大鼠容量超负荷早期心室压力变化和心肌原癌基因c-fos、c-jun、c-myc、egr-1的表达

目的:检测大鼠心脏容量超负荷(volume overload,VOL)时左心室内压变化诱导心肌原癌基因c-fos、c-jun、c-myc 、egr-1 mRNA表达的时间变化规律。方法:测定VOL 、假手术对照组大鼠术后30 min、1、4、6、12及48 h左心室收缩压(LVSP)和舒张末压(LVEDP),用狭缝杂交检测2组各时间左室心肌原癌基因表达,用密度仪进行定量分析。结果:VOL大鼠术后LVSP显著低于假手术组(P＜0.05),48 h最低;术后30 min LVEDP高于假手术组(P＜0.05),12 h最高,48 h与假手术组相近(P＞0.05);假手术组和阴性对照各原癌基因无阳性表达,AVF后1 h检测到c-fos 、c-jun、egr-1 mRNA表达,4h检测到c-myc表达,均于4h出现表达峰值,此后减弱,48h c-fos无表达,c-jun表达较弱,c-myc和egr-1仍有较高表达。结论:VOL大鼠LVEDP增加,首先诱导c-fos、c-jun、egr-1表达,c-myc表达较晚,c-myc、c-jun、 egr-1表达持续时间较长,可能反映了活体VOL刺激心肌原癌基因表达时间变化规律;当室壁受到一定程度和一定时间的负荷牵拉后心肌原癌基因即开始表达,负荷的强弱可能并非重要因素。     

Effects of lipopolysaccharide on a macrophage-like cell line (HS-P) from a rat histiocytic sarcoma.

Lipopolysaccharide (LPS) is a major modulator of macrophage functions. To characterize a newly established rat histiocytic sarcoma-derived cell line (HS-P), immunophenotypic changes and cellular growth responses of HS-P cells exposed to LPS were investigated and compared with those of MT-9 cells isolated from a rat malignant fibrous histiocytoma. MT-9 cells have somewhat histiocytic features, because occasional cells react to rat macrophage-specific antibodies. Addition of LPS to cultured HS-P cells increased the numbers of cells immunopositive to ED1 (rat macrophage-specific antibody) and ED2 (rat histiocyte-specific antibody) and stimulated the phagocytosis of latex beads, whereas LPS-treated MT-9 cells did not show such immunophenotypic changes. LPS-treated HS-P cells showed enhanced immunolabelling of alpha-smooth muscle actin, suggesting a possible modulation of macrophages towards myofibroblastic cells. To evaluate cellular growth after the addition of LPS or fetal bovine serum, DNA synthesis was examined by measuring tritiated thymidine incorporation, and the mRNA expression of c- jun and c- myc (immediate early genes in the cell cycle) was examined by Northern blot analysis. In HS-P cells, the addition of serum greatly increased DNA synthesis and induced high expression of c- jun and c- myc; in contrast, LPS markedly depressed DNA synthesis and reduced the expression of c- jun and c- myc. HS-P cells were more sensitive than MT-9 cells to the growth-promoting effect of serum and the growth-inhibiting effect of LPS. The study demonstrated that HS-P cells are highly LPS-responsive, indicating that they would be useful for studies of macrophage functions. Copyright Harcourt Publishers Ltd.     

Transforming growth factor-beta2 induces bronchial epithelial mucin expression in asthma

The transforming growth factor (TGF)-beta family is important for tissue repair in pathological conditions including asthma. However, little is known about the impact of either TGF-beta1 or TGF-beta2 on asthmatic airway epithelial mucin expression. We evaluated bronchial epithelial TGF-beta1 and TGF-beta2 expression and their effects on mucin expression, and the role of TGF-beta1 or TGF-beta2 in interleukin (IL)-13-induced mucin expression. Epithelial TGF-beta1, TGF-beta2, and mucin expression were evaluated in endobronchial biopsies from asthmatics and normal subjects. The effects of TGF-beta1 or TGF-beta2 on mucin MUC5AC protein and mRNA expression, and the impact of IL-13 on epithelial TGF-beta1, TGF-beta2, and MUC5AC were determined in cultured bronchial epithelial cells from endobronchial brushings of both subject groups. In biopsy tissue, epithelial TGF-beta2 expression levels were higher than TGF-beta1 in both asthmatics and normals. TGF-beta2, but not TGF-beta1, was increased in asthmatics compared with normals, and significantly correlated with mucin expression. TGF-beta2, but not TGF-beta1, increased mucin expression in cultured epithelial cells from both subject groups. IL-13 increased the release of TGF-beta2, but not TGF-beta1, from epithelial cells. A neutralizing TGF-beta2 antibody partially inhibited IL-13-induced mucin expression. These data suggest that TGF-beta2 production by asthmatic bronchial epithelial cells may increase airway mucin expression. IL-13-induced mucin expression may occur in part through TGF-beta2 up-regulation.     

Cell density governs the ability of human bronchial epithelial cells to recognize serum and transforming growth factor beta-1 as squamous differentiation-inducing agents.

Sparse (75 to 2000 cells/cm2) density cultures of normal human bronchial epithelial cells uniformly undergo terminal squamous differentiation when incubated in medium containing serum (fetal bovine serum [FBS]) or transforming growth factor beta-1 (TGF-beta 1). It was found that the cell density of the culture affects the probability that a cell will respond to these differentiation-inducing agents. Thus whereas irreversible inhibition of DNA synthesis occurs in sparse cell-density cultures within 24 hours after exposure, only a transient (less than 36 hours) depression in DNA synthesis was seen in high (more than 10,000 cells/cm2) density cultures. In addition, although phase microscopic image analysis revealed that virtually all of the cells displayed a squamous morphology within 1 hour after exposure to FBS or TGF-beta 1, observations made 48 to 72 hours later showed the presence of clusters of small prolate spheroid-shaped cells surrounded by many involucrin-positive squamous-appearing cells. Only the small cells were capable of DNA synthesis and cell division as determined by autoradiography and time-lapse photomicrographic images. These replicating cells immediately undergo squamous differentiation if they are subcultured and reinoculated at low cell density and incubated in medium supplemented with FBS or TGF-beta 1. Therefore the probability that a human bronchial epithelial cell will be refractive to FBS- or TGF-beta 1 induced terminal squamous differentiation is solely a function of the cell density of the culture.     

Regulated expression of the IL-31 receptor in bronchial and alveolar epithelial cells, pulmonary fibroblasts, and pulmonary macrophages.

Interleukin-31 (IL-31), an IL-6 cytokine family member, is proposed to play a role in animal models of airway hyperreactivity. It is produced by activated T cells and signals via a heterodimeric receptor complex composed of IL-31Ralpha and OSMRbeta. Only low levels of IL-31Ralpha expression have been demonstrated in pulmonary epithelial cell lines, however, and little is known about the ability to regulate its expression and signaling. Therefore, primary cultures of human bronchial and alveolar epithelial cells, pulmonary fibroblasts, pulmonary macrophages, and established lines of immortalized bronchial epithelial cells (HBE) and alveolar carcinoma cells (A549) were analyzed by RT-PCR, immunoblotting, and thymidine incorporation. Distinct, cell type-specific regulation of IL-31Ralpha expression was detected. Transforming growth factor-beta (TGF-beta) enhanced IL-31Ralpha mRNA expression in primary cultures and established lines of epithelial cells, but not in macrophages. In contrast, interferon-gamma (IFN-gamma) induced IL-31Ralpha mRNA expression in macrophages. IL-31Ralpha protein expression was below detection threshold in primary epithelial cell cultures but was detectable in A549 cells and increased with TGF-beta treatment. In HBE and A549 cells, TGF-beta pretreatment increased IL-31-mediated Stat3 and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. In A549 cells, TGF-beta magnified IL-31-dependent suppression of proliferation. The data suggest that increased IL-31Ralpha expression correlates with an enhanced response to IL-31.     

硒、碘对大鼠海马神经细胞原癌基因c-fos、c-jun表达的影响

目的 探讨硒、碘对培养大鼠海马神经细胞原癌基因c-fos、c-jun表达的影响。方法 以无血清培养大鼠民神经细胞为实验对象,培养液中加入不同浓度的碘,硒,用盖玻片培养法。在培养的1,3,5,7和10d时对海马神经细胞进行c-fos、c-jun的mRNA原位分子杂交和蛋白产物的免疫组织化学SABC法染色,对杂交信号和免疫阳性产物进行计算机图像分析。结果 硒可明显促进海马神经细胞原癌基因c-fos、c-jun mRNA及其蛋白的表达。硒、碘合用的促进作用最明显,尤其是c-jun mRNA表达。结论 碘和硒可通过促进海马神经细胞即刻早期基因c-fos、c-jun表达,特别是c-jun表达,进而影响神经功能发育。     

A newly established metastatic breast tumor cell line with integrated amplified copies of ERBB2 and double minute chromosomes

A continuous line of human mammary tumor cells, called 21MT, has been established in culture from a pleural effusion of a 36-year-old woman with metastatic breast cancer. The cells are epithelial as shown by morphology and expression of keratins and are mammary tumor cells as shown by expression of the HMFG-2 antigenic determinant. The cells grow well both in DFCI-1, a partially defined medium containing pituitary extract and 1% fetal bovine serum, and in alpha-minimum essential medium (alpha-MEM) supplemented with 10% serum, epidermal growth factor (EGF), insulin, and hydrocortisone. Karyotypic analysis of cells at early passage has shown the presence of rearranged (marker) chromosomes as well as aneuploidy with a net DNA content in the tetraploid range, confirmed by DNA cytofluorography, as well as double minute chromosomes in about 5% of the cells. Southern blots have revealed a 40-fold amplification of the ERBB2 gene and a 50-fold overexpression of its mRNA. The amplification of ERBB2 DNA was localized by in situ hybridization to one of the marker chromosomes but not to the double minutes. It is inferred, therefore, that at least two genes have been amplified in these cells.     

Mechanical stress triggers selective release of fibrotic mediators from bronchial epithelium

Transforming growth factor-beta (TGF-beta) and endothelin (ET) are found in elevated amounts in the airways of individuals with asthma. The cellular source of these peptides and their role in mediating the airway fibrosis of chronic asthma are unknown. In response to mechanical stresses similar to those occurring in vivo during airway constriction, bronchial epithelial cells increase the steady-state level of mRNA for both ET-1 and ET-2, followed by increased release of ET protein. Mechanical stress also enhances release of TGF-beta2 from a preformed cell-associated pool. TGF-beta2 and ET act individually and, more importantly, synergistically to promote fibrotic protein synthesis in reporter fibroblasts. To confirm the role of these intermediates in stress-induced fibrosis, conditioned medium from mechanically stressed bronchial epithelial cells was shown to elicit fibrotic protein synthesis in reporter fibroblasts; this effect was significantly inhibited by combined treatment with ET receptor antagonists and a neutralizing antibody to TGF-beta2. These data are consistent with a primary pathogenic role for mechanical stress-induced release of both TGF-beta2 and ET in the subepithelial fibrosis that characterizes chronic asthma.     

Glomerular epithelial cells transform to myofibroblasts: early but not late removal of TGF-beta1 reverses transformation

Studies were carried out to determine whether epithelial-mesenchymal transformation (EMT), well described in renal tubular epithelial cells, also occurs in glomerular epithelial cells and whether it is reversible. To this effect, cultured glomerular epithelial cells were incubated with TGF-beta(1) and their transformation into myofibroblasts was studied. At 4 days, the cells altered their phenotype, as shown by a change in shape, an increase in intracellular staining for alpha-smooth muscle actin (alpha-SMA), a decrease in membrane staining for cytokeratin, and an increase in matrix deposition. Changing the medium after 4 days by excluding TGF-beta(1) and adding fetal bovine serum (FBS) [as a source of epidermal growth factor (EGF) and other growth factors] caused the cells to revert to their original epithelial phenotype. By contrast, when the medium was changed in the same manner after 8 days of exposure to TGF-beta(1), the cells did not revert but remained myofibroblastic. Staining the cells for expression of EGF receptor before and after exposure to TGF-beta(1) caused this receptor, originally present on the plasma membrane, to become partly intracellular after 4 days of TGF-beta(1) exposure and completely intracellular after 8 days of TGF-beta(1) exposure. Kidney sections from 2 models of renal mass reduction were stained. Loss of the epithelial marker (podocalyxin) staining and the acquisition of alpha-SMA staining was observed in the glomeruli. It is concluded that EMT takes place in glomerular epithelial cells in vivo and in vitro. In cultured glomerular epithelial cells, the process can be reversed by early, but not late intervention. It seems that TGF-beta(1) exposure progressively downregulates the EGF receptor on the membrane, rendering the cell refractory to EGF signals critical for maintaining the epithelial phenotype.