共查询到20条相似文献,搜索用时 62 毫秒
1.
Olga L Huk David J Zukor John Antoniou Alain Petit 《Journal of orthopaedic research》2003,21(1):81-87
The demonstration that one of the mechanisms of action of bisphosphonates (BPs) is the induction of osteoclast and macrophage apoptosis, suggests a potent therapeutic role for the BPs and other apoptosis-modulating agents in the management of periprosthetic osteolysis. The purpose of this study was to improve our understanding of the basic underlying molecular events leading to the inhibitory effect of pamidronate on the macrophage response to ultra-high-molecular-weight polyethylene (UHMWPE) particles. Murine J774 macrophages were incubated for 0-72 h in the presence of UHMWPE particles and/or pamidronate. TNF-alpha release was measured by ELISA while poly(ADP-ribose)polymerase (PARP) expression was measured by Western blot. DNA was analyzed on agarose. The appearance of PARP fragment and the fragmentation of DNA were used as markers of apoptosis. We observed a dose-dependent response to UHMWPE particles with TNF-alpha release reaching 4, 10, and 19 times control with 10, 25, and 125 particles/macrophage, respectively. UHMWPE particles (25 particles/macrophage) stimulate TNF-alpha release by a factor of 10, 7, and 6 after 24, 48, and 72 h, respectively, indicating a rapid stimulating effect of UHMWPE particles on TNF-alpha release. Our results also showed that at 10 particles/macrophage, pamidronate inhibits UHMWPE-induced TNF-alpha release by 12%, 14%, and 23% respectively after 24, 48, and 72 h (p<0.05 vs. 24 and 48 h). With 25 particles/macrophage, the inhibition of TNF-alpha reached 9%, 12%, and 15% after 24, 48, and 72 h (p<0.05 vs. 24 h), respectively. There is no significant difference between the inhibition by pamidronate of TNF-alpha release induced by 125 particles/macrophages at 24, 48, and 72 h. When cells are pre-incubated for 48 h with pamidronate prior to addition of UHMWPE particles for 24 h, we observed an increased inhibition of TNF-alpha compared to the co-incubation protocol. The inhibiting effect of pamidronate reaches 56% when pre-incubated with macrophages prior to incubation with 10 particles of UHMWPE/macrophage (p<0.05 vs. co-incubation).Co-incubation of pamidronate with UHMWPE particles also led to the appearance of the proteolytic PARP fragment after 24 h incubation. We also demonstrated the stimulation of DNA fragmentation (DNA laddering) after 48-72 h with pamidronate. The proteolytic cleavage of PARP, an early event in the induction of apoptosis, precedes the inhibition of UHMWPE particle-induced TNF-alpha release by pamidronate whereas the fragmentation of DNA, a late apoptotic event, parallels this inhibition. Our results suggest the induction of macrophage apoptosis is associated with the inhibitory effect of pamidronate on TNF-alpha release. There is a need for the development of medical management of periprosthetic osteolysis. The demonstration that drugs such as pamidronate induce specific apoptosis-related pathways in macrophages contributes data for a rational approach in the treatment and/or prevention of periprosthetic osteolysis. 相似文献
2.
Cytotoxicity and macrophage cytokine release induced by ceramic and polyethylene particles in vitro 总被引:5,自引:0,他引:5
Catelas I Petit A Marchand R Zukor DJ Yahia L Huk OL 《The Journal of bone and joint surgery. British volume》1999,81(3):516-521
Although the response of macrophages to polyethylene debris has been widely studied, it has never been compared with the cellular response to ceramic debris. Our aim was to investigate the cytotoxicity of ceramic particles (Al2O3 and ZrO2) and to analyse their ability to stimulate the release of inflammatory mediators compared with that of high-density polyethylene particles (HDP). We analysed the effects of particle size, concentration and composition using an in vitro model. The J774 mouse macrophage cell line was exposed to commercial particles in the phagocytosable range (up to 4.5 microns). Al2O3 was compared with ZrO2 at 0.6 micron and with HDP at 4.5 microns. Cytotoxicity tests were performed using flow cytometry and macrophage cytokine release was measured by ELISA. Cell mortality increased with the size and concentration of Al2O3 particles. When comparing Al2O3 and ZrO2 at 0.6 micron, we did not detect any significant difference at the concentrations analysed (up to 2500 particles per macrophage), and mortality remained very low (less than 10%). Release of TNF-alpha also increased with the size and concentration of Al2O3 particles, reaching 195% of control (165 pg/ml v 84 pg/ml) at 2.4 microns and 350 particles per cell (p < 0.05). Release of TNF-alpha was higher with HDP than with Al2O3 particles at 4.5 microns. However, we did not detect any significant difference in the release of TNF-alpha between Al2O3 and ZrO2 at 0.6 micron (p > 0.05). We saw no evidence of release of interleukin-1 alpha or interleukin-1 beta after exposure to ceramic or HDP particles. 相似文献
3.
Gibon E Ma T Ren PG Fritton K Biswal S Yao Z Smith L Goodman SB 《Journal of orthopaedic research》2012,30(4):547-553
The biological mechanisms leading to periprosthetic osteolysis involve both chemokines and the monocyte/macrophage cell lineage. Whether MCP-1 plays a major role in macrophage recruitment in the presence of wear particles is unknown. We tested two hypotheses: (1) that exogenous local delivery of MCP-1 induces systematic macrophage recruitment and (2) that blockade of the MCP-1 ligand-receptor axis decreases macrophage recruitment and osteolysis in the presence of ultra high molecular weight polyethylene (UHMWPE) particles. Six groups of nude mice were used. We used non-invasive imaging to assay macrophage recruitment and osteolysis. A murine macrophage cell line and primary wild type and CCR2 knockout murine macrophages were used as the reporter cells. Particles were infused into the femoral canal. Bioluminescence and immunohistochemical staining were used to confirm the migration of reporter cells. Locally infused MCP-1 induced systemic macrophage trafficking to bone. Injection of MCP-1 receptor antagonist significantly decreased reporter cell recruitment to bone infused with UHMWPE particles and decreased osteolysis. Systemic migration of reporter cells to infused particles was decreased when the reporter cells were deficient in the CCR2 receptor. Interruption of the MCP-1 ligand-receptor axis appears to be a viable strategy to mitigate trafficking of macrophages and osteolysis due to UHMWPE particles. 相似文献
4.
BACKGROUND: Two main pathways of apoptosis in mammalian cells have been described: the death receptor pathway and the mitochondrial pathway. Two different cell types have been identified for Fas-mediated apoptosis, each using almost exclusively one of two different signaling pathways. Human prostatic carcinoma cell line, PC3 is sensitive to Fas-mediated apoptosis, but relation of receptor and mitochondrial pathways is not clear. METHODS: Cell viability was estimated by calcein assay. Apoptosis was determined by preparation of DNA ladder. Expression of Fas-associated death domain-dominant negative (FADD-DN) and Bcl-2, activation of caspases, PARP, DFF45, Bid cleavage, and cytochrome c release were assessed using Western blotting techniques. [(35)S] Methionine-labeled caspase-3 was transcribed in vitro and translated using the TNT kit (Promega). A vector containing caspase-3 was prepared by the ligation of EcoR I/BamHI flanked PCR fragment of full size caspase-3 cDNA into pBlusckript II SK(+/-) (Stratagen). RESULTS: Overexpression of both FADD-DN and Bcl-2 genes prevent Fas-mediated apoptosis in PC3. As predicted, overexpression of FADD-DN prevented activation of caspase-8 and Bid cleavage and attenuated the release of cytochrome c and activation of caspases -2, -7, and -9. Bcl-2 overexpression did not affect caspase-8 activation and cleavage of Bid but blocked the release of cytochrome c and activation of mitochondria localized caspases -2, -7, and-9. Overexpression of FADD-DN and Bcl-2 affected the activation of caspase-3 and PARP cleavage differently: FADD-DN attenuated the activation of caspase-3 and PARP cleavage whereas Bcl-2 overexpression prevented caspase-3 activation and completely blocked cleavage of PARP. CONCLUSIONS: These data suggest that activation of caspase-8 is necessary but not sufficient to complete Fas-mediated apoptosis in PC3 cells without activation of the mitochondrial pathway. In addition, caspase-3 activation after Fas-receptor ligation involves two steps and is dependent on mitochondrial activation. 相似文献
5.
Nicolaas de Jonge Dick F van Wichen Joyce van Kuik Hans Kirkels Jaap R Lahpor Frits H J Gmelig-Meyling Jan G van den Tweel Roel A de Weger 《The Journal of heart and lung transplantation》2003,22(9):1028-1036
BACKGROUND: Left ventricular assist device (LVAD) implantation in patients with end-stage heart failure results in impressive hemodynamic improvement. The effects on myocardial apoptosis and its mediators are unknown. METHODS: Myocardial biopsies from 17 patients at the time of LVAD implantation and after explantation, at the time of heart transplantation (HTx), were examined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) reaction and with antibodies against Fas ligand (FasL), Fas, tumor necrosis factor (TNF)-alpha receptor 1 (TNF-R1), TNF-alpha receptor 2 (TNF-R2), TNF-alpha, TNF-alpha-converting enzyme (TACE), poly(ADP-ribose) polymerase (PARP), poly(ADP-ribose) (PAR), caspase-3 and FLICE inhibitory protein (FLIP). RESULTS: Apoptosis incidence was low: 0.8% (range 0% to 3%) positive cardiomyocytes nuclei before support, and 0.1% (range 0% to 0.6%) after support (p < 0.01). This was accompanied by low expression of caspase-3 and high expression of the DNA repair enzyme, PARP. Its product, PAR, increased after support. Mediators and receptors inducing apoptosis as well as FLIP were widely present before and after support. CONCLUSIONS: Despite the abundant presence of mediators and receptors inducing apoptosis, the incidence of apoptosis itself was low before and after mechanical support. The abundant expression of FLIP may suggest an important role for this protein in the inhibition of cardiomyocyte death. 相似文献
6.
Max D Kauther Hagen S Bachmann Laura Neuerburg Martina Broecker-Preuss Gero Hilken Florian Grabellus Gabriele Koehler Marius von Knoch Christian Wedemeyer 《BMC musculoskeletal disorders》2011,12(1):186
Background
Periprosthetic osteolysis is a major cause of aseptic loosening in joint arthroplasty. This study investigates the impact of CT (calcitonin) deficiency and CT substitution under in-vivo circumstances on particle-induced osteolysis in Calca -/- mice.Methods
We used the murine calvarial osteolysis model based on ultra-high molecular weight polyethylene (UHMWPE) particles in 10 C57BL/6J wild-type (WT) mice and twenty Calca -/- mice. The mice were divided into six groups: WT without UHMWPE particles (Group 1), WT with UHMWPE particles (Group 2), Calca -/- mice without UHMWPE particles (Group 3), Calca -/- mice with UHMWPE particles (Group 4), Calca -/- mice without UHMWPE particles and calcitonin substitution (Group 5), and Calca -/- mice with UHMWPE particle implantation and calcitonin substitution (Group 6). Analytes were extracted from serum and urine. Bone resorption was measured by bone histomorphometry. The number of osteoclasts was determined by counting the tartrate-resistant acid phosphatase (TRACP) + cells.Results
Bone resorption was significantly increased in Calca -/- mice compared with their corresponding WT. The eroded surface in Calca -/- mice with particle implantation was reduced by 20.6% after CT substitution. Osteoclast numbers were significantly increased in Calca -/- mice after particle implantation. Serum OPG (osteoprotegerin) increased significantly after CT substitution.Conclusions
As anticipated, Calca -/- mice show extensive osteolysis compared with wild-type mice, and CT substitution reduces particle-induced osteolysis.7.
皮质酮诱导的大鼠Leydig细胞凋亡中caspase-3活化途径的研究 总被引:1,自引:0,他引:1
目的我们新近的研究表明,超生理剂量的皮质酮(大鼠体内的糖皮质激素)可通过激活caspase-3诱导大鼠Leydig细胞凋亡。本研究拟评价在皮质酮诱导的大鼠Leydig细胞凋亡过程中,caspase-3的激活是否有其上游的caspase-8平和 caspase-9的参与。方法采用荧光分光光度法榆测经皮质酮处理的大鼠Leydig细胞中caspase-8活性,以DNA梯状电泳条带作为评价细胞凋亡的指标,观察caspase-8抑制剂是否能够抑制细胞凋亡,采用RTPCR枪测皮质酮诱导的大鼠Leydig细胞中caspase-9的mRNA水平。结果在皮质酮诱导的大鼠Leydig细胞中出现caspase-8活性增高,以12h最为址著,升高的caspase-8的活性可被caspase-8抑制剂抑制,并导致Leydig细胞的凋亡过程被阻断。Leydig细胞中caspase-9的mRNA水平在皮质酮作用下上升,同样以12h最为显著。结论皮质酮诱导的大鼠Leydig细胞调亡与caspase-8和caspase-9有关。 相似文献
8.
Messmer UK Briner VA Pfeilschifter J 《Journal of the American Society of Nephrology : JASN》2000,11(12):2199-2211
Endothelial cell damage of glomeruli and kidney arterioles seems to play a pivotal role in several pathologic situations, such as Gram-negative sepsis, glomerulonephritis, and acute renal failure. Bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) have been identified as potent inducers of apoptotic cell death in bovine glomerular endothelial cells. Both agents elicited apoptotic DNA laddering within 12 to 24 h. Basic fibroblast growth factor (bFGF) was generally described as a protective factor for endothelial cells against radiation-, TNF-alpha-, and UV-light-induced programmed cell death. Therefore, whether bFGF also affects apoptosis of microvascular endothelial cells was questioned. Surprising was that simultaneous treatment of glomerular endothelial cells with bFGF and either LPS or TNF-alpha left LPS-induced death unaffected, whereas TNF-alpha-induced death induction was potentiated, amounting to 48.9+/-6.3% versus 22.4+/-4.3% DNA degradation with TNF-alpha alone. Comparably, acidic FGF also selectively potentiated TNF-alpha-induced apoptosis. In mechanistic terms, bFGF synergistically increased TNF-alpha-induced mitochondrial permeability transition, the release of cytochrome c from mitochondria to the cytosol, and upregulation of the proapoptotic protein Bak and significantly enhanced activation of caspase-8 protease activity. In contrast, stress-activated protein kinase and nuclear factor kappaB activation, which represent primary signals of TNF/TNF receptor interaction, downregulation of the antiapoptotic protein Bcl-x(L), and caspase-3-like protease activation, were unaffected. As bFGF did not affect LPS-induced apoptotic cell death, bFGF also left LPS-induced Bak upregulation and Bcl-x(L) downregulation unaffected. The results point to a selective bFGF-mediated enhancement of distinct proapoptotic pathways induced by TNF-alpha in glomerular endothelial cells. 相似文献
9.
微小假体磨损颗粒诱导置入物旁骨溶解的扫描电镜观察 总被引:2,自引:0,他引:2
为比较不同人工关节微小磨损颗粒对假体-骨界面处骨整合及骨整合及骨溶解的影响,探讨假体松动的机制。方法本实验采用扫描电镜技术对钛事金、钴-铬-钼与聚乙烯(ultrahighmolecularweightpolyethylene,UHMWPE)三种微小颗粒诱导的假体周围骨组织改变进行超微结构观察。结果研究发现,直径2.5μm的Ti-6Al-4V颗粒诱导置入物旁骨吸收或骨溶解的程度明显低于相同直径的Co 相似文献
10.
Hiroyuki Shindo Shintaro Warabi Hitoshi Taneda Hirohiko Azuma 《Journal of orthopaedic science》1997,2(2):93-97
Osteolysis associated with artificial joint arthroplasty seems to be the result of particles of wear debris (from ultra-high
molecular weight polyethylene (UHMWPE), polymethylmethacrylate (PMMA), and metals), causing a macrophage response, Silicon
particles, as residual contaminants embedded in the surface of both textured metal implants and polyethylene sockets, may
be a factor in osteolysis. We present the case of a 57-year-old woman who had massive and aggressive osteolysis. The osteolytic
lesion was isolated in the greater trochanter region 5 years after she had had primary cementless total hip arthroplasty (Cobalt-chromium
alloy). There were no signs of mechanical loosening, but she experienced moderate pain. Under a polarized microscope and scanning
electron micrography, a biopsied specimen from the osteolytic lesion revealed conglomerates of UHMWPE particles of various
sizes between proliferated synovium-like cells. Quantative energy-dispersive X-ray analysis focused on the conglomerates of
UHMPWE particles demonstrated a marked presence of silicon. Although the definitive causative factor for a the osteolysis
was regarded as a foreign-body reaction induced by UHMWPE particles, the presence of silicon was interesting in terms of the
pathogenesis of osteolysis associated with artificial joint surgery. 相似文献
11.
12.
Purpose
The purpose of the study was to determine if nerve growth factor (NGF) stimulation induces apoptosis in the BE(2)C neuroblastoma cell line in vitro.Methods
The LPCX retroviral vector was used to achieve stable transduction of NGF complementary DNA into BE(2)C neuroblastoma cells. Wild-type and NGF-transduced cells were then incubated with varying concentrations of NGF for varying periods. A laddering assay was performed to determine the presence of DNA fragments characteristic of apoptosis. The expression of various cleaved and total caspases was determined by Western immunoblotting.Results
p75 receptor expression in the NGF-transduced cell line was equivalent to that in the wild-type cell line, but Trk A receptor expression was significantly decreased in BE(2)C-NGF cells. DNA laddering assay demonstrated that only BE(2)C-NGF cells underwent apoptosis after stimulation with exogenous NGF. BE(2)C-NGF cells have increased expression of cleaved caspase-3 when compared with wild-type cells. Cleaved caspase-3 expression is further increased with exogenous NGF stimulation in the transduced cells.Conclusion
This study confirms that NGF stimulation of BE(2)C neuroblastoma cells can induce apoptosis through activation of the caspase cascade in vitro. The differential expression of the receptors Trk A and p75 between the wild-type and NGF-transduced cell lines may explain the differing effects observed. 相似文献13.
Periprosthetic osteolysis is a dominant factor in the success or failure of total hip prostheses. Polyethylene wear debris has been implicated in the process of bone resorption and subsequent implant loosening. The present study is the first to examine the effect of ultra high molecular weight polyethylene (UHMWPE) wear debris produced by a hip simulator on calvarial bone resorption in vitro. (45)Ca release was measured in cultured mouse calvarial bone samples. Although short-term exposure to UHMWPE particles (2 h) decreased (45)Ca release, longer-term exposure for 1-2 days increased release in a dose-dependent manner. After one-day exposure to 7.5 x 10(6) particles per mL, 18% more (45)Ca was released from cultured calvarial bone than from control samples. It was concluded that UHMWPE wear particles either directly or indirectly stimulated osteoclasts to activate bone resorption. Polyethylene wear debris contributes to the osteolytic process at the bone-implant interface. 相似文献
14.
Wear of orthopaedic implants generates particles capable of inducing bone resorption and aseptic loosening of the implant. The present study shows the combined effect of particles and cell activation on macrophage (THP-1) and osteoclast (HD-11EM) release of reactive oxygen and nitrogen species, providing insight into mechanisms that can lead to osteolysis. In the absence of cell activation, exposure of either cell type to submicron zirconia or latex particles did not elicit an increase in reactive oxygen and nitrogen species production. Suboptimal stimulation with 4 beta-phorbol-12-myristate-13-acetate (PMA) plus particles resulted in a synergistic release of superoxide (O2-), however, and a low-level production of nitric oxide small middle dot by THP-1 macrophages. Similarly, particle stimulation of tumor necrosis factor-alpha-activated THP-1 cells increased O2- release. Our findings show the synergistic effect of cell activation and wear particles on O2- production by activated macrophages and osteoclasts, suggesting O2- involvement in mediating osteolysis. 相似文献
15.
人工关节磨损颗粒激活骨—假体界面巨噬细胞生物学机制探讨 总被引:12,自引:1,他引:11
目的 观察无菌性松动人工关节骨-假体界面磨损颗粒刺激下巨噬细胞(MΦ)膜Ca^2+通道的变化,分析胞内游离钙离子浓度([Ca^2+]i)变化在其激活机制中的作用。方法 体外培养获得滑膜细胞系;以CD68单抗免疫组化(SABC法)等方法确定其中巨噬细胞样细胞(MCs)形态及其吞噬行为发生时间;滴加1.5mg/ml(W/V)Ti合金、Co-Cr合金或超高分子聚乙烯(UHMWPE)颗粒悬液等,动态监测M 相似文献
16.
Hypoxia/re-oxygenation injury induces apoptosis in renal tubule cells, but its underlying molecular pathways are not fully
elucidated. Activation of caspase-2 has recently been proposed as a novel mechanism of apoptosis in fibroblasts. In this study
we examined whether hypoxia/re-oxygenation injury induces apoptosis in proximal tubule cells by activation of caspase-2. Porcine
proximal tubule (LLC-PK1) cells were subjected to hypoxia/re-oxygenation injury in the presence or absence of caspase inhibitors.
Apoptosis was detected by DNA laddering, flow cytometry, and immunocytochemistry for Bax and cytochrome c. The activity of caspases-2, 8 and 9 was measured. Apoptosis was evident after hypoxia/re-oxygenation and was best prevented
by pretreatment with caspase-2 inhibitor. Hypoxia/re-oxygenation resulted in a dramatic increase in caspase-2 activity (32-fold,
in comparison with a 16-fold increase in caspase-8 activity and a tenfold increase in caspase-9 activity). Immunocytochemistry
revealed Bax activation and translocation to mitochondria and cytochrome c release into the cytosol following hypoxia/re-oxygenation, both of which were significantly suppressed by pretreatment with
caspase-2 inhibitor. These results indicate that hypoxia/re-oxygenation injury in cultured proximal tubule cells induced apoptosis
by activation of caspase-2, which is required for the mitochondrial translocation of Bax. 相似文献
17.
Advanced glycation end products induce mitochondrial pathway apoptosis in glomerular mesangial cells
Objective To investigate whether advanced glycation end products (AGEs) can induce the expression of Ros, JC-1 and its apoptosis-related proteins in glomerular mesangial cells under high glucose environment, induce apoptosis and injury of glomerular mesangial cells. Methods Rat glomerular mesangial cell line HBZY-1 was cultured in vitro. The cells were cultured with different concentrations of AGEs for 0, 12, 24 and 48 hours respectively. MTT assay was used to observe the cell proliferation ability. After the optimal time and concentration of AGEs were selected, the caspase enzyme inhibitor Z-VAD-fmk and reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) were cultured and the apoptosis rate was detected by cell death detection apoptosis ELISA plus and Annexin V-FITC/PI kit. JC-1 staining was used to detect the changes of mitochondrial membrane potential (MMP). Cell ROX deep red flow cytometry was used to detect the total ROS level. The expression of anti-apoptotic protein Bcl-2, pro-apoptotic protein BAX, caspase-9, caspase-3 and poly ADP-ribose polymerase (PARP)-activated fragments was detected by Western blotting. Results AGEs could decrease the activity of glomerular mesangial cells in a time and concentration-dependent manner, and induce cell death. The percentage of apoptotic cells in glomerular mesangial cells was significantly increased after treatment with 250 mg/L AGEs for 24 h (P<0.01), and Z-VAD-fmk could significantly alleviate AGEs-induced glomerular mesangial cell apoptosis (P<0.01). Compared with the control group, AGEs increased the level of intracellular reactive oxygen species and decreased MMP in a time-dependent manner, and the two time points that AGEs significantly caused the change were 1 h and 2 h (all P<0.01). AGEs also reduced the expression of antiapoptotic protein Bcl-2 and increased the expression of pro-apoptotic protein Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP (all P<0.01). Compared with AGEs group, NAC could significantly stabilize MMP (P<0.01), increase Bcl-2 expression (P<0.01), and decrease the expression of BAX, cleaved caspase-9, cleaved caspase-3 and cleaved PARP (all P<0.01). Conclusion AGEs induce mitochondrial pathway apoptosis in glomerular mesangial cells by increasing intracellular ROS level and destroying MMP. 相似文献
18.
BACKGROUND: In previous studies we have shown that epidermal growth factor (EGF) at concentrations between 50 and 100 ng/mL induced apoptosis in wild-type SK-N-SH neuroblastoma cells. We hypothesize that this apoptotic event separates EGF-induced neuroblastoma cell growth into a biphasic concentration-dependent process, due to activation of different signaling cascades. METHODS: Cells were incubated in concentrations of EGF ranging from 5 to 250 ng/mL for 3 days, and cell proliferation was determined by the MTT assay. Cells incubated with EGF 5, 100, or 250 ng/mL for 17 h were also assayed for apoptosis by DNA laddering. Western immunoblots were performed on whole cell lysates prepared from cells incubated with EGF (5-250 ng/mL) for 17 h. Antibodies against cleaved caspase3, p-AKT, p-GSK-3beta, p-BAD, p-RAF, p-ERK, and p-P38 were used as probes. RESULTS: A triphasic, concentration-dependent response was observed following incubation of cells with EGF. Cell proliferation was increased by EGF 5 ng/mL (P < 0.05), decreased by EGF 100 ng/mL, and increased when incubated with EGF 250 ng/mL (P < 0.05). DNA laddering only occurred after treatment with EGF 100 ng/mL. The expressions of p-ERK, p-RAF, p-BAD, and p-GSK-3beta were increased at EGF concentrations of 5-10 ng/mL. At 50-100 ng/mL EGF, the expression of cleaved caspase3 was increased. Maximal p-P38 expression was at 50 ng/mL EGF. At EGF concentrations of 150-250 ng/mL, the expressions of p-AKT and p-GSK-3beta were elevated. CONCLUSIONS: Neuroblastoma cell growth induced by EGF exhibited a triphasic pattern; cell growth was increased at EGF concentrations 5-20 and 150-250 ng/mL, but decreased at 50-100 mg/mL. Apoptosis was induced at 50-100 ng/mL EGF. Each growth phase activated different signaling molecules. 相似文献
19.
Differential effects of tumor necrosis factor-alpha and interleukin-1beta on cell death in human articular chondrocytes 总被引:2,自引:0,他引:2
Caramés B López-Armada MJ Cillero-Pastor B Lires-Dean M Vaamonde C Galdo F Blanco FJ 《Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society》2008,16(6):715-722
OBJECTIVE: The death of chondrocytes by apoptosis is characteristic of degenerative joint diseases, such as osteoarthritis (OA). Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been shown to play an important role in the development of OA. In this study we analyzed the effects of TNF-alpha and IL-1beta on cell death in normal human chondrocytes. METHODS: Normal human chondrocytes were isolated from knee cartilage obtained at autopsy from 30 adult cadaveric donors. The cells were stimulated with TNF-alpha (10 ng/ml) or IL-1beta (5 ng/ml) in the presence or absence of Ro 31-8220 (Ro: a structurally related analog of bisindolylmaleimide that inhibits mitogen-activated protein kinase phosphatase 1 [MKP-1]) (Ro; 10 microM), an MKP-1 inhibitor, which induces apoptosis in chondrocytes. Apoptosis was evaluated by flow cytometry (propidium iodide) and nuclear morphology was evaluated with 4',6'-dianidino-2-phenylindole dihydrochloride. The expressions of caspase-8, -7 and -3 and Bcl-2 were analyzed by Western blot and the activation of caspase-3 and -8 was measured by flow cytometry. Prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay. RESULTS: At 24 h the percentage of apoptotic (hypodiploid) nuclei induced by TNF-alpha+Ro was higher than the level induced by Ro alone. The combination of IL-1beta (5 ng/ml) with Ro did not show a synergistic effect. A morphological analysis demonstrated that treatment with TNF-alpha+Ro resulted in a large number of cells with condensed nuclei and DNA fragmentation. Western blot studies indicated that IL-1beta+Ro did not induce the time-dependent activation of caspase-8, -7 and -3 as seen with TNF-alpha+Ro. As quantified by flow cytometry, TNF-alpha+Ro induced a higher level of caspase-3 and -8 activation than that seen with IL-1beta+Ro. Pre-incubation for 2h with caspase inhibitors for caspase-3, -7, -8 and pan-caspase significantly decreased the hypodiploid DNA peak induced by treatment with TNF-alpha+Ro at 24 h. Indomethacin increased the cell death induced by IL-1beta+Ro; however, apoptosis induced by TNF-alpha+Ro was not modified by indomethacin. CONCLUSIONS: These results confirm that TNF-alpha and IL-1beta regulate apoptosis differently in this human chondrocyte model and that the differing effects of these cytokines are PGE2-independent. Indomethacin potentiates the effect of IL-1 on cell death and this may explain the reported effect of indomethacin on the progression of joint destruction. 相似文献
20.
John A.J. Broomfield Tamer T. Malak Geraint E.R. Thomas Antony J.R. Palmer Adrian Taylor Sion Glyn-Jones 《The Journal of arthroplasty》2017,32(4):1186-1191