The gap junctional intercellular communication is no prerequisite for the stabilization of xenobiotic metabolizing enzyme activities in primary rat liver parenchymal cells in vitro

Summary In primary monocultures of adult rat liver parenchymal cells (PC), the activities of the xenobiotic metabolizing enzymes microsomal epoxide hydrolase (mEH_b), soluble epoxide hydrolase (sEH), glutathione S-transferases (GST), and phenolsulfotransferase (ST) were reduced after 7 d to values below 33% of the initial activities. Furthermore, the gap junctional intercellular communication (GJIC), measured after microinjection by dye transfer, decreased from 90% on Day 1 to undetectable values after 5 d in monoculture. Co-culture of PC with nonparenchymal rat liver epithelial cells (NEC) increased (98% on Day 1) and stabilized (82% on Day 7) the homotypic GJIC of PC. Additionally, most of the measured xenobiotic metabolizing enzyme activities were well stabilized over 1 wk in co-culture. Because GJIC is one of several mechanisms playing an important role in cell differentiation, the importance of GJIC for the stabilization of xenobiotic metabolizing enzymes in PC was investigated. PC in monoculture were, therefore, treated with 2% dimethyl sulfoxide (DMSO), a differentiation promoting factor, and 1,1,1-trichloro-2,2,-bis (p-chlorophenyl) ethane (DDT) (10 μg/ml), a liver tumor promotor and inhibitor of GJIC, was given to co-cultures of PC with NEC. DMSO significantly stabilized (68% on Day 7), while DDT significantly inhibited (8% on Day 7) homotypic GJIC of PC in the respective culture systems. In contrast, the activities of mEH_b, sEH, GST, and ST were not affected in the presence of DMSO or DDT. These results lead to the assumption that the differentiation parameters measured in this study (i.e., homotypic GJIC and the activities of xenobiotic metabolizing enzymes) are independently regulated in adult rat liver PC.     

Characterization and co-culture of novel nontransformed cell lines derived from rat endometrial epithelium and stroma

Summary Normal and neoplastic growth of epithelial cells depends on mutual interactions between epithelial and stromal cells. As a tool for the study of the underlying molecular mechanisms, we have developed temperature-sensitive, nontransformed cell lines derived from rat uterine epithelium and stroma by transfecting primary cultures with a temperature-sensitive mutant of the SV40 large T antigen. The epithelial and stromal cell lines obtained shared relevant morphological characteristics with the primary cells from which they were derived. Immunocytochemical analysis showed that the epithelial cell lines expressed the intermediate filament cytokeratin, whereas the stromal lines expressed the intermediate filament vimentin. Alkaline phosphatase activity was present in all cell lines examined. All cell lines were anchorage dependent and did not form foci. One epithelial cell line expressed oxytocin mRNA, a gene product recently shown to be highly expressed in vivo in the uterine epithelium at term. If grown on Matrigel, this cell line formed domelike structures, a further characteristic of its differentiated phenotype. In an attempt to reconstitute an endometrium in vitro, epithelial cells were seeded on top of a layer of stromal cells. Paraffin cross sections showed that this in vitro system consisted of a bilayer structure. Four to five cuboidal epithelial cells were typically anchored atop one stromal cell, forming an endometriumlike tissue. The present in vitro system should provide a useful model for further studies on endometrial functions and epithelial/stromal cell interactions at a molecular level.     

Protein enrichment of sweet potato residue by solid-state cultivation with mono- and co-cultures of amylolytic fungi

The degree of protein enrichment of sweet potato residue by different amylolytic moulds in solid-state cultivation was much greater than that obtained using amylolytic yeasts. The optimum initial moisture content for protein enrichment was about 65% (w/w). Adding nitrogen sources to the culture twice (at the start of the incubation and after 24 h) considerably improved the final protein content. A co-culture of amylolytic mycelial fungi yielded a product with 32% (w/w) crude protein after 4 days' incubation at 30°C. In a column reactor, the highest temperatures reached were 42°C and 40°C and the minimum O₂ concentrations were 1.5% and 2.5% of full saturation in the central and bottom layers, respectively.     

An in vitro model for cell-cell interactions

Summary Heterotypic cell-cell interactions appear to be involved in the control of development and function in a wide variety of tissues. In the vasculature, endothelial cells and mural cells (smooth muscle cells or pericytes) make frequent contacts, suggesting a role for intercellular interactions in the regulation of vascular growth and function. We have previously grown endothelial cells and mural cells together in mixed cultures and found that heterocellular contact led to endothelial growth inhibition. However, this mixed culture system does not lend itself to the examination of the effects of contact on the phenotype of the individual cell types. We have therefore developed a co-culture system in which cells can be co-cultured across a porous membrane, permitting intercellular contact while maintaining pure cell populations. Co-culture of endothelial cells and smooth muscle cells across membranes with pore sizes of 0.02, 0.4, 0.6, and 0.8μm maintained the two cell types as homogeneous populations, whereas smooth muscle cells migrated across the membrane through pores of 2.0μm. Vascular cell co-culture across membranes with 0.8-μm pores resulted the inhibition of endothelial cell proliferation and the generation of conditioned media which inhibited endothelial cell growth. The arrangement of the cells in this co-culture system mimics thein vivo orientation of vascular cells in which mural cells are separated from the abluminal surface of the endothelium by a fenestrated internal elastic lamina or basement membrane. Because this co-culture system maintains separable populations of cells in contact or close proximity allowing for biochemical and molecular analyses of pure populations, it should prove useful for the study of cell-cell interactions in a variety of systems.     

海洋动物来源微生物的共培养研究进展

近年来,由于一些新疾病的发生和细菌耐药性的出现,微生物来源次级代谢产物的筛选重复率越来越高,微生物一些代谢基因在现有实验室条件下无法表达,所以需要发现新的微生物资源,同时找到激活微生物代谢产物基因的方法。海洋动物体内蕴含着大量的共附生微生物资源,可以产生很多具有生物活性的化合物,是潜在的药用资源。本文综述了近年来海洋动物(海鞘、海绵、珊瑚和海葵等)来源的微生物进行共培养的研究策略,包括共培养菌株的选择、共培养条件、群体感应和信号分子对共培养菌株的影响,以及不同种类微生物间的共培养实例。共培养与单培养相比,增加了次级代谢产物的种类,提高了次级代谢产物的生物活性或产量。共培养的研究有助于发现新的海洋动物来源微生物的活性天然产物,为海洋药物的开发提供新思路。     

Effects of hydroxybenzyl alcohols on melanogenesis in melanocyte-keratinocyte co-culture and monolayer culture of melanocytes

In mammalian skin, melanocyte proliferation and melanogenesis can be stimulated by keratinocytes, fibroblasts and other regulatory factors. To determine whether hydroxybenzyl alcohols (HBAs) show more inhibitory in melanocytes cultured alone or in melanocytes co-cultured with keratinocytes, we developed a murine melanocyte–keratinocyte co-culture model to investigate the pigmentation regulators in company with other melanogenic inhibitors and stimulators. It was found that the effects of HBAs and melanogenic factors were more evident in melanocytes co-cultured with keratinocytes. Keratinocytes may play a synergistic role in melanocyte melanogenesis and influence the pigment production. The tests in the co-culture model also imply that the inhibitory effects of HBAs on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity. HBAs showed a low cytotoxicity. The eventual results proved that HBAs are promising and safe agents for skin whitening in melanocyte alone and in co-culture systems. The co-culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.     

Fibroblasts rescue oral squamous cancer cell from metformin-induced apoptosis via alleviating metabolic disbalance and inhibiting AMPK pathway

Metformin is an antidiabetic drug widely used for the treatment of type 2 diabetes. Growing evidence suggests that it may exert antitumor effects in vivo and in vitro. However, even with the promising potency on defeating cancer cells, the pre-clinical and epidemiological studies of metformin on various kinds of cancers are not satisfactory, and the reasons and underlying mechanisms remain unknown. Since cancer is a complex system, dependent on a promoting microenvironment, we hypothesize that the interactions between cancer cells and their neighborhood fibroblasts are essential for metformin resistance. To test this, we used a cell co-culture model closely mimicking the in vivo interactions and metabolic exchanges between normal stromal cells (NOFs) and oral squamous cancer cells (OSCC). Here we show that while metformin can significantly inhibit cell growth and induce apoptosis of OSCC cultured alone in a dose-dependent manner through activating p-AMPK^T172 and modulating Bcl-2, Bax, and cleaved PARP. However, when OSCC are co-cultured with NOFs the metformin effects on OSCC cells are annihilated. NOFs are rescuing OSCC from metformin – induced apoptosis, at least partially, through inhibiting the activity of AMPK and PARP, maintaining mitochondrial membrane potential and increasing the oxidative stress. Our results indicate that metformin effects on oral cancer cells are modulated by the microenvironment and that this has to be taken into consideration in the context of developing a new combination of drugs for oral cancer treatment.     

Inhibitory effect of curcumin on the invasion of rat ascites hepatoma cells in vitro and ex vivo

Curcumin, a yellow pigment in turmeric, is a food factor withantioxidative activity. The effect of curcumin on the proliferation and invasion of the rat ascites hepatoma AH109Acells was studied in vitro and ex vivo assay systems. Especially, a co-culture system of the hepatoma cellswith mesothelial cells derived from rat mesentery was employed to investigate the invasive motility. Curcumin suppressed thehepatoma slipping motility in a dose-dependent manner up to 5 M and thereafter maintained the effect up to 20 M, whereas this substance exerted little influence on the proliferation of the hepatoma cells at the same concentrations. Sera obtained from rats orally given curcumin also inhibited the AH109A cellular invasive movement when added to the culturemedium. Hepatoma cells previously cultured with hypoxanthineand xanthine oxidase showed a highly invasive activity. Curcumin and curcumin-loaded rat sera suppressed this reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with hypoxanthine, xanthine oxidase and either of curcumin samples. These resultssuggest that the antioxidative property of curcumin may beinvolved in its anti-invasive action.     

与成纤维细胞共培养的肺泡II型上皮细胞的生物学特性

建立胎鼠肺泡II型上皮细胞(AECII)与肺成纤维细胞(LF)共培养模型,观察与LF共培养下AECII的生物学特性。倒置相差显微镜观察AECII形态和基本生长情况;RT-PCR和流式细胞术分别检测肺泡表面活性蛋白-C(SP-C)、水通道蛋白5(AQP5)mRNA及蛋白质表达;流式细胞术检测细胞周期及Ki67表达。结果显示,与LF共培养时,AECII能较好地保留其细胞形态,SP-CmRNA及其蛋白质表达明显增加,而AQP5mRNA及其蛋白质表达则明显减少;LF促进AECII增殖,使G2/M、S期细胞及表达Ki67 细胞的比率明显增多。结果提示,AECII与LF共培养时,能更好地保留其细胞形态、分化及增殖特性。     

Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells

Abstract In vitro derivation of oocytes from embryonic stem (ES) cells has the potential to be an important tool for studying oogenesis as well as advancing the field of therapeutic cloning by providing an alternative source of oocytes. Here, we demonstrate a novel, two-step method for inducing mouse ES cells to differentiate into oocyte-like cells using mouse ovarian granulosa cells. First, primordial germ cells (PGCs) were differentiated within the embryonic body (EB) cells around day 4 as defined by the expression of PGC-specific markers and were distinguished from undifferentiated ES cells. Second, day 4 EB cells were co-cultured with ovarian granulosa cells. After 10 days, these cells formed germ cell colonies as indicated by the expression of the two germ cell markers Mvh and SCP3. These cells also expressed the oocyte-specific genes Fig α, GDF-9 , and ZP1-3 but not any testis-specific genes by RT-PCR analysis. EB cultured alone or cultured in granulosa cell-conditioned medium did not express any of these oocyte-specific markers. In addition, EB co-cultured with Chinese hamster ovary (CHO) cells or cultured in CHO cell-conditioned medium did not express all of these oocyte-specific markers. Immunocytochemistry analysis using Mvh and GDF-9 antibodies confirmed that some Mvh and GDF-9 double-positive oocyte-like cells were generated within the germ cell colonies. Our results demonstrate that granulosa cells were effective in inducing the differentiation of ES cell-derived PGCs into oocyte-like cells through direct cell-to-cell contacts. Our method offers a novel in vitro system for studying oogenesis; in particular, for studying the interactions between PGCs and granulosa cells.