Effect of Copper on Levels of Collagen and Alkaline Phosphatase Activity from Chondrocytes in Newborn Piglets In Vitro

The effects of different concentrations of copper on collagen content and alkaline phosphatase (AKP) activity from chondrocytes in newborn piglets were measured. Chondrocytes were cultured in media containing 15% fetal calf serum supplemented with 0, 15.6, 31.2, and 62.5?μmol/L copper in a 12-well culture plate. Collagen content and AKP activity from the chondrocyte extracellular matrix increased significantly in the culture media with 15.6, 31.2, and 62.5?μmol/L copper and was the highest at 31.2?μmol/L copper (P?相似文献   

Effect of Copper on the Expression of IGF-1 from Chondrocytes in Newborn Piglets In Vitro

The experiment was performed to evaluate the influence of copper supplementation on insulin-like growth factor-1 (IGF-1) mRNA expression in chondrocytes of newborn pigs. Chondrocytes were isolated and cultured in media containing 15% fetal calf serum supplemented with 0, 15.6, 31.2, and 62.5 μmol/L copper in 90-mm culture plate. After 0, 12, 24, and 48 h, total RNA was isolated from chondrocytes. Then, IGF-1 mRNA expression was determined by semiquantitative RT-PCR. The results showed that the expression of IGF-1 mRNA, adjusted for β-actin expression, was increased in the culture media added to 15.6, 31.2, and 62.5 μmol/L copper, respectively. In the present experiment, the optimal copper concentration and optimal culture time for the expression of IGF-1 mRNA were 31.2 μmol/L and 48 h, respectively.     

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.     

Effect of sulfate concentration on glycosaminoglycan synthesis in explant cultures of bovine articular cartilage

The effects of the sulfate- and FCS concentration on the rate of synthesis and the biochemical properties of glycosaminoglycans, synthesized in bovine articular cartilage in vitro, were studied. 20% FCS in the culture medium stimulated the rate of synthesis. In media without FCS, the rate of synthesis decends from day 0 on. The differences in incorporation rates of [35S]-sodium sulfate and 1,6-[3H]-glucosamine-HCl into glycosaminoglycans in serum free media containing 9 microM and 900 microM sulfate were used to discuss the inorganic sulfate concentration in cartilage. In 9 microM sulfate medium, the newly synthesized glycosaminoglycans contain higher levels of unsulfated disaccharides than the endogenous glycosaminoglycans. In each culture medium, the ratio 6-sulfated disaccharides to 4-sulfated disaccharides of the newly synthesized glycosaminoglycans becomes higher after 3 days in culture. The glycosaminoglycan synthesis is underestimated, when chondrocytes are cultured in media containing less than 200 microM sulfate.     

Pharmacological disruption of insulin-like growth factor 1 binding to IGF-binding proteins restores anabolic responses in human osteoarthritic chondrocytes

Insulin-like growth factor 1 (IGF-1) has poor anabolic efficacy in cartilage in osteoarthritis (OA), partly because of its sequestration by abnormally high levels of extracellular IGF-binding proteins (IGFBPs). We studied the effect of NBI-31772, a small molecule that inhibits the binding of IGF-1 to IGFBPs, on the restoration of proteoglycan synthesis by human OA chondrocytes. IGFBPs secreted by human OA cartilage or cultured chondrocytes were analyzed by western ligand blot. The ability of NBI-31772 to displace IGF-1 from IGFBPs was measured by radiobinding assay. Anabolic responses in primary cultured chondrocytes were assessed by measuring the synthesis of proteoglycans in cetylpyridinium-chloride-precipitable fractions of cell-associated and secreted ³⁵S-labeled macromolecules. The penetration of NBI-31772 into cartilage was measured by its ability to displace ¹²⁵I-labeled IGF-1 from cartilage IGFBPs. We found that IGFBP-3 was the major IGFBP secreted by OA cartilage explants and cultured chondrocytes. NBI-31772 inhibited the binding of ¹²⁵I-labeled IGF-1 to IGFBP-3 at nanomolar concentrations. It antagonized the inhibitory effect of IGFBP-3 on IGF-1-dependent proteoglycan synthesis by rabbit chondrocytes. The addition of NBI-31772 to human OA chondrocytes resulted in the restoration or potentiation of IGF-1-dependent proteoglycan synthesis, depending on the IGF-1 concentrations. However, NBI-31772 did not penetrate into cartilage explants. This study shows that a new pharmacological approach that uses a small molecule inhibiting IGF-1/IGFBP interaction could restore or potentiate proteoglycan synthesis in OA chondrocytes, thereby opening exciting possibilities for the treatment of OA and, potentially, of other joint-related diseases.     

In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera

Media (TCM-199 and Ham's F-10); sera (fetal calf serum, FCS, and buffalo estrous serum, BES); and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol 1 ug/ml) were tested to determine the efficiency of in vitro maturation and fertilization of buffalo follicular oocytes. Immature good quality cumulus-oocyte complexes (COCs) were randomly assigned to 1 of 4 experiments. Each experiment consisted of 6 treatment groups. Oocytes cultured for 24 hours in medium (TCM-199 or Ham's F-10) containing 10% FCS or BES had a significantly higher maturation rate than those in medium alone (P < 0.05). However, the maturation rate was higher in medium supplemented with 10% FCS than with 10% BES. Addition of hormones alone or in combination with sera further improved the maturation rate, but no significant difference was observed in the maturation rate among the 3 hormone-treated groups. Immature oocytes matured in the various cultures were fertilized with frozen-thawed buffalo spermatozoa. Our findings show that hormone and/or serum supplementation of TCM-199 did not improve the fertilization rate. Supplementation of Ham's F-10 with LH alone or in combination with LH + FSH + E(2) and with FCS significantly improved the fertilization rate of oocytes while medium with FSH, E(2) or no hormones did not (P < 0.05); same media supplemented with BES resulted in lower fertilization rates both in the presence or absence of hormones. The results indicate that the culture medium has a marked effect on the fertilization rate of buffalo oocytes. Ham's F-10 + LH + FSH + E(2) supplemented with FCS was the most efficacious culture system of those studied for the in vitro maturation of buffalo oocytes.     

Prostaglandin E2 up-regulates insulin-like growth factor binding protein-3 expression and synthesis in human articular chondrocytes by a c-AMP-independent pathway: Role of calcium and protein kinase A and C

Insulin-like growth factor-1, IGF-1, is believed to be an important anabolic modulator of cartilage metabolism and its bioactivity and bioavailability is regulated, in part, by IGF-1 binding protein 3 (IGFBP-3). Prostaglandin E₂ (PGE₂) stimulates IGF-1 production by articular chondrocytes and we determined whether the eicosanoid could regulate IGFBP-3 and, as such, act as a modifier of IGF-1 action at a different level. Using human articular chondrocytes in high density primary culture, Western and Western ligand blotting to measure secreted IGFBP-3 protein, and Northern analysis to monitor IGFBP-3 mRNA levels, we demonstrated that PGE₂ provoked a 3.9 ± 1.1 (n = 3) fold increase in IGFBP-3 mRNA and protein. This effect was reversed by the Ca⁺⁺ channel blockers, verapamil and nifedipine, and the Ca⁺⁺/calmodulin inhibitor, W-7. The Ca⁺⁺ ionophore, ionomycin, mimicked the effects of PGE₂ as did the phorbol ester PMA, which activates Ca⁺⁺-phospholipid-dependent protein kinase C (PKC). Cyclic AMP mimetics, such as forskolin, IBMX, Ro-20-1724, and Sp-cAMP, inhibited the expression and synthesis of the binding protein. PGE₂ did not increase the levels of cAMP or protein kinase A (PKA) activity in chondrocytes. The PGE₂ secretagogue, IL-1β, down-regulated control levels of IGFBP-3 which could be completely abrogated by pre-incubation with the tyrosine kinase inhibitor, erbstatin, and partially reversed (50 ± 8%) by KT-5720, a PKA inhibitor. These observations suggested that PGE₂ does not mediate the effect of its secretagogue and that IL-1β signalling in chondrocytes may involve multiple kinases of diverse substrate specificities. Dexamethasone down-regulated control, constitutive levels of IGFBP-3 mRNA and protein eliminating the previously demonstrated possibility of cross-talk between glucocorticoid receptor (GR) and PGE₂ receptor signalling pathways. Taken together, our results suggest that PGE₂ modulates IGFBP-3 expression, protein synthesis, and secretion, and that such regulation may modify human chondrocyte responsiveness to IGF-1 and influence cartilage metabolism. © 1996 Wiley-Liss, Inc.     

Mutual regulation between IGF-1R and IGFBP-3 in human corneal epithelial cells

The insulin-like growth factor type 1 receptor (IGF-1R) is part of the receptor tyrosine kinase superfamily. The activation of IGF-1R regulates several key signaling pathways responsible for maintaining cellular homeostasis, including survival, growth, and proliferation. In addition to mediating signal transduction at the plasma membrane, in serum-based models, IGF-1R undergoes SUMOylation by SUMO 1 and translocates to the nucleus in response to IGF-1. In corneal epithelial cells grown in serum-free culture, however, IGF-1R has been shown to accumulate in the nucleus independent of IGF-1. In this study, we report that the insulin-like growth factor binding protein-3 (IGFBP-3) mediates nuclear translocation of IGF-1R in response to growth factor withdrawal. This occurs via SUMOylation by SUMO 2/3. Further, IGF-1R and IGFBP-3 undergo reciprocal regulation independent of PI3k/Akt signaling. Thus, under healthy growth conditions, IGFBP-3 functions as a gatekeeper to arrest the cell cycle in G0/G1, but does not alter mitochondrial respiration in cultured cells. When stressed, IGFBP-3 functions as a caretaker to maintain levels of IGF-1R in the nucleus. These results demonstrate mutual regulation between IGF-1R and IGFBP-3 to maintain cell survival under stress. This is the first study to show a direct relationship between IGF-1R and IGFBP-3 in the maintenance of corneal epithelial homeostasis.     

Production of nerve growth-stimulating factor(s) from chick embryo heart cells : Use of Cytodex 3 microcarriers and serum-free media

Medium conditioned by embryonic chick heart cells is known to support extensive neurite outgrowth from autonomic and sensory neurons. In the present report we describe the use of microcarrier cell culture with serum-free media to scale up the production of the nerve growth-stimulating factors. A growth medium composed of DME /F10 supplemented with insulin, transferrin, human serum albumin and fibronectin in combination with a low molecular weight (MW) fraction of fetal calf serum (FCS) or a mixture of FGF, dexamethasone, calmodulin and thrombin supported the heart cell proliferation at a rate similar to that of medium with 10% FCS. Furthermore, the level of successively accumulated nerve growth activity measured in a bioassay with sympathetic ganglia proved to be nearly equivalent to what was obtained when cells were grown in medium containing serum. The results confirm the potential of microcarrier cell culture in serum-free media for the production and subsequent recovery of a specific cell product.     

Enhanced Efficiency of In Vitro Rootstock Micro-propagation Using Silica-Based Nanoparticles and Plant Growth Regulators in Myrobalan 29C (Prunus cerasifera L.)

Different experiments were conducted to establish and optimize an efficient in vitro micropropagation protocol for Myrobalan 29C rootstocks. Disinfection of initial explants with AgNPs (2.5%) reduced the needed amount of NaClO (5.0%) by half. The highest rates of induced active buds were obtained in the DKW (90.63%), MS (86.67%), modified MS (82.22%), and WPM (78.15%) culture media supplemented with BAP (2.22 μmol L^?1)?+?GA₃ (2.88 μmol L^?1)?+?IBA (0.05 μmol L^?1)?+?Fe-EDDHA (228.72 μmol L^?1). The highest quality of the proliferated shoots (5.0) was also achieved using DKW medium. Inclusion of GA₃ (5.76 μmol L^?1), Fe-EDDHA (114.36–228.72 μmol L^?1), or BAP (2.22 μmol L^?1) were also able to enhance the rate of shoot multiplication. Compared to the agar-solidified culture system, the established shoots proliferated more efficiently when immersed by bioreactor in the liquid DKW culture medium on a regular basis. Exogenous application of silica-based nanoparticles (NPs) including the chemically synthesized silica NPs (TSiO₂ NPs, 1.0 ppm), rice husk derived biogenic silica NPs (RSiO₂ NPs, 10.0 ppm), or amine modified silica NPs (ASiO₂ NPs, 10.0 ppm) to the multiplication medium increased the number of regenerated lateral shoots by 520%, 360%, and 349%, respectively. Proliferated shoots with well-developed root system were obtained from the rooting medium supplemented with 19.68 μmol L^?1 IBA. Our results indicated that the rootstocks of Myrobalan 29C could be efficiently propagated under in vitro condition providing proper culture medium and optimal concentrations of additives and plant growth regulators were adopted.

     

Defined media for vitrification,warming, and rehydration: effects on post-thaw protein synthesis and viability of in vitro derived ovine embryos

The purpose of this study was to assess the viability (rates of re-expanding and hatching in vitro), of in vitro derived ovine blastocysts using vitrification and warming/rehydration media containing fetal calf serum (20% FCS) or polyvinyl alcohol (0.1% PVA), and the incorporation of labelled methionine in protein synthesised during the first 4h after cryopreservation. In experiment 1, after 60 h culture in TCM-199 supplemented with 10% FCS, the hatching rates of blastocysts that had been vitrified, warmed, and rehydrated in media containing only PVA (p/p) were significantly (P<0.05) lower than those vitrified in medium containing PVA with warming and rehydration in medium containing FCS (p/s). Blastocysts that were vitrified in medium containing FCS and warmed and rehydrated in medium with PVA (s/p) had hatching rates that were significantly lower (P<0.01) than those vitrified, warmed, and rehydrated in media with only FCS (s/s). After warming, the number of dead cells in the p/p group was significantly (P<0.05) lower than in all other groups. In experiment 2, the [35S]methionine uptake by embryonic cells of the s/p group was significantly (P<0.01) higher than in other groups. The incorporation of labelled methionine into newly synthesised proteins was significantly lower in the p/p group (P<0.01) than in all other groups. No differences in the newly synthesised proteins were observed between groups. In conclusion, these results suggest that it is possible to replace serum with defined macromolecules in vitrification and warming/rehydration media for in vitro derived ovine blastocysts but this leads to a decrease in viability and a reduction in protein synthesis after warming.     

Modulations of the epithelial phenotype and functional activity of cultured bovine tracheal gland cells: dependence on the culture medium and passage number.

Bovine tracheal gland (BTG) cells in culture show an epithelial-fibroblastoid transition after several passages. To investigate these BTG cell phenotype changes, we studied the effects of both the culture medium and passage number on the expression of epithelial cytoskeletal proteins and glandular serous cell markers. We also analyzed the intracellular cAMP level in the basal state and after adrenergic stimulation. Three culture media were used: 1) serum-free defined medium (SFDM); 2) medium supplemented with 2% Ultroser G; and 3) medium supplemented with 10% fetal calf serum (FCS). Using immunofluorescence microscopy, we showed that, in the first 4 passages whatever the culture conditions, BTG cells expressed immunoreactivities to cytokeratin filaments and desmoplakins I and II, whereas vimentin filaments were not detected. After four passages, BTG cells cultured in 10% FCS or 2% Ultroser G became progressively fibroblastoid and showed immunoreactivities to both vimentin and cytokeratin intermediate filaments. No immunoreactivity to vimentin filaments was observed on BTG cells cultured in a SFDM. Using biochemical analysis, we showed that basal levels of cAMP in cultured BTG cells and lysozyme secretion by these cells vary according to the culture medium and passage number. It was higher in BTG cells cultured in a SFDM compared to that recovered from cells cultured in medium supplemented with Ultroser G or FCS. Whatever the culture medium, BTG cells responded to stimulation by isoproterenol. However, the results of stimulation in a SFDM were higher than in Ultroser G or FCS supplemented medium. We conclude that the BTG epithelial cell organization and the regulation of biosynthesis of secretory proteins by these cells in culture depend on both the culture medium and passage number.(ABSTRACT TRUNCATED AT 250 WORDS)     

视黄酸和亚硒酸钠对肝癌细胞某些表型逆转的加和作用

 人肝癌细胞株SMMC-7721经1μmol/L视黄酸和或2.5μmol/L亚硒酸钠处理后,膜上纤维连接蛋白沉着量逐日上升,且较相应天数的对照组细胞增加,而甲胎蛋白分泌量和~3H-TdR参入率被明显抑制。视黄酸和亚硒酸钠同时处理的联合组作用强度接近于两者单独使用时作用强度的加和。对以上结果和视黄酸及亚硒酸钠使肝癌细胞接触抑制恢复及表型逆转的关系作了讨论。     

IGFBP-3 Inhibits the Proliferation of Neural Progenitor Cells

Insulin like growth factor-1 (IGF-1) plays an important role in the proliferation and differentiation of neural progenitor cells. The effects of IGF-1 can be regulated by insulin like growth factor binding protein-3 (IGFBP-3) which can either inhibit or stimulate the proliferation of cells depending on the expression of proteases that can release IGF-1 from IGF1-IGFBP3 complex. Although IGF-1 is essential for the development of brain, both IGFBP-3 and IGF-1 are elevated in the brains of children younger than 6 months of age. Likewise, IGFBP-3 is also upregulated following cerebral ischemia and hypoxia. However, the role of IGFBP-3 in neurogenesis is not clear. Using an in vitro culture system of rat neural progenitor cells, we demonstrate that IGFBP-3 specifically regulates the IGF-1 mediated neural progenitor cell proliferation via down regulation of phopho-Akt, and cyclin D1. In addition, IGFBP-3 also decreased the content of nestin in the neural progenitor cells indicating its potential role in neurogenesis.