Influence of Matrigel-overlay on constitutive and inducible expression of nine genes encoding drug-metabolizing enzymes in primary human hepatocytes

1.?Previous studies reported that rat hepatocytes overlaid with extracellular matrix components (Matrigel) maintain the expression and responsiveness of drug-metabolizing enzymes. However, whether Matrigel provides similar advantages in human hepatocytes remains largely uncertain.

2.?The influence of Matrigel-overlay on the constitutive and phenobarbital- and oltipraz-inducible expression of nine biotransformation enzymes, cytochrome P450s 1A1, 1A2, 2B6, 3A4, and glutathione S-transferases A1, A2, M1, T1, P1, in primary human hepatocytes was evaluated.

3.?Hepatocytes from five livers were maintained on a rigid collagen substratum with or without Matrigel overlay and treated for 48?h with two doses of each inducer. Quantitative RT-PCR, and for selected genes, immunoblot and enzyme activity analyses, demonstrated that human hepatocytes overlaid with Matrigel showed consistently higher constitutive and inducible expression of biotransformation genes. 4. Phenobarbital-mediated responsiveness of cytochrome P450 2B6, a potential indicator of hepatocyte differentiation status, was markedly higher in overlaid relative to non-overlaid hepatocytes. 5. It is concluded that an Matrigel overlay facilitates the maintenance and induction of xenobiotic metabolizing enzymes in primary cultures of human hepatocytes.     

Gene expression profiling in human lung fibroblast following cadmium exposure.

Cadmium is a naturally occurring metallic element with food and smoking being the main sources of exposure in the non-occupationally exposed population. Chronic exposure to cadmium leads to tumors in a number of tissues including lung. In the present study we investigated genes whose expression is modified by Cd exposure in human lung fibroblast WI38-VA13 cells. We employed a cDNA microarray hybridization method to identify changes in the gene expression profile. Thirty five genes were identified as cadmium-responsive. Their level of expression differed significantly from controls (significance analysis of microarray; SAM, q<5%). The largest groups of gene products affected by cadmium exposure were those involved in cell cycle, immunity and defense, nucleoside metabolism and signal transduction. Repressed expression of E2f1, Tubb and Actg2 following cadmium exposure may contribute to the cell cycle arrest. Down-regulation of Eno1 indicates a potential for causing protooncogene expression and possibly for cadmium-induced carcinogenicity. These results may contribute to better understand the toxic mechanism of cadmium toxicity. Moreover, the gene expression profile of cadmium could provide potential biomarkers for cadmium exposure.     

同型半胱氨酸对内皮细胞基质金属蛋白酶-1表达的影响

目的:观察同型半胱氨酸(Hcy)对血管内皮细胞基质金属蛋白酶-1(MMP-1)基因表达的影响.方法:体外培养人脐静脉血管内皮细胞株CRL-1730,加入不同浓度Hcy后,将细胞分为空白对照组、生理浓度组、低浓度组、中浓度组和高浓度组,提取细胞总RNA,逆转录-聚合酶链反应半定量检测MMP-1 mRNA的表达.分析各组MMP-1 mRNA的表达水平.结果:空白对照组、生理浓度组6、24 h的MMP-1 mRNA表达量低于1 h(P＜0.01),而6h与24 h的表达量差异无统计学意义(P＞0.05).低、中、高浓度组MMP-1 mRNA的表达量随时间延长而逐渐下降(P＜0.01).不同Hcy浓度组MMP-1 mRNA的表达量的比较,差异有统计学意义(F组间=131.806,P＜0.01).Hcy浓度与MMP-1 mRNA的表达量呈正相关(r=0.307,P＜0.05).结论:高浓度Hcy使血管内皮细胞MMP-1的基因表达呈剂量依赖式上调.     

氯沙坦抑制糖基化终末产物刺激培养的人内皮细胞外基质基因的表达

目的 探讨氯沙坦防治糖尿病血管病变的作用机理。方法 用反转录-聚合酶链反应(RT-PCR)和Northern印迹杂交方法测定了0.1～10 μmol·L^-1浓度的氯沙坦对经体外制备的糖基化终末代谢产物（AGEs）处理培养的人脐静脉内皮细胞（HUVECs）转化生长因子（TGF）-β₁和纤连蛋白（FN）基因表达的影响。结果 由AGEs处理的HUVECs TGF-β₁和FN mRNA表达较正常对照组明显增高；与AGEs组相比，TGF-β₁和FN mRNA的表达在氯沙坦1.0 μmol·L^-1时分别降低了29%和23%，10 μmol·L^-1时降低了56%和62%。结论 氯沙坦通过抑制AGEs刺激的内皮细胞TGF-β₁和FN mRNA表达，从而抑制细胞外基质的生成，防止血管重构可能是其防治糖尿病血管并发病的机理之一。     

氯沙坦抑制糖基化终末产物刺激培养的人内皮细胞外基质基因的表达

目的　探讨氯沙坦防治糖尿病血管病变的作用机理。方法　用反转录 聚合酶链反应 (RT PCR)和Northern印迹杂交方法测定了 0 .1～ 10 μmol·L- 1浓度的氯沙坦对经体外制备的糖基化终末代谢产物(AGEs)处理培养的人脐静脉内皮细胞 (HUVECs)转化生长因子 (TGF) β1和纤连蛋白 (FN)基因表达的影响。结果　由AGEs处理的HUVECsTGF β1和FNmRNA表达较正常对照组明显增高 ;与AGEs组相比 ,TGF β1和FNmRNA的表达在氯沙坦 1.0 μmol·L- 1时分别降低了 2 9%和 2 3%,10 μmol·L- 1时降低了 5 6 %和 6 2 %。结论　氯沙坦通过抑制AGEs刺激的内皮细胞TGF β1和FNmRNA表达 ,从而抑制细胞外基质的生成 ,防止血管重构可能是其防治糖尿病血管并发病的机理之一。     

Role of human airway smooth muscle in altered extracellular matrix production in asthma

1. The underlying abnormality in asthma is not fully understood; however, inflammation, airway remodelling and bronchial hyperresponsiveness are key factors. The plasma exudate from the microvascular leakage plays a significant role in remodelling, which includes extracellular matrix (ECM) protein deposition/breakdown and airway smooth muscle (ASM) hyperplasia/hypertrophy. 2. The ECM is an intricate network of macromolecules that forms the 'scaffolding' of the airways. This scaffolding not only acts as mechanical support that plays a crucial role in the maintenance of airway function and structure, but it is also a dynamic and complex network that has the potential to influence cellular function, including migration, differentiation and proliferation of a number of cell types. 3. In asthmatic airways, the profile of ECM proteins is altered. The deposition of collagen I, III, V, fibronectin, tenascin, hyaluronan, versican and laminin alpha2/beta2 is increased, whereas the deposition of collagen IV and elastin is decreased. 4. This imbalance in the ECM profile within the asthmatic airway could be due to: (i) increased de novo synthesis of ECM proteins; (ii) decreased activity of its degrading enzymes, namely matrix metalloproteinases (MMP); or (iii) upregulation of the tissue-specific inhibitors of metalloproteinases (TIMP). 5. One of the characteristic features of asthma is an increase in the amount of ASM within the airways. The ECM proteins/MMP/TIMP in and around the smooth muscle may play a contributory role in this increased growth. 6. The role of current asthma treatments in the prevention or reversal of airway ECM changes is an area that has only recently become of interest, with the majority of the in vivo work focusing on the effects of corticosteroids. 7. The evidence presented in this review indicates that the ASM may influence its own environment/proliferation through the production of ECM proteins, MMP and TIMP. Further studies are needed to fully understand the role of the ASM in the production of ECM proteins, MMP and TIMP andtheir potential influence in the mechanisms underlying asthma.     

Toxicogenomic profile of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the murine fetal heart: modulation of cell cycle and extracellular matrix genes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and similar environmental contaminants have been demonstrated to be potent cardiovascular teratogens in developing piscine and avian species. In the present study, we investigated the effects of TCDD on gene expression during murine cardiovascular development. C57Bl6N pregnant mice were dosed with 1.5, 3.0, or 6.0 microg TCDD/kg on gestational day (GD) 14.5, and microarray analysis was used to characterize the global changes in fetal cardiac gene expression on GD 17.5. TCDD significantly altered expression of a number of genes involved in xenobiotic metabolism, cardiac homeostasis, extracellular matrix production/remodeling, and cell cycle regulation. Interestingly, while the AhR-responsive genes Cyp1A1, Cyp1B1, Ugt1a6, and Ahrr, were all induced by TCDD in the fetal murine heart, other AhR-responsive genes, Cyp1a2, Nqo1, and Gsta1, were not. Quantitative real-time polymerase chain reactions confirmed the changes in expression of several G1/S-type cyclins and extracellular matrix-related genes. These results demonstrate the global changes in cardiac gene expression that result from TCDD exposure of the fetal murine heart and implicate genes involved in cell cycle and extracellular matrix regulation in TCDD-induced cardiac teratogenicity and functional deficits.     

Gene expression in two hepatic cell lines, cultured primary hepatocytes, and liver slices compared to the in vivo liver gene expression in rats: possible implications for toxicogenomics use of in vitro systems.

Microarray technology allows the simultaneous analysis of mRNA expression levels of thousands of genes. In the field of toxicogenomics, this technology could help to identify potentially unsafe compounds based on the changes in mRNA expression patterns they induce. Rodent in vivo and in vitro systems are currently the experimental models of choice for predictive toxicology, especially in early phases of development. This study characterizes several hepatic in vitro systems based on mRNA expression profiles, comparing them to gene expression in liver tissue. The in vitro systems investigated comprise two rat liver cell lines (BRL3A and NRL clone 9), primary hepatocytes in conventional monolayer or in sandwich culture, and liver slices. The results demonstrate that liver slices exhibit the strongest similarity to liver tissue regarding mRNA expression, whereas the two cell lines are quite different from the whole liver. We were able to identify genes with strong changes in expression levels in all or at least one of the in vitro systems relative to whole liver. In particular, for some cytochrome P450s the differences observed on the mRNA expression level were paralleled by protein expression and enzymatic activity. In addition, the effect of time in culture was assessed. We were able to show a profound effect of the duration of culture. Expression patterns change most rapidly soon after cell isolation and culture initiation and stabilize with time in culture. The findings are discussed with respect to the usefulness of the various hepatic in vitro systems for microarray-based toxicological testing of compounds.     

Induction of unscheduled DNA synthesis in primary human urothelial cells by the mycotoxin ochratoxin A.

Ochratoxin A (OTA) is a widespread contaminant in human staple food. Exposure of humans to this mycotoxin is a matter of concern because OTA is a known rodent carcinogen. As the urothelium is one target tissue of this mycotoxin, primary cultured human urothelial cells (HUC) from adults and children were used to analyze the induction of unscheduled DNA synthesis (UDS) by OTA. HUC were isolated from the ureters or renal pelves of two nephrectomized adults and of two children with ureteropelvic junction stenosis and cultured under serum-free conditions. After a confluency of 70-80% was reached, cell proliferation was suppressed by arginine-deficient medium (ADM), and UDS was assessed autoradiographically by 3H-thymidine incorporation upon exposure to OTA (10-2000 nM), ethyl methanesulfonate (EMS, 5 mM, positive control), or dimethyl sulfoxide (DMSO, 0.2%, solvent control). In control cultures the level of UDS was low. Exposure to EMS resulted in an induction of UDS (2-to 5-fold compared to control), thus allowing the sensitive detection of repair resulting from induction of DNA lesions in all four specimens, and demonstrating that repair of EMS-induced DNA lesions can take place under the chosen culture conditions. In two HUC cultures derived from adults, a significant induction of UDS was observed in the concentration range of 50-500 nM OTA. The highest fraction of cells in repair (CIR) was found at 50 nM OTA for the HUC from the older male (50% CIR). The maximum response in the other specimens from the adult female and the 7-year-old boy were seen at OTA concentrations of 500 and 250 nM, respectively. In contrast to all other specimens, no significant induction of UDS by OTA was found in the HUC cultures derived from an infant's urothelium. Signs of cytotoxicity were observed above 500 nM OTA in all cultures. The varying susceptibility toward OTA observed in vitro may hint at varying predispositions of individuals in vivo.     

An improved primary human nasal cell culture for the simultaneous determination of transepithelial transport and ciliary beat frequency

Objectives The aim was to establish a preclinical in‐vitro system of the nasal mucosa for the simultaneous evaluation of nasal absorption and effects on ciliary activity. Methods Human nasal epithelial cells were grown in collagen‐coated transport inserts with transparent polyethylene terephthalate membranes (3 μm). Transepithelial transport and ciliary beat frequency values were measured every 15 min for 1 h. Key findings The apparent permeability coefficients (P_app) for atenolol (mainly paracellular transport) and propranolol (transcellular transport) amounted to 0.1 ± 0.1 and 23.7 ± 0.6 × 10^?6 cm/s, respectively, illustrating that the system can be used to discriminate between high permeability and low permeability compounds. Transport of talinolol (substrate for the P‐glycoprotein efflux carrier) did not reveal polarity (0.3 ± 0.2 and 0.2 ± 0.1 × 10^?6 cm/s for absorptive and secretory transport, respectively) and was not affected by verapamil (10 μm), suggesting the absence of P‐glycoprotein in the nasal cell culture. No significant effects of atenolol, propranolol and talinolol on ciliary beat frequency were observed (98 ± 20% of the control condition after 60 min). Chlorocresol significantly decreased the ciliary activity but this decrease was not accompanied by effects on the transepithelial transport of atenolol, propranolol and talinolol. Conclusions A new system was developed which offers possibilities as a fast screening tool for studying the potential of compounds for nasal drug administration, since permeability and a possible cilio‐toxic effect can be assessed simultaneously.     

Assessment of hepatocytes and liver slices as in vitro test systems to predict in vivo gene expression.

The use of in vitro systems to predict in vivo responses to chemical agents provides the benefits of requiring fewer animals, reducing variability between samples, requiring less test material, and enabling higher throughput. In the present study rat tissue slices and primary hepatocytes were compared as in vitro systems to predict in vivo changes in gene expression in response to treatment with known liver toxicants or inducers. Five compounds (phenobarbital, carbon tetrachloride, Wy-14,634, alpha-napthylisothiocyanate, and tacrine) were chosen for their established and diverse mechanisms of hepatoxicity or microsomal induction. Expression profiles from male Sprague-Dawley rats or in vitro systems treated for 24 h were measured by DNA oligonucleotide microarrays containing 8700 probe sets. Qualitative comparison of expression revealed a >80% concordance between in vivo liver and both in vitro systems; however, the responsiveness of both in vitro systems to compound-induced changes in gene expression was far less than that of in vivo. Furthermore, both in vitro systems appeared similar in their ability to reproduce compound-induced changes in gene expression observed in vivo.     

BMP-7对TGF-β_1诱导人肾小管上皮细胞外基质表达的影响

目的观察骨形态发生蛋白-7(BMP-7)对TGF-β1诱导人近端肾小管上皮细胞(HK-2)细胞外基质(ECM)成分表达的影响,以探讨其延缓肾间质纤维化病变的作用机制。方法将体外培养的HK-2细胞分为空白对照组,3μg.L-1TGF-β1组,BMP-7组,TGF-β1加不同浓度BMP-7组。免疫荧光检测FN的表达;RT-PCR和Western blot分别检测FN、ColⅠα1mRNA及蛋白的表达。结果免疫荧光检测结果显示,3μg.L-1TGF-β1刺激HK-2细胞48h后,FN表达明显增强;加入200μg.L-1BMP-7干预后荧光明显减弱。RT-PCR和Western blot结果显示,TGF-β1刺激HK-2细胞48h后,FN、ColⅠα1mRNA及蛋白表达均明显高于对照组(P<0.01);BMP-7(100～400μg.L-1)可在mRNA及蛋白水平呈剂量依赖性下调TGF-β1诱导的FN、ColⅠα1的表达。结论BMP-7能抑制TGF-β1诱导的HK-2细胞FN、ColⅠα1的合成。表明BMP-7抑制肾小管间质纤维化的进展、改善肾功能的作用,部分是通过抑制肾小管上皮细胞分泌细胞外基质实现的。     

三维细胞培养技术在肿瘤研究领域的应用

传统二维细胞培养具有易操作、费用低、可大量应用的优点,在细胞培养领域广泛使用。但是由于细胞呈平面生长,无法构建出一个立体的微环境,且缺乏各种细胞因子的作用,因而与体内细胞状态存在差距;而三维细胞培养可以在细胞基因表达、基质分泌及细胞功能活动等方面更好地模拟体内细胞状态。该文着重论述三维细胞培养技术在肿瘤研究中的应用,包括构建肿瘤微环境、观察肿瘤生物学行为、肿瘤血管生成、研究肿瘤耐药性等,为肿瘤研究工作者提供参考。     

Gene expression patterns as potential molecular biomarkers for malignant transformation in human keratinocytes treated with MNNG, arsenic, or a metal mixture.

In previous studies, treatment with 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) enhanced malignant transformation of immortal human epidermal (RHEK-1) keratinocytes. In contrast, arsenic (As) alone or in a mixture of As, cadmium (Cd), chromium (Cr), and lead (Pb) inhibited this process. Microarray analysis showed unique gene expression patterns in RHEK-1 exposed to MNNG, As, or the metal mixture. From this analysis, we have selected 16 genes potentially involved in the enhancement or inhibition of transformation. These 16 genes, nine (IFN inducible protein 9-27, MAA A32, CCLB protein, integrin beta4, XRCC1, K8, K18, MT3, MAPKK6) of which were altered in a chemical-specific manner and seven (MIC1, bikunin, MTS1, BMP4, RAD23A, DOC2, vimentin) of which were commonly affected by the MNNG and As or mixture treatments, were examined for expression in detail by real-time RT-PCR. Qualitatively, both microarray and real-time RT-PCR analyses gave comparable results for 15 of 16 genes, i.e., genes were consistently induced or suppressed under the different treatment regimens when measured by either technique. Of the seven genes altered in their expression by multiple chemical treatments, five showed patterns consistent with a role in the transformation process, i.e., they were oppositely regulated in MNNG-transformed RHEK-1 cells (designated as OM3) as compared to the nonmalignant As- and mixture-exposed cells. Through time-course studies, we also identified markers whose expression correlates with acquisition of transformation-associated characteristics in OM3. Identification of a battery of genes altered during progressive transformation of RHEK-1 should aid in developing a mechanistic understanding of this process, as well as strengthening the utility of these genes as biomarkers.     

Primary human sinonasal epithelial cell culture model for topical drug delivery in patients with chronic rhinosinusitis with nasal polyposis

Objectives The primary human sinonasal epithelial cell culture (HSNEC) allows for in‐vitro modelling of mucosal responses to topical therapy. Cultures grown from healthy donors may underestimate changes in individuals with chronic sinonasal disease thereby yielding inaccurate results with respect to this large patient population. The purpose of this study was to analyse HSNECs derived from patients with chronic rhinosinusitis with nasal polyposis (CRSwNP) to determine whether expected disease dependent variables salient to topical drug delivery persist in culture. Methods Cultures were grown from patients with CRSwNP. Ciliary beat frequency (CBF) (basal and stimulated), permeability (trans and paracellular), inflammatory response, and glucocorticoid dose response were measured and compared with healthy controls. Key findings Methylcholine stimulated CBF was greater in CRSwNP versus controls (ΔCBF_60min 7.25 ± 1.02 vs 0.89 ± 1.04 Hz, respectively). Paracellular permeability was greater in CRSwNP versus controls (basolateral dextran_120min 18.97 ± 3.90 vs 11.31 ± 4.35 µg/ml, respectively). Lipopolysaccharide (0.1 mg/ml) stimulated interleukin‐6 (IL‐6) and IL‐8 secretion was increased in CRSwNP versus controls (IL‐6 Δbaseline 1738.72 ± 654.82 vs 1461.61 ± 533.51%, respectively; IL‐8 Δbaseline 137.11 ± 0.83 vs 111.27 ± 0.67%, respectively). CRSwNP cultures were more sensitive than controls to dexamethasone (1 µg/ml) dependent IL‐6 and IL‐8 suppression. Conclusions HSNECs derived from patients with CRSwNP retained their primary phenotype with respect to ciliary function, epithelial permeability, irritant induced inflammatory cytokine secretion, and glucocorticoid dose response.     

人绒毛膜促性腺激素对绒癌细胞系JEG-3表达基质金属蛋白酶-19的影响

目的 观察人绒毛膜促性腺激素(hCG)对滋养层细胞基质金属蛋白酶MMP-19表达的影响.方法 用逆转录多聚酶链反应(RT-PCR)检测方法 ,观察不同浓度hCG于不同处理时间,对来源于人绒癌组织的JEG-3细胞系表达基质金属蛋白酶MMP-19的影响.将JEG-3细胞分别用0.05、0.5、5、25 u·mL-1 hCG处理48 h.结果 JEG-3细胞中,MMP-19 mRNA的含量降低;但降低程度与hCG作用浓度的关系不明显.25 u·mL-1 hCG处理对JEG-3细胞中MMP-19表达的影响有一定的时间效应,以温育30 h以上的作用较明显.结论 hCG可抑制细胞系JEG-3中MMP-19的表达,并可能通过该机制影响滋养层细胞的侵袭性.