子（ＥＧＦ）对小鼠胚胎成纤维细胞Ｃ_３Ｈ／１０Ｔ_１／２ＣＬ_８（简称Ｃ_３Ｈ／１０）细胞周期的影响。结果表明：ＥＧＦ使Ｓ期提前,细胞周期缩短。进一步探讨了ＥＧＦ对细胞周期影响的机制,发现ＥＧＦ可活化在细胞周期调节中起重要作用的Ｐ３４~（ｃｄｃ２）激酶（简称ＣＤ２Ｋ）,使ＣＤ２Ｋ在周期中活性高峰出现的时间提前,提示ＥＧＦ对细胞周期的影响可能通过作用于ＣＤ２Ｋ实现的。     

Enhancement of Interferon Production in a Mouse Cell Line, a High-Yielding Source of Mouse Interferon

Priming with interferon prior to poly(I).poly(C) treatment of a mouse cell line, MO 57/2, followed by sequential administration of metabolic inhibitors, resulted in the production of high yields of mouse interferon.     

建立对未分化细胞特异性标记的小鼠胚胎干细胞系

构建pRex-1-EGFP表达载体,电穿孔转染小鼠ES细胞,用增强绿色荧光蛋白对起源于3．5d胚泡内细胞团的小鼠胚胎干细胞进行特异性标记,用荧光显微观察EGFP的表达以及RT-PCR方法检测Rex-1基因在未分化和分化中ES细胞中的表达情况。结果显示,EGFP基因成功转入小鼠ES细胞,并在未分化的ES细胞中高效表达;细胞开始分化后,EGFP的表达开始下降。由Rex-1基因启动子控制下的EGFP稳定表达的小鼠ES细胞系,对哺乳动物早期发育过程的研究以及对筛选能够调节上述过程的小分子化合物具有重要意义。     

小鼠鳞癌细胞培养液中溶骨因子的分离纯化

以7、12,二甲基(α)苯基蒽诱导建立了小鼠鳞癌细胞株,接种至裸鼠中可致高血钙。其细胞培养液经超滤浓缩100倍后,通过DEAE层析、Ultrogel AcA54层析、麦胚凝集素琼脂糖层析及HPLC(C_(18))。初步分离纯化了溶骨因子。这一溶骨因子在体外可显著刺激小鼠颅骨片中~(45)Ca的释出,伴有PGE2的生成及腺苷酸环化酶活性的增高,其分子量约15,000道尔顿。     

小鼠3T3-L1前脂肪细胞系的特异性标记(英文)

用增强绿色荧光蛋白特异性标记小鼠 3T3 L1前脂肪细胞系 .构建paP2 promoter EGFP载体 ,电穿孔转染小鼠 3T3 L1前脂肪细胞 ,显微荧光观察和RT PCR确认aP2基因的内源表达 .EGFP基因转入 3T3 L1前脂肪细胞 ,观察到细胞分化过程中EGFP表达和脂肪积累 .RT PCR分析表明 ,EGFP代表了稳定而真实的aP2基因的内源性表达 .建立了由脂肪组织特异表达基因aP2的表达控制的EGFP标记的小鼠 3T3 L1前脂肪细胞系 ,目前尚未见用同样方法对前脂肪细胞进行特异性标记 .该细胞系将为脂肪细胞分化机理研究以及为抗肥胖症和抗糖尿病药物筛选提供有力工具 .     

Polarization Optical Properties of the Pancreatic Acinar Cell of the Mouse

The birefringence of fresh and fixed mouse pancreatic acinar tissue was studied, utilizing whole mounts of pancreas from which the mesentery had been removed. Fresh pancreas in Tyrode's solution demonstrated positive birefringence with respect to the radial axis (the axis radiating from the nucleus as spokes from a wheel). Formol fixation reversed the sign of birefringence to negative with respect to the radius. The magnitude of birefringence increased with longer fixation. Neutral formol also reversed the sign to radially negative, but the magnitude did not increase with longer fixation. Tissue fixed in 2 per cent osmium tetroxide or potassium permanganate demonstrated strongly negative birefringence with respect to the radius. The cytoplasm of tissue fixed in acetic acid, though finely granular, still possessed radially positive birefringence. Birefringent areas could be seen in tissue fixed in potassium dichromate, but the sign of birefringence could not be determined. Ethyl alcohol, chromic acid, picric acid, and mercuric chloride all produced a brilliant cytoplasm in which no birefringence could be demonstrated. Freezing markedly decreased the radially positive birefringence of fresh tissue. Fresh tissue placed in increasing concentrations of glycerol demonstrated increasing radially positive birefringence. When formol-fixed tissue was placed in glycerol, the radially negative birefringence decreased. Osmium tetroxide-fixed tissue in 50 per cent glycerol was isotropic. The granularity of ethyl alcohol-fixed tissue disappeared in 50 per cent glycerol, and radially positive birefringence was evident. Frozen tissue showed increasing radially positive birefringence in increasing concentrations of glycerol. The results are discussed in relation to theories of fixation.     

小鼠膜联蛋白Ⅱ基因剔除重组子的构建及小鼠无膜联蛋白Ⅱ细胞系的建立

为了便于对膜联蛋白II(annexinII)的进一步研究以及今后进一步发展膜联蛋白II-/-动物模型,构建了膜联蛋白II基因封闭重组子(pPNT/annexinII/LacZ),筛选了细胞(L5178Y)克隆,并获得了膜联蛋白II-/-稳定细胞克隆(D4,E2)。所获膜联蛋白II-/-L5178Y克隆有待于以PCR做进一步鉴定。     

A Novel Mesoderm-Specific cDNA Isolated from a Mouse Embryonal Carcinoma Cell Line

A novel mesoderm-specific cDNA clone has been isolated by differential screening of cDNA library from an embryonal carcinoma (EC) cell line MC12. The cDNA clone 121a is about 2.5 kb in length and apparently encodes a putative polypeptide of 335 amino acids which may be secreted or membrane anchored glycoprotein since it has a possible signal sequence and a potential N-linked glycosylation site. In situ hybridization using mouse embryos revealed that 121a expression was confined to mesoderm and its derivatives such as allantois, the mesodermal layer of amnion, chorion and yolk sac, somites, heart, etc. These findings suggest that 121 a may be essential for mesodermal differentiation or function, although nothing definite is known. Conservation of 121a homolog in mammals and even in Drosophila seems to support this presumption. Fluorescence in situ hybridization successfully localized 121a to B1 band of mouse chromosome 6.     

Isolation of an Insulin-Responsive Preadipose Cell Line and a Mammary Tumor Virus-Producing, Dome-Forming Epithelial Cell Line from a Mouse Mammary Tumor

Two functional tissue culture cell lines, MTD and MTF cell lines, have been isolated from a mouse mammary tumor. MTD cells are epithelial and retain the ability to transport fluid leading to the formation of three-dimensional fluid-filled multicellular structures called "domes" or "hemicysts". Another property of MTD cells is the production of murine mammary tumor virus (MTV). Release of MTV into the culture medium was verified by immunological, electrophoretic and enzymatic analyses. Addition of dexamethasone in the culture medium enhanced both the formation of domes and the production of MTV. Thus, MTD cells retain the morphological and functional properties of the original mammary tumor cells.
MTF cells show the fibroblastic morphology in subconfluent cultures. After reaching confluence, however, these cells gradually accumulated triglycerides in the cytoplasm and eventually assumed the morphology of fat cells. This adipose conversion was greatly enhanced by the presence of insulin in the culture medium. The morphological resemblance of adipose-converted MTF cells to the mammary fat cells suggests that the MTF cell line was derived from the mammary fat pad stroma. These functional cell lines will be useful to study cell differentiation as well as cell-to-cell interactions in the mammary gland.     

Role of Calcium in Volume-Activated Chloride Currents in a Mouse Cholangiocyte Cell Line

Volume-activated Cl(-) channels (VACCs) play vital roles in many cells including cholangiocytes. Previously, we characterized the VACCs in mouse cholangiocytes. Since calcium plays an important role in VACC regulation in many cells, we have studied the effect of calcium modulation on the regulatory volume decrease (RVD) and VACC currents in mouse bile duct cells (MBDCs). Cell volume measurements were assessed by a Coulter counter with cell sizer, and conventional whole-cell patch-clamp techniques were used to study the role of calcium on RVD and VACC currents. Cell volume study indicated that MBDCs exhibited RVD, which was inhibited by 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) but not by removal of extracellular calcium. During hypotonic challenge, MBDCs exhibited an outwardly rectified current, which was significantly inhibited by administration of classical chloride channel inhibitors such as NPPB and tamoxifen. Chelation of the intracellular calcium with BAPTA-AM or removal of extracellular calcium and calcium channel blocker had no significant effect on VACC currents during hypotonic challenge. In addition to VACC, MBDC had a calcium-activated chloride channel, which was inhibited by NPPB. The present study is the first to systemically study the role of calcium on the VACC and RVD in mouse cholangiocytes and demonstrates that a certain level of intracellular calcium is necessary for RVD but the activation of VACC during RVD does not require calcium. These findings suggest that calcium does not have a direct regulatory role on VACC but has a permissive role on RVD in cholangiocytes.     

Characterization of the Phosphoinositide-Linked Dopamine Receptor in a Mouse Hippocampal-Neuroblastoma Hybrid Cell Line

Abstract: Previous studies have established that dopamine (DA) can stimulate phosphoinositide (PI) metabolism in the CNS and in the periphery. The present study summarizes our attempt to find a cell line that expresses this dopaminergic system. We describe that the stable clonal HN33.11 cell line, established by fusion of mouse hippocampal cells with neuroblastoma cells (N18TG2) that originate from A/J mouse, natively expresses the D₁ DA receptor system that couples to PI hydrolysis. In this cell line, 500 µM DA or SKF38393 produced 43 and 75% increases in inositol phosphate (IP) accumulations, respectively. In contrast, noradrenaline or 5-hydroxytryptamine did not affect IP accumulations. The formation of IP that was stimulated by DA or SKF38393 was selectively blocked by the D₁ DA receptor antagonist SCH23390 with IC₅₀ values of 13 and 16 µM. This response was not mediated by the D_1A DA receptor and was cyclic AMP-independent, as HN33.11 cells did not express this receptor, and DA or SKF38393 was unable to stimulate the formation of cyclic AMP. In Ca²⁺-free/100 µM EGTA medium, basal IP level was reduced by 31.5%, but SKF38393-stimulated PI hydrolysis was not affected. SKF38393-stimulated IP accumulation was also not affected by pertussis toxin (PTX) treatment (200 ng/ml), suggesting that this dopaminergic response is mediated by PTX-insensitive G proteins. Co-immunoprecipitation studies indicated that in membranes of HN33.11 cells, D₁-like binding sites are coupled to Gα_q protein. Blockade of SKF38393-induced PI hydrolysis with antiserum against phospholipase C (PLC) isozymes, performed in permeabilized cells, as well as co-immunoprecipitation studies implicate PLCβ3 and PLCβ4 in this dopaminergically mediated PI hydrolysis cascade. The results indicate that HN33.11 cells express a D₁-like DA receptor that couples to PLCβ3/4 via Gα_q protein. These cells may therefore be a useful model system for investigating this receptor system.     

Characterization of Whole Cell Chloride Conductances in a Mouse Inner Medullary Collecting Duct Cell Line mIMCD-3

The chloride conductance of inner medullary collecting duct cells (mIMCD-3 cell line) has been investigated using the whole cell configuration of the patch clamp technique. Seventy-seven percent of cells were chloride selective when measured with a NaCl-rich bathing solution and a TEACl-rich pipette solution. Seventy-five percent of chloride-selective cells (90/144) had whole cell currents which exhibited an outwardly-rectifying (OR) current-voltage (I/V) relationship, while the remaining cells exhibited a linear (L) I/V relationship. The properties of the OR and L chloride currents were distinct. OR currents (mean current densities at ±60 mV of 66 ± 5 pA/pF and 44 ± 3 pA/pF), were time- and voltage-independent with an anion selectivity (from calculated permeability ratios) of SCN⁻ (2.3), NO⁻ ₃ (1.8), ClO⁻ ₄ (1.7), Br⁻ (1.7), I⁻ (1.6), Cl⁻ (1.0), HCO⁻ ₃ (0.5), gluconate⁻ (0.2). Bath additions of NPPB, flufenamate, glibenclamide (all 100 μm) and DIDS (500 μm) produced varying degrees of block of OR currents with NPPB being the most potent (IC₅₀ of approximately 50 μm) while DIDS was the least effective. Linear chloride currents had similar current densities to the OR chloride currents and were also time- and voltage-independent. The anion selectivity sequence was SCN⁻ (2.5), NO⁻ ₃ (1.9), Br⁻ (1.4), I⁻ (1.1), Cl⁻ (1.0), ClO⁻ ₄ (0.5), HCO⁻ ₃ (0.5), gluconate⁻ (0.3). In contrast to the OR conductance, glibenclamide was the most potent and DIDS the least potent blocker of L currents. An IC₅₀ of >100 μm was observed for NPPB block. Neither OR of L chloride currents were affected by acutely or chronically increased intracellular cAMP and were not affected when intracellular Ca²⁺ levels were increased or decreased. The molecular identity and physiological role of OR and linear currents in mIMCD-3 cells are discussed. Received: 13 June 1995/Revised: 15 September 1995     

Hepatitis B Virus Infection of a Mouse Hepatic Cell Line Reconstituted with Human Sodium Taurocholate Cotransporting Polypeptide

Hepatitis B virus (HBV) enters hepatocytes via its receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). So far, HBV infection has been achieved only in human hepatic cells reconstituted with hNTCP and not in cells of mouse origin. Here, the first mouse liver cell line (AML12) which gains susceptibility to HBV upon hNTCP expression is described. Thus, HBV infection of receptor-expressing mouse hepatocytes does not principally require a human cofactor but can be triggered by endogenous murine determinants.