共查询到20条相似文献,搜索用时 11 毫秒
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Chi Zhang Ser-Mien Chia Siew-Min Ong Shufang Zhang Yi-Chin Toh Danny van Noort Hanry Yu 《Biomaterials》2009,30(23-24):3847-3853
3D-microfluidic cell culture systems (3D-μFCCSs) support hepatocyte functions in vitro which can be further enhanced by controlled presentation of 100–200 pg/ml TGF-β1, thus mimicking the roles of supporting cells in co-cultures. Controlled presentation of TGF-β1 is achieved by either direct perfusion or in situ controlled release from gelatin microspheres immobilized in the 3D-μFCCS. Primary hepatocytes cultured for 7 days with the in situ controlled released TGF-β1 exhibited up to four-fold higher albumin secretion and two-fold higher phase I/II enzymatic activities, significantly improving the sensitivity of hepatocytes to acetaminophen-mediated hepatotoxicity, compared to hepatocytes cultured with directly perfused TGF-β1 or without TGF-β1. The controlled presentation of TGF-β1 enhanced hepatocyte functions in microfluidic systems without the complications of co-cultures, allowing for simplifications in drug testing and other hepatocyte-based applications. 相似文献
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Cheng-zhen Liang Hao Li Yi-qing Tao Li-hua Peng Jian-qing Gao Jing-jun Wu Fang-cai Li Jian-ming Hua Qi-xin Chen 《Acta biomaterialia》2013,9(12):9423-9433
Low back pain is frequently caused by nucleus pulposus (NP) degeneration. Tissue engineering is a powerful therapeutic strategy which could restore the normal biomechanical motion of the human spine. Previously we reported that a new nanostructured three-dimensional poly(lactide-co-glycolide) (PLGA) microsphere, which is loaded with dexamethasone and growth factor embedded heparin/poly(l-lysine) nanoparticles via a layer-by-layer system, was an effective cell carrier in vitro for NP tissue engineering. This study aimed to investigate whether the implantation of adipose-derived stem cell (ADSC)-seeded PLGA microspheres into the rat intervertebral disc could regenerate the degenerated disc. Changes in disc height by plain radiograph, T2-weighted signal intensity in magnetic resonance imaging (MRI), histology, immunohistochemistry and matrix-associated gene expression were evaluated in normal controls (NCs) (without operations), a degeneration control (DC) group (with needle puncture, injected only with Dulbecco’s modified Eagle’s medium), a PLGA microspheres (PMs) treatment group (with needle puncture, PLGA microspheres only injection), and PLGA microspheres loaded with ADSCs treatment (PMA) group (with needle puncture, PLGA microspheres loaded with ADSC injection) for a 24-week period. The results showed that at 24 weeks post-transplantation, the PM and PMA groups regained disc height values of ~63% and 76% and MRI signal intensities of ~47% and 76%, respectively, compared to the NC group. Biochemistry, immunohistochemistry and gene expression analysis also indicated the restoration of proteoglycan accumulation in the discs of the PM and PMA groups. However, there was almost no restoration of proteoglycan accumulation in the discs of the DC group compared with the PM and PMA groups. Taken together, these data suggest that ADSC-seeded PLGA microspheres could partly regenerate the degenerated disc in vivo after implantation into the rat degenerative intervertebral disc. 相似文献
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Ryoichi Miyashita Li Chen Hisashi Oshiro Hiroyuki Uchino Futoshi Shibasaki 《Neuroscience letters》2012
We have previously shown that when siRNA against Int6 (siRNA-Int6) was used, hypoxia-inducible factor 2α (HIF2α) activity was stabilized even under normoxic conditions, and the expression of several angiogenic factors was increased. In neuronal tissues, the mechanism underlying angiogenesis remains largely unknown. In the current study, we investigate the role of the tumor suppressor Int6/eIF3e in the regulation of the expression of angiogenic factors in neuronal cells. In addition, we test whether siRNA-Int6 reduces cold-induced brain damage in rats. We used human neuroblastoma SHSY5Y cells transfected with either siRNA-Int6, or a negative control siRNA. Real-time PCR and supersensitive multiplex assay were used to detect gene and protein expression of several angiogenic factors after transfection. For the animal studies, Wistar rats were subjected to brain damage by cold injury, and 50 μg siRNA-Int6, 100 μg siRNA-Int6, or negative control was administrated. At day 7 post-treatment, brain sections were stained and image analysis system was used to determine the damaged area. Our experiments using SHSY5Y cells revealed a significant effect of siRNA-Int6 on the expression of HIF2α but not HIF1α, both at 8 and 24 h after transfection. The siRNA-Int6 led to significant up-regulation of angiogenic factors, including vascular endothelial growth factor and platelet-derived growth factor-B, both at the mRNA and protein levels. Furthermore, our animal studies revealed significantly reduced area of cold-induced damage in rats receiving siRNA-Int6, compared to negative controls. Our findings indicate that Int6 act as a hypoxia-independent master switch of angiogenesis in neuronal cells, and that inhibition of Int6 by siRNA may be an effective therapeutic strategy in treating ischemic diseases such as brain ischemia and injury. 相似文献
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Mast cell function is a critical component of allergic reactions. Mast cell responses mediated by the high-affinity immunoglobulin E receptor FcεRI can be enhanced by co-activation of additional receptors such as CC chemokine receptor 1 (CCR1). To examine the downstream effects of FcεRI-CCR1 costimulation, rat basophilic leukemia cells stably transfected with CCR1 (RBL-CCR1 cells) were sensitized and activated with antigen and/or the CCR1 ligand CC chemokine ligand (CCL) 3. Gene and protein expression were determined at 3h and 24h post-activation, respectively, using GeneChip and Luminex bead assays. Gene microarray analysis demonstrated that 32 genes were differentially regulated in response to costimulation, as opposed to stimulation with antigen or CCL3 alone. The genes most significantly up-regulated by FcεRI-CCR1 costimulation were Ccl7, Rgs1, Emp1 and RT1-S3. CCL7 protein was also expressed at higher levels 24h after dual receptor activation, although RGS1, EMP1 and RT1-S3 were not. Of the panel of chemokines and cytokines tested, only CCL2, CCL7 and interleukin (IL)-6 were expressed at higher levels following costimulation. IL-6 expression was seen only after FcεRI-CCR1 costimulation, although the amount expressed was very low. CCL7, CCL2 and IL-6 might play roles in mast cell regulation of late-phase allergic responses. 相似文献
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A variety of neurological diseases are characterized by disturbances of the blood–brain barrier (BBB) and its transporters. We recently introduced fibroblast growth factor treated cortical organotypic slice cultures of mice as a model for in vitro studies of the blood–brain barrier and have now further characterized the maintenance and function of transport-proteins typically expressed in the endothelium of cerebral blood vessels. The glucose transporter GLUT-1 is present in blood vessels of slice cultures derived from postnatal day 4 to 21 mice after 3 days in vitro. The endothelial multidrug resistance P-glycoprotein (P-gp) which is involved in the control of pharmacological substance transport across the blood–brain barrier is also maintained in blood vessels, most prominently in slice cultures derived from postnatal day 14 and 21 mice. To assess P-gp function, we tested rhodamine 123 transport in presence or absence of the P-gp inhibitor verapamil. Rhodamine 123-fluorescence accumulated rapidly in the vascular lumen both in acute slices and in slices cultured for 3 days in vitro. Our results provide evidence that endothelial transporters and their functional properties can be maintained in organotypic cortical slices cultures, thus making it an attractive model system for the study of the blood–brain barrier. 相似文献
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Maude L. Cuchiara Kelsey L. Horter Omar A. Banda Jennifer L. West 《Acta biomaterialia》2013,9(12):9258-9269
Hematopoietic stem cells (HSCs) are currently utilized in the treatment of blood diseases, but widespread application of HSC therapeutics has been hindered by the limited availability of HSCs. With a better understanding of the HSC microenvironment and the ability to precisely recapitulate its components, we may be able to gain control of HSC behavior. In this work we developed a novel, biomimetic PEG hydrogel material as a substrate for this purpose and tested its potential with an anchorage-independent hematopoietic cell line, 32D clone 3 cells. We immobilized a fibronectin-derived adhesive peptide sequence, RGDS; a cytokine critical in HSC self-renewal, stem cell factor (SCF); and a chemokine important in HSC homing and lodging, stromal derived factor 1α (SDF1α), onto the surfaces of poly(ethylene glycol) (PEG) hydrogels. To evaluate the system’s capabilities, we observed the effects of the biomolecules on 32D cell adhesion and morphology. We demonstrated that the incorporation of RGDS onto the surfaces promotes 32D cell adhesion in a dose-dependent fashion. We also observed an additive response in adhesion on surfaces with RGDS in combination with either SCF or SDF1α. In addition, the average cell area increased and circularity decreased on gel surfaces containing immobilized SCF or SDF1α, indicating enhanced cell spreading. By recapitulating aspects of the HSC microenvironment using a PEG hydrogel scaffold, we have shown the ability to control the adhesion and spreading of the 32D cells and demonstrated the potential of the system for the culture of primary hematopoietic cell populations. 相似文献
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Jonitz A Lochner K Tischer T Hansmann D Bader R 《International journal of molecular medicine》2012,30(3):666-672
To prevent de-differentiation of chondrocytes in vitro, the 3D environment, growth factors and different oxygen concentrations were considered. In this in vitro study, we quantified the influence of insulin-like growth factor (IGF)-1 and/or transforming growth factor (TGF)-β1 under differing oxygen (5/21% O(2)) levels on the proliferation and synthesis rates of hyaline extracellular matrix (ECM) components in chondrogenic pellet cultures. Human chondrocytes isolated from articular cartilage were transferred into conical tubes to form pellets. Pellets were stimulated with TGF-β1 and/or IGF-1. After 2 and 5 weeks of cultivation the DNA concentration and expression of pro-collagen type 1, type 2 and aggrecan were analysed. Under hypoxia the DNA content remained stable. In contrast, under normoxia, cells showed an increase of DNA concentration after stimulation with TGF-β1/IGF-1 and TGF-β1. Nevertheless, DNA contents under normoxia did not reach the values of hypoxic-cultivated cells. Under both culture conditions a reduced synthesis of pro-collagen type 1 could be determined. Although the expression of pro-collagen type 2 was significantly higher under normoxia, a decrease in the case of TGF-β1/IGF-1- and IGF-1-stimulated cells was observed. Under hypoxia pro-collagen type 2 contents remained stable or increased for TGF-β1/IGF-1-stimulated cells. Furthermore, incubation with growth factors resulted in aggrecan accumulation under hypoxia, while a reduced expression under normoxia could be determined for TGF-β1/IGF-1- and IGF-1-stimulated cells. Our results demonstrate that the treatment with growth factors causes differences in the expression of ECM compounds within pellet cultures. While under normoxia TGF-β1 alone leads to a positive effect of the expression of hyaline cartilage-specific ECM components, an additive effect of both growth factors was only determined under hypoxia. 相似文献
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The development and formation of the neuronal architectonics of organotypic structures of the sensorimotor cortex in rats aged 7–8 days were studied in roller cultures. Free-floating slices were cultured for 2–3 weeks. Serial paraffin sections of cultured tissue were stained with fast cresyl violet as described by Nissl. Initially planar sections of the cortex were found to change their configuration during the process of cultivation and were transformed into spherical structures, retaining the major histotypic features of cortical formations. Radially orientated pyramidal cells and fusiform neurons formed a cortical rudiment, not discriminated into layers, over the whole surface of the spherical tissue structures. In free-floating slices of the sensorimotor cortex of rats aged 7–8 days in roller cultivation, histogenetic processes continued, leading to the formation of histotypic cortical structures similar to the phylogenetically more ancient allocortical formations of the forebrain.Translated from Morfologiya, Vol. 125, No. 1, pp. 12–15, January–February, 2004. 相似文献
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Mehtap Nisari Tolga Ertekin ?zlem ?z?elik ?erife ??nar Selim Do?anay Niyazi Acer 《Surgical and radiologic anatomy : SRA》2012,34(9):825-832
Purpose
Brain development in early life is thought to be critical period in neurodevelopmental disorder. Knowledge relating to this period is currently quite limited. This study aimed to evaluate the volume relation of total brain (TB), cerebrum, cerebellum and bulbus+pons by the use of Archimedes’ principle and stereological (point-counting) method and after that to compare these approaches with each other in newborns.Methods
This study was carried out on five newborn cadavers mean weighing 2.220?±?1.056?g with no signs of neuropathology. The mean (±SD) age of the subjects was 39.7 (±1.5) weeks. The volume and volume fraction of the total brain, cerebrum, cerebellum and bulbus+pons were determined on magnetic resonance (MR) images using the point-counting approach of stereological methods and by the use of fluid displacement technique.Results
The mean (±SD) TB, cerebrum, cerebellum and bulbus+pons volumes by fluid displacement were 271.48?±?78.3, 256.6?±?71.8, 12.16?±?6.1 and 2.72?±?1.6?cm3, respectively. By the Cavalieri principle (point-counting) using sagittal MRIs, they were 262.01?±?74.9, 248.11?±?68.03, 11.68?±?6.1 and 2.21?±?1.13?cm3, respectively. The mean (±?SD) volumes by point-counting technique using axial MR images were 288.06?±?88.5, 275.2?±?83.1, 19.75?±?5.3 and 2.11?±?0.7?cm3, respectively. There were no differences between the fluid displacement and point-counting (using axial and sagittal images) for all structures (p?>?0.05).Conclusion
This study presents the basic data for studies relative to newborn’s brain volume fractions according to two methods. Stereological (point-counting) estimation may be accepted a beneficial and new tool for neurological evaluation in vivo research of the brain. Based on these techniques we introduce here, the clinician may evaluate the growth of the brain in a more efficient and precise manner. 相似文献11.
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Helen E. Gruber Gretchen L. Hoelscher Jane A. Ingram Synthia Bethea H. James Norton Edward N. Hanley Jr. 《Experimental and molecular pathology》2014
Chemokines act as important secondary inflammatory mediators which are released by cells in response to a variety of stimuli. Chemokines bind to cell surface receptors and act as second-order cytokines with specialized functions in inflammation. The role of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) (also called CCL5 (chemokine (C–C motif) ligand 5)) has received little attention to date in disc tissue. Microarray analyses of lumbar disc annulus tissue revealed that RANTES expression was significantly upregulated in more degenerated Thompson grades IV and V discs compared to expression levels in grades I, II and III discs (p = 0.032). Immunolocalization confirmed the presence of RANTES in the annulus and nucleus of the disc, and localized the RANTES receptors CCR1, CCR3 and CCR5 to cells in the disc. In vitro studies with IL-1-β and TNF-α challenges, both proinflammatory cytokines resulted in elevated levels of RANTES in conditioned media (p < 0.01); TNF-α exposure, however, produced significantly greater levels than did IL-1alpha (p < 0.0001), suggesting a differential regulation by TNF-α. Local production of RANTES in vivo by annulus and nucleus cells, and in vitro induction of RANTES by proinflammatory cytokines suggest that disc cells are primary effector cells as well as target cells, and thus can mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling. 相似文献
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《International journal of immunopharmacology》1997,19(1):39-47
In previous reports in this series, we described the growth conditions, morphology and supernatant activities of human thymic epithelial cell cultures. The human thymic epithelial cell supernatant (HTES) contained IL-6, G-CSF and M-CSF activities and exhibited a strong enhancing effect on thymocyte pro-liferative response to mitogens, which was identified as IL-6 related. The responding thymocyte population was apparently identified as PNA, mature T cells. In order to simplify further analysis of HTES activities, we selected to use a well-defined mature murine T cell clone which has a Th2 phenotype (8-5 clone). HTES induced 8-5 cell proliferation without the presence of antigen, antigen presenting cells (APC) or mitogens. This enhancing effect of HTES was completely blocked with anti hIL-6 antibody but could not be reproduced by rhIL-6 alone. Hence, IL-6 is a necessary but insufficient factor in mediating this effect. HTES induced proliferation was accompanied by endogenous IL-4 secretion from 8-5 cells. Furthermore, the proliferation was blocked by anti mIL-4 antibody, implicating IL-4 as an autocrine growth factor in this system. HTES increased also the expression of IL-2 receptor. In addition, rhIL-2 and rmIL-4 each had a synergistic effect on the proliferative response of 8-5 cells to HTES. A similar synergistic activity was demonstrated when rhIL-6 was used instead of HTES, suggesting that IL-6 regulates some of HTES activities. Our findings indicate that HTES activities, of which IL-6 is only part, are mediated via the induction of autocrine growth factors and by the regulation of T cell growth factor receptor expression. 相似文献
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Olmedilla MP Lebourg M Ivirico JE Nebot I Giralt NG Ferrer GG Soria J Ribelles JG 《Journal of biomaterials applications》2012,27(3):299-309
Two series of 3D scaffolds based on ε-caprolactone were synthesized. The pore size and architecture (spherical interconnected pores) was the same in all the scaffolds. In one of the series of scaffolds, made of pure ε-polycaprolactone, the volume fraction of pores varied between 60% and 85% with the main consequence of varying the interconnectivity between pores since the pore size was kept constant. The other scaffolds were prepared with copolymers made of a ε-caprolactone-based hydrophobous monomer and hydroxyethyl acrylate, as the hydrophilic component. Thus, the hydrophilicity and, presumably, the adhesion properties varied monotonously in the copolymer series while porosity was kept constant. A suspension of human chondrocytes in culture medium was injected in the 3D scaffolds and cultured in static conditions up to 28 days. SEM and immunofluorescence assays allowed characterizing cells and extracellular matrix inside the scaffolds after different culture times. To do that, cross sections of the scaffolds were observed by SEM and confocal microscopy. The quantity of cells inside the scaffolds decreases with a decrease of the volume fraction of pores, due to the lack of interconnectivity between the cavities. The scaffolds up to a 30% of hydrophilicity behave in a similar way than the hydrophobous; a further increase of the hydrophilicity rapidly decreases cell viability. In all the experiments production of collagen type I, type II, and aggrecan was found, and some cells were Ki-67 positive, showing that some cells are adhered to the pore walls and maintain their dedifferentiated phenotype even when cultured in three-dimensional conditions. 相似文献
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Gascoigne NR Alam SM Lin CM McGuire MV Marine S Niederberger N Redpath S Sim BC Travers PJ Yachi P Zal MA Zal T 《Immunologic research》2000,21(2-3):225-231
The kinetics of the interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) has an important
role in determining thymocyte-positive and-negative selection in the thymus, as well as in T cell activation. The α chain
of the TCR is the major player in determining how the TCR fits onto the MHC ligand, and thus has a major role in determining
whether a T cell develops as class I or class II restricted. In this article, we summarize recent data from our laboratory
and otherson the role of poly-morphism in the Vα combining site in determining MHC class restriction, and on kinetic parameters
in thymocyte selection. 相似文献
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Wang L Liu T Wang Y Cao L Nishioka M Aguirre RL Ishikawa A Geng L Okada N 《Virchows Archiv : an international journal of pathology》2007,451(5):959-966
Desmocollin 3 (Dsc3) and desmoglein 3 (Dsg3) are both transmembrane glycoproteins that belong to the cadherin family of calcium-dependent
cell adhesion molecules. β-Catenin is a member of the cadherin–catenin complex that mediates homotypic cell–cell adhesion
and is also an important molecule in the wnt signaling pathway. In this study, we examined the simultaneous expression level
of Dsc3, Dsg3, and β-catenin in oral squamous cell carcinomas (OSCCs) and normal oral epithelia using immunohistochemistry.
There was a significant correlation (p < 0.05) among the following variables in OSCCs: reduced or loss of expression of Dsc3, Dsg3, and β-catenin compared to normal
oral epithelium, reduced or loss of expression of Dsc3 and histological grade (moderately or poorly differentiated), and reduced
or loss of expression of β-catenin and lymph node metastasis. Furthermore, a positive correlation was found between reduced
or loss of β-catenin staining and reduced or loss of Dsc3 staining in lymph node metastatic cancer tissue (r = 0.734, p < 0.05). These results suggest an abnormal expression of Dsc3, Dsg3, and β-catenin induced in the progression of oral carcinomas
and that the Dsc3 expression level might be related to the regulation of β-catenin in lymph node metastasis and cell proliferation
in OSCCs. 相似文献
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The blood–brain barrier (BBB), formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. Lipopolysaccharide (LPS) is known to alter the integrity of the BBB in sepsis, although the underlying mechanism remains unknown. The aim of this study was to elucidate the molecular mechanisms underlying the disruption of the BBB in LPS-induced sepsis and to determine whether the activation of AMP-activated protein kinase (AMPK) prevents LPS-induced BBB dysfunction. The exposure of human brain microvascular endothelial cells (HBMECs) to LPS (1 μg/ml) for 4 to 24 h a week dramatically increased the permeability of the BBB in parallel with the lowered expression levels of occludin and claudin-5, which are essential to maintain tight junctions in HBMECs. In addition, LPS significantly increased the reactive oxygen species (ROS) productions. All effects induced by LPS in HBVMCs were reversed by adenoviral overexpression of superoxide dismutase, inhibition of NAD(P)H oxidase by apocynin or gain-function of AMPK by adenoviral overexpression of constitutively active mutant (AMPK-CA) or by 5-amino-4-imidazole carboxamide riboside (AICAR). Finally, the upregulation of AMPK by either AMPK-CA or AICAR abolished the levels of LPS-enhanced NAD(P)H oxidase subunit protein expressions. We conclude that AMPK activation improves the integrity of the BBB disrupted by LPS through suppressing the induction of NAD(P)H oxidase-derived ROS in HBMECs. 相似文献
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