心室成纤维细胞条件培养液促进成纤维细胞胶原合成和增殖

采用心室成纤维细胞条件培养液培养心室成纤维细胞,通过测定[^3H]－脯氨酸（[^3H]－proline）的掺入率来了解心室成纤维细胞总胶原合成速率,通过测定[^3H]－胸腺嘧啶核苷（[^3H]－TdR）的掺入率以及c-fos基因的表达丰度来了解心室成纤维细胞的增殖速率。结果显示：心室成纤维细胞条件培养液（FCGM）能增加细胞自身的[^3H]－proline的掺入率和[^3H]－TdR的掺入率,并具有剂量依赖性;FCGM也能促进细胞自身c-fos基因的表达,刺激后1h达高峰。ETA受体拮抗剂BQ123能部分阻断FCGM增加成纤维细胞胶原合成的增殖作用,而AT1受体拮抗剂CV11974和α肾上腺素受体拮抗剂regitin无此效果。结果提示：心室成纤维细胞具有自分泌功能,能分泌内皮素等生物活性物质,促进成纤维细胞胶原的合成和增殖。     

Effects of selective prostaglandin EP2 and EP4 receptor agonists on bone resorption and formation in fetal rat organ cultures

Prostaglandin E2 (PGE2) can stimulate bone resorption and formation through receptors which activate adenylyl cyclase. We examined the effects of selective EP2 and EP4 agonists (EP2A and EP4A) on the release of previously incorporated 45Ca from fetal rat long bones and the incorporation of [3H]-proline or [3H]-thymidine (TdR) in fetal rat calvaria to assess the relative effects of these selective agonists on bone formation and resorption. Only EP4A was effective in increasing 45Ca release. Both agonists increased [3H]-TdR incorporation and [3H]-proline incorporation into calvariae, particularly in the presence of cortisol.     

Glial conditioned media inhibit the proliferation of cultured rat cerebellar astrocytes

Conditioned medium (CM) obtained from rat cerebellar astrocytes cultured in a serumcontaining medium was able to inhibit [³H]thymidine incorporation into proliferating astrocytes, when compared to fresh medium. This effect could be attributed to two fractions of the CM with different molecular weights. The low molecular weight fraction (M_r<1,000) inhibited the cellular transport of the labeled precursor, without significantly affecting cell proliferation. The high molecular weight fraction (M_r>10,000) showed a strong inhibitory effect on astrocyte proliferation, which was documented using different assay techniques: i) [³H]thymidine incorporation performed in conditions preventing the effects of CM on transport; ii) [³H]thymidine autoradiography; iii) determination of the DNA content of the cultures. The inhibitory activity was present in media conditioned by non proliferating astrocytes treated with the antimitotic cytosine arabinoside, but not in media conditioned by neuron-enriched cultures nor in a chemically defined (N2) CM. The antiproliferative activity of astrocyte CM could be due either to a rapid depletion of mitogenic factors present in serum, or, to a secretion of growth inhibitory factor(s) by cultured astrocytes.Special Issue Dedicated to Dr. Abel Lajtha.     

Synergistic effect of amino acids on production of lymphocyte proliferation-suppressing substances by rat intestinal epithelial cells

Conditioned culture medium of rat small intestinal epithelial cells suppressed proliferation of spleen lymphocytes stimulated with concanavalin A (approx. 10% of its control [³H]thymidine incorporation) whereas conditioned phosphate-buffered saline of the epithelial cells did not. On the other hand, conditioned saline of the epithelial cells exposed to a mixture of total 22 amino acids at their concentrations in the culture medium suppressed the proliferation (approx. 45% of its control [³H]thymidine incorporation). Neither conditioned saline of the epithelial cells exposed to other medium components nor lysates of freshly harvested epithelial cells suppressed the proliferation. Thus, amino acids synergistically stimulated intestinal epithelial cells to produce substances with the ability to suppress lymphocyte proliferation.     

LH biosynthesis and secretion in rat anterior pituitary cell cultures: stimulation of LH glycosylation and secretion by GNRH and an agonistic analogue and blockade by an antagonistic analogue.

In rat anterior pituitary cell cultures GnRH (1nM) stimulated a progressive increase in LH release into the medium from 1 to 8 h of incubation, while cellular LH showed a corresponding decrease. GnRH (1nM) neither modified the uptake nor the incorporation of [³H]-glucosamine and [³H]-proline into total protein. The incorporation of [³H]-proline into cellular LH also was unaffected by GnRH. In contrast, GnRH stimulated a 3 to 4-fold increase in [³H]-glucosamine incorporation into cellular LH. The agonistic analogue, [des GlyNH₂¹⁰]-LHRH ethylamide, mimicked the GnRH effects and was 5 to 6 times more potent than GnRH. The antagonistic analogue, [D-Phe², D-Phe⁶]-LHRH blocked the GnRH-stimulated effects. These results suggest that GnRH and agonistic analogues may preferentially regulate turnover or synthesis of the carbohydrate moiety of LH.     

The effect of insulin on collagen production in isolated chondrosarcoma chondrocytes

Insulin stimulated the incorporation of [³H]-proline into collagen of freshly isolated chondrosarcoma chondrocytes. In addition, insulin enhanced incorporation of radiolabeled precursors into general protein, RNA, and proteoglycans. The stimulatory effects on collagen and non-collagen protein occurred with 2 h while the effects on RNA and proteoglycan were observed at 5 h and 8 h, respectively. All responses were obtained with physiological concentrations (1–2 nM) and were proportional to concentrations to 2 uM. These results demonstrate that insulin, in addition to exerting a general anabolic action on chondrocytes, also stimulates the incorporation of [³H]-proline into a specific protein, i.e., collagen. The latter effect should provide a useful means to probe insulin's mechanism of action.     

A correction: inhibitory activity in the conditioned medium of embryonic chick bones is due to thymidine

Earlier studies from this laboratory suggested that embryonic chick bones in organ culture released into the culture medium a specific inhibitor of bone cell proliferation as defined by inhibition of [3H]TdR incorporation into DNA. Dialysis and membrane ultrafiltration experiments suggested that the inhibitory substance (IS) had a molecular weight between 6000 and 14,000. However, subsequent studies on the purification of IS have revealed that the inhibitory activity in bone-conditioned medium is of lower molecular weight and has several properties in common with thymidine (TdR): (1) IS coeluted with [3H]TdR upon gel filtration chromatography on Sephadex G-10. (2) IS bound to charcoal but not to cation or anion exchange resins. (3) Bone-conditioned medium decreased incorporation of [3H]TdR into the free [3H]TdR pool of cells in monolayer culture. (4) Conditioned medium inhibited [3H]TdR incorporation into [3H]thymidine monophosphate in a reaction catalyzed by thymidine kinase. The equivalent concentration of TdR in conditioned medium as estimated by thymidine kinase assay was sufficient to account for the reduction in [3H]TdR incorporation into bone cell DNA. No evidence was found for a specific inhibitor of bone cell proliferation other than TdR. Hence we conclude that the inhibitory effect of IS is due to dilution of [3H]TdR by nonradioactive TdR. Furthermore, media conditioned by several tumor cell lines also contained a low-molecular-weight component which inhibited [3H]TdR incorporation. The results suggest that organ- and cell-conditioned media can contain significant concentrations of TdR which can artifactually inhibit [3H]TdR incorporation in cell proliferation assays.     

Genistein abolishes nucleoside uptake by cardiac fibroblasts

Genistein, a soy isoflavone, is reported to exert significant beneficial action in several human disorders, which has generated immense interest in the mechanisms underlying its effects on diverse cellular processes. It has anti-proliferative action on many cell types, an effect generally attributed to tyrosine kinase inhibition. In this study, genistein was found to cause total inhibition of [³H]-thymidine incorporation into DNA and a modest reduction in [³H]-proline incorporation into protein in primary cultures of cardiac fibroblasts. The decrease in [³H]-thymidine incorporation was not associated with a decrease in cell proliferation but correlated exactly with low intracellular levels of [³H]-thymidine. Genistein dramatically reduced [³H]-thymidine but not [³H]-proline uptake by these cells in which the equilibrative nucleoside transporter may be the major route of nucleoside uptake. The effect was irreversible and was demonstrable in pulmonary fibroblasts as well. The findings suggest that nucleoside uptake mechanisms may be a novel target of genistein action in cardiac fibroblasts and point to serious limitations in using genistein to assess the role of tyrosine kinase in cell proliferation by the standard technique of [³H]-thymidine incorporation.     

Detection of glucocorticoid sensitive secretory proteins from human melanoma cells

Summary NEL-M1 human melanoma cells have been established to grow in Ham's F12 medium in the absence of serum, hormones, and exogenous growth factors. Conditioned medium from NEL-M1 cultures stimulates growth of these same cells whereas glucocorticoids retard growth in the presence and absence of conditioned medium. Because recent reports indicate that glucocorticoids inhibit the synthesis of growth factors from a variety of cell types, we hypothesized that glucocorticoids may be inhibiting growth of NEL-M1 cells by either suppressing the synthesis of the autocrine growth factor or regulating other secretory proteins that may inhibit the activity of the autocrine growth factor. Initial studies were done to clearly show that NEL-M1 cells were growth inhibited, both in vivo and in vitro, when exposed to the synthetic glucocorticoid, triamcinolone acetonide (TA). When NEL-M1 cells were injected into nude mice and treated with TA (100 μg per mouse per week) a 67% reduction in tumor mass was observed compared to the control group over a 5-wk growth period. Additional studies show that in a serum-free defined medium TA (100 nM) inhibited growth of MEL-M1 cells by 56% after 6 d in culture. At this same time TA was shown to affect the expression of several proteins secreted from these cells. TA treatment resulted in the appearance of a 125 000 molecular weight protein, suppression of the synthesis-secretion of three proteins (37 000, 57 000, and 76 000 molecular weight) and enhanced expression of a 60 000-molecular weight protein. However when NEL-M1 cells were cultured in conditioned medium from TA-treated cells, a stimulation in both [³H]thymidine incorporation into DNA and cell proliferation was observed. When the conditioned medium was fractionated by Amicon ultrafiltration, the growth stimulatory activity was found in the <10 000 molecular weight fraction. These results demonstrate that glucocorticoids, as a single agent, inhibit the growth of NEL-M1 human melanoma cells. However, this growth inhibition by glucocorticoids may not be through the regulation of the synthesis-secretion of the autocrine growth factor. Furthermore, the data suggest that the glucocorticoid-sensitive secretory proteins may not be directly involved in the in vitro regulation of NEL-M1 cellular growth.     

染料木黄酮对成骨细胞增殖分化的影响

目的：研究染料木黄酮对体外培养乳鼠颅盖骨成骨细胞增殖分化的影响。方法：取乳鼠颅盖骨,采用胶原-胰蛋白酶消化法,进行颅骨成骨细胞培养,取第二代成骨细胞,添加10^-5～10^-7mol／L染料木黄酮,在CO2孵箱中培养48h和72h后MTT比色法测定细胞增殖,培养72h采用^3H-TdR和^H-Pro掺入实验测定DNA和胶原合成。用试剂盒检测细胞裂解液碱性磷酸酶(ALP)活性。结果：染料木黄酮明显增加成骨细胞MTT的吸光度值、^3H-TdR和^3H-Pro的掺入,增加成骨细胞碱性磷酸酶活性。结论：染料木黄酮促进体外培养的乳鼠颅盖骨成骨细胞DNA和胶原的合成,促进增殖和分化。     

Direct oxidation of lymphocytes by chromic acid does not induce blastogenesis

Blastogenic and cytotoxic effects of hexavalent chromium were evaluated by direct, 2 and 20 min oxidation of lymphocytes by 10.0 to 0.0005 mM CrO₃ at 0°C. Oxidized cells exhibited concentration-dependent cytotoxicity and the inhibition of tritiated thymidine incorporation rates. When lymphocytes were oxidized first by 1.0 mM periodic acid (H₅IO₆) and thereafter by 1.0 mM CrO₃, the viability and [³H]-TdR incorporation rates of sequentially oxidized cells were identical to the corresponding indicators of lymphocytes oxidized only by CrO₃. The reversal of the oxidation sequence restored [³H]-TdR incorporation to control levels and increased cell survival. It is therefore concluded that direct interaction of hexavalent CrO₃ with plasma membrane of lymphocytes results in concentration-dependent cytotoxicity and the inhibition of [³H]-TdR incorporation, but it does not induce blastogenesis.     

Degradation of extracellular thymidine by cultured hepatocytes from rainbow trout (Salmo gairdneri)

1. Trout hepatocytes cultured as attached monolayers had low rates of [3H]-thymidine ([3H]-TdR) incorporation during replicative or repair synthesis of DNA. 2. Within 2 hr, most [3H]-TdR was metabolized by trout hepatocytes to a major product that eluted in advance of intact [3H]-TdR on Sephacryl S-200 columns. 3. Metabolism of [3H]-TdR by trout hepatocytes rapidly destroyed its ability to label replicating indicator cultures of proliferating rat hepatocytes. 4. These studies demonstrate that [3H]-TdR tracer assays for DNA synthesis cannot be reliably used in cultured trout hepatocytes which catabolize thymidine much more rapidly than do rat hepatocytes.     

Endocardial endothelial celsl stimulate proliferation and collagen synthesis of cardiac fibroblasts

Given that vascular endothelial cells play an important role in the modulation of vascular structure and function, we hypothesized that endocardial endothelial cells (EECs) may have a modulator role in regulating the cardiac interstitial cells. Endocardial endothelial cells were isolated from freshly collected pig hearts and cardiac fibroblasts were isolated from 3- to 4-d-old Wistar rats. Fibroblasts were cultured in the presence or absence of conditioned medium from EECs. Proliferation of cardiac fibroblasts was measured by the incorporation of [³H]-Thymidine and collagen synthesis was assayed by the incorporation of [³H]-proline. To determine the involvement of signaling mediators, in separate experiments, cardiac fibroblasts were incubated with BQ123 (selective ET_A receptor antagonist), PD142893 (nonselective ET_A/ET_B receptor antagonist), Bis-indolylmaleimide (PKC inhibitor), PD 098059 (MEK inhibitor), or neutralizing anti-transforming growth factor (TGF)-β-antibody. Endocardial endothelium-derived factors endothelin (ET)-1, TGF-β, and Angiotensin (Ang)-II in the conditioned medium were assayed by enzyme-linked immunosorbent assay using commercially available kits. We report here evidence that suggest that endocardial endothelial cells stimulate both proliferation and collagen synthesis of cardiac fibroblasts. The response seems to be mediated by endothelin through its ET_A receptor. Our results also indicate that protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways are essential for the EEC-induced proliferation of cardiac fibroblasts.     

Stimulation of DNA synthesis,cell multiplication,and ornithine decarboxylase in 3T3 cells by multiplication stimulating activity (MSA)

Multiplication stimulating activity (MSA) has been purified from the conditioned media of rat liver cells in culture by a modification of the procedure of Dulak and Temin. Purified MSA stimulates [³H] thymidine incorporation into DNA in subconfluent, serum starved 3T3 cells. Cell cycle analysis by the flow microfluorometer shows that the [³H] thymidine incorporation data reflects DNA synthesis. MSA also stimulates the multiplication of serum starved subconfluent 3T3 cells. MSA is approximately 10-fold less active in 3T3 cells than in chick embryo fibroblasts in stimulating [³H] thymidine incorporation into DNA. MSA causes a 2–10-fold increase in ornithine decarboxylase (ODC) activity in 3T3 cells and the dose response curve parallels the dose response curve for [³H] thymidine incorporation into DNA. The Km of ODC for ornithine is 0.12 mM. There is a 30% decrease in the activity of ornithine transaminase (OTA) during the time period in which MSA causes an increase in ODC activity. Insulin also stimulates [³H] thymidine incorporation into DNA, cell multiplication and ODC activity over the same concentration range as shown for MSA, however, the extent of stimulation by insulin is less than that observed following MSA addition.     

Effect of the neuropeptide substance P on the rat bone marrow-derived osteogenic cells in vitro

Substance P containing, thin, sensory nerve fibres have been demonstrated in bone and bone marrow. However the role of substance P in bone tissue is not fully understood. We investigated the effects of substance P on the growth and development of rat bone marrow-derived osteogenic cells in vitro. To examine this, the marrow-derived osteogenic cells were treated from 3rd to 6th day of subculture with substance P at concentrations 10(-10), 10(-9) and 10(-8)M. [(3)H]-thymidine, L-2,3-[(3)H]-proline incorporation, protein accumulation, alkaline phosphatase activity, and calcium deposition were measured in cultures. Substance P slightly stimulated [(3)H]-thymidine incorporation at 10(-10) M. Protein accumulation and L-2,3-[(3)H]-proline incorporation were enhanced in a dose dependent manner. Simultaneous application of spantide, a substance P receptor antagonist, could not block substance P-induced L-2,3-[(3)H]-proline incorporation probably because of statistically significant effect of spantide itself. Calcium deposits were significantly lower (about 30%) in cultures treated with SP. This effect was probably due in part by the fall in alkaline phosphatase activity which in substance P treated cultures was decreased about 17%. Our results indicate that substance P could be one of the factors modulating bone metabolism.     

Overexpression of ovine insulin-like growth factor-I stimulates autonomous autocrine or paracrine growth in bovine mammary-derived epithelial cells.

To test the hypothesis that insulin-like growth factor-I (IGF-I) affects the growth of bovine mammary epithelial cells through an autocrine and/or paracrine pathway, a cell line (MD-IGF-I) was originated from MAC-T cells by cotransfection with a construct containing the cDNA for an ovine exon 2-encoded prepro-IGF-I under control of the mouse mammary tumor virus-long terminal repeat promoter. Clone MD-IGF-I contained multiple copies of the plasmid integrated into the genome, expressed the highest level of IGF-I mRNA, and secreted radioimmunoactive IGF-I into the medium. The mitogenic activity of MD-IGF-I cells was stimulated 80% by dexamethasone (DEX). The total DNA in MD-IGF-I cells was 2.5-fold higher than that in parental MAC-T cells in the presence of DEX. Conditioned medium from MD-IGF-I cells, induced with DEX, stimulated [3H]thymidine incorporation into DNA of MAC-T cells and uninduced MD-IGF-I cells. These data provide evidence that IGF-I was secreted into medium by MD-IGF-I cells. It is suggested that IGF-I can stimulate the growth of mammary epithelial cells by an autocrine and/or paracrine mode of action. The MD-IGF-I cell line may be a suitable system to study translational and posttranslational modifications of IGF-I peptides.     

Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

The effects of monocyte-conditioned medium and interleukin 1 on the synthesis of collagenous and non-collagenous proteins by mouse bone and human bone cells in vitro

Cultural adherent human mononuclear cells produce factor(s) which stimulate the release of calcium from new-born mouse calvaria in organ culture. This stimulation of bone resorption is accompanied by an inhibition of the incorporation of [³H]proline into collagen which is independent of increased prostaglandin production by the bone. When human osteoblast-like cells are treated with conditioned medium from human mononuclear cells, collagen accounts for a decreased proportion of the protein synthesised. This effect on matrix synthesis is not accompanied by an inhibitory action of the monocyte-conditioned medium preparations on net cell proliferation. In human osteoblast-like cell cultures, partially purified human interleukin 1 also inhibits the production of the bone-specific protein osteocalcin in a dose-dependent fashion. These observations are consistent with the hypothesis that products of human monocytes similar to, or identical with, human interleukin 1 may be important regulators of bone metabolism and may contribute to the bone loss seen in diseases such as chronic rheumatoid arthritis.     

Heat shock modulates the ability of A-549 cell line from human lung adenocarcinoma to regulate the intensity of DNA and protein biosynthesis by an autocrine mechanism]

A-549 cells of human lung adenocarcinoma were subjected to heat shock (30 min, 44 degrees C) which caused substantial decreases in the rates of biosynthesis of the great bulk of cellular proteins with simultaneous increases in the synthesis rates of the 70 kDa protein predominantly localized in cell cytosol. By the 6th hour after the heat shock cessation this protein synthesis reached its maximum; by the 18th hour it was no longer detectable, while the protein itself was not denatured. During the recovery after the heat shock the ability of the serum-free culture medium conditioned by A-549 cells in autocrine regulation of [3H]thymidine incorporation into DNA and [3H]leucine incorporation into proteins changed also. The conditioned medium obtained within 1-3 hours after the heat shock did not influence the intensity of DNA synthesis, while the medium obtained 4-48 hours after the heat shock stimulated this process, the maximal effect (3.3-fold stimulation) being observed in the case of the 48-hour conditioned medium. Temporary (1 hour) acidification of the conditioned media down to pH 2.0 resulted in complete inhibition of the stimulating activity. Besides, these media acquired an ability to inhibit [3H]thymidine incorporation into the DNA of tracer cells. Study of effects of conditioned media on the rate of [3H]leucine incorporation into A-549 cell proteins revealed that the media obtained 1-4 hours after the heat shock inhibited this process, while the media obtained 6-18 hours thereafter stimulated it 1.2-2.1-fold. In the test systems under study temporary acidification of the media increased their stimulating influence on [3H]leucine incorporation into cellular proteins.     

Comparative studies between freshly isolated and spontaneously immortalized bovine granulosa cells: Protein secretion,steroid metabolism,and responsiveness to growth factors

A bovine granulosa cell line (BGC-1) has been obtained by spontaneous immortalization of primary cultures. BGC-1 cells have retained some characteristics of primary cultures, such as the hormonal regulation of fibronectin biosynthesis. In the present study we have compaed BGC-1 cells and primary cultures of bovine granulosa cells in terms of protein secretion, steroid metabolism, and mitogenic responses to growth factors. The pattern of protein secretion in BGC-1 cells was qualitatively similar to that of primary cultures. The main differences were a higher proportion of fibronectin and the relative amounts of several other unidentified proteins. Progesterone levels in BGC-1 cultures were undetectable. When BGC-1 cells and primary cultures were incubated with [³H]-pregnenolone, the former showed a lower conversion rate to progesterone. In contrast, the conversion rate of [³H]-progesterone to 5α-reduced metabolites was markedly increased in BGC-1 cells. We also examined the effects of epidermal growth factor (EGF), insulin like growth factor-1 (IGF-I), and transforming growth factor-b? (TGF-b?) on DNA synthesis under serum-free conditions. Both primary cultures and BGC-1 cells exhibited a stimulatory response to EGF and IGF-I on [³H]-thymidine incorporation. Neither BGC-1 cells nor primary cultures showed a significant response to TGF-b? when added alone. However, in the presence of a combination of EGF and IGF-I, TGF-b? displayed an inhibitory effect on primary cultures while it stimulated DNA synthesis in BGC-1 cells even further. The addition of conditioned medium from BGC-1 cells (BGC-1-CM) stimulated DNA synthesis on primary cultures to a greater extent than the addition of conditioned medium from primary cultures. These results suggest that BGC-1 cells may be a useful model to study the regulation of granulosa cell function during the period previous to the preovulatory stage of follicular development. The differential responses of the immortalized cells to growth regulators may offer some clues on the mechanisms that control cell proliferation in normal tissues. © 1995 Wiley-Liss, Inc.