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1.
研究HGF拮抗剂NK4在前列腺癌中的作用。将包含NK4cDNA的表达载体pBudCE4.1-EGFP-NK4转染到DU145细胞中。体外实验检测白分泌的NK4对肿瘤细胞增殖、迁移、转移及凋亡的影响。体内实验裸鼠分为三组,分别皮下种植DUl45、空质粒转染的Dul45和NK4转染的DUl45细胞,检测皮下肿瘤的大小、细胞凋亡及细胞增殖情况。体外实验结果显示转染NK4的DUl45细胞可以分泌NK4蛋白。自分泌的NK4抑制HGF诱导的肿瘤细胞增殖、迁移及转移,促进凋亡(P〈0.01)。NK4可以调节HGF受体c-Met及其下游ERKl与Akt1/2蛋白的活性。体内实验显示,NK4转染DUl45细胞组的肿瘤生长及细胞增殖受到抑制,同时肿瘤细胞凋亡增加。本实验显示包含NK4cDNA的表达载体转染前列腺癌细胞可以有效地调节肿瘤细胞的增殖、迁移、侵袭及凋亡。NK4作用于HGF/c-Met可以作为前列腺癌治疗的一个有效靶点。 相似文献
2.
OBJECTIVE: To determine whether insulinlike growth factor-I (IGF-I) and hepatocyte growth factor (HGF) cooperate to induce migration and invasion of human colorectal carcinoma (CRC) cells and whether the effects of IGF-I and/or HGF are mediated through activation of the urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system, a central mediator of tumor-cell migration and invasion. SUMMARY BACKGROUND DATA: CRC cells must invade through the basement membrane of the colon and migrate to form metastases. CRC cells are known to overexpress IGF-I receptor (IGF-IR), c-Met, and uPAR, 3 cell-surface receptors known to mediate cell migration and invasion. We hypothesized that IGF-IR and c-Met cooperate to induce migration and invasion in CRC cells and that this signaling is dependent on uPAR. METHODS: KM12L4 human CRC cells were treated with IGF-I, HGF, or IGF-I + HGF in transwell migration and invasion chambers; cells that had migrated or invaded were counted. To determine the role of c-Met in IGF-I-induced migration and invasion, c-Met was inhibited by infection of cells with an adenovirus containing a c-Met ribozyme; transwell assays were then repeated. To determine the role of the uPA/uPAR system in IGF-I-induced CRC cell migration and invasion, transwell assays were repeated after pretreating cells with the uPA inhibitor amiloride or with neutralizing antibodies to uPA and uPAR. RESULTS: IGF-I and HGF, alone or in combination, increased cell migration and invasion. The c-Met ribozyme inhibited IGF-I- and HGF-mediated migration and invasion, indicating that c-Met is essential for these processes. uPA and uPAR inhibition blocked IGF-I- and HGF-mediated migration and invasion, suggesting that uPAR is downstream of IGF/IGF-IR and HGF/c-Met in the signaling pathways that mediate cell migration and invasion. CONCLUSIONS: IGF-I and HGF cooperate to induce migration and invasion of CRC cells, and c-Met and uPA/uPAR are required for IGF-I-mediated migration and invasion. In our in vitro model of CRC migration and invasion, uPA and uPAR appear to be downstream of IGF-IR and c-Met and are required for migration and invasion. Elucidation of the pathways that contribute to tumor progression and metastasis should provide a foundation for the rational development and use of targeted therapies for CRC. 相似文献
4.
BACKGROUND: Hepatocyte growth factor scatter factor (HGF/SF) elicits a number of biological activities including invasion and migration through activation of its tyrosine kinase receptor c-Met. Over expression of c-Met has been implicated in prostate cancer development and progression. This study examined the effect of a ribozyme transgene, designed to inhibit human c-Met expression, and its impact on in vitro invasion and migration in prostate cancer. METHODS: A transgene (Met 560) consisting of U1 snRNA, hammerhead ribozyme, and antisense was cloned into a modified pZeoU1-EcoSpe vector and transfected into DU-145 cells. The effect of HGF/SF was tested on prostate cancer cells whose expression of c-Met had been blocked by way of a ribozyme transgene. RESULTS: Met 560 stable transfectants (DU-145(+/+)) manifested a complete loss of c-Met expression at mRNA and protein levels. In contrast, control plasmid (DU-145(+/-)) and wild-type DU-145 cells (DU-145(-/-)) had similar levels of c-Met expression. HGF/SF significantly increased the in vitro invasiveness (mean 47.71 +/- SE 7.75; P < 0.01 vs. control 24.14 +/- 1.34), and migration (mean 48.44 +/- SE 3.51; P < 0.01 vs. control 22.95 +/- 1.47) of DU-145(-/-) cells, respectively. Similarly, HGF/SF also increased the invasion (62.33 +/- 6.34; P < 0.001 vs. control 24.5 +/- 2.35) and migration (46.14 +/- 2.26; P < 0.01 vs. control 21.82 +/- 1.62) of DU-145(+/-) cells. In contrast, DU-145(+/+) cells had lost its response to HGF/SF induced invasion (22.33 +/- 2.08; P > 0.05 vs. control 23.5 +/- 2.11) and migration (24.12 +/- 0.86; P > 0.05 vs. control 23.27 +/- 0.81). CONCLUSIONS: Targeting the HGF/SF receptor by way of a hammerhead ribozyme encoding antisense to c-Met, is an effective method to reduce the invasive or migration potential in prostate cancer, and may have important therapeutic implications. 相似文献
5.
In osteoarthritis (OA), hepatocyte growth factor (HGF) is supposed to play a role in cartilage repair. Because the development of osteophytes is a major characteristic of OA and thought to be part of an attempted repair process, the purpose of this study was to determine whether HGF may be involved in osteophyte formation. HGF levels in synovial fluids from 41 patients assessed by enzyme immunosorbant assay were correlated with disease severity and osteophyte formation, evaluated by anteroposterior weight-bearing radiographs. Detection of HGF, c-Met, and CD68 in cartilage and synovial tissues was assessed by immunohistochemistry. Effects of HGF on the secretion of TGF-beta1 and BMP-2 by chondrocytes, fibroblast-like synovial cells (FLS), and macrophages as well as HGF-induced secretion of MCP-1 by FLS and chondrocytes were determined by ELISA. HGF was detected in all synovial fluids and concentrations correlated highly with disease severity and osteophyte formation (p < 0.001). Immunohistochemistry revealed weak synovial staining for HGF, whereas increasing numbers of HGF expressing chondrocytes were detected depending on disease severity. In addition, an increased number of macrophages in synovial specimens was observed, which was likewise severity dependent. In a series of subsequent in vitro studies, HGF remarkable induced MCP-1 secretion by FLS in a dose-dependent manner. No effect on TGF-beta1 and BMP-2 secretion by FLS and chondrocytes was evident upon HGF stimulation, whereas secretion of these growth factors by PMA-differentiated THP-1 cells was significantly increased by HGF. The results indicate that HGF may facilitate osteophyte development by promoting MCP-1-mediated entry of monocytes/macrophages into the OA-affected joint and/or by stimulating macrophage-derived growth factors. 相似文献
7.
Background: As a renotropic cytokine, hepatocyte
growth factor (HGF) prevents acute renal failure and accelerates renal
regeneration. HGF initiates its biological effects by interaction with
specific transmembrane receptors, the c-Met proto-oncogene, possessing an
intracellular tyrosine kinase domain. We tested the hypothesis of whether
the complex biological effects of HGF in renal proximal tubular cells are
mediated by different intracellular signalling cascades and/or different
receptors. Methods: PT-1 cells, a proximal tubular
cell line derived from rabbit kidney, were cultured under defined
serum-free conditions to examine the biological effects of exogenously
added HGF. By specific assays, we determined HGF binding and its effects on
cell proliferation, migration, scattering and tubulogenic differentiation.
To investigate whether HGF action could be inhibited by protein tyrosine
kinase inhibitors (PTKIs), cells were incubated with HGF and different
concentrations of herbimycin A, genestein, methyl-2,5-dihydroxycinnamate
(MDC) and geldanamycin. All PTKIs are known inhibitors of
pp60 c-src, a non-receptor tyrosine kinase involved
in cell growth control. Results: HGF bound with high
affinity to cell membrane receptors and displayed multiple biological
effects. Compared with serum-free controls, HGF increased the number of
microvilli 1.5-fold, enhanced cell proliferation and migration 1.8-fold,
and stimulated the formation of tubular structures 2.3-fold. Consistent
with the known tyrosine kinase activity of the c-Met receptor, the
mitogenic and motogenic effects of HGF were inhibited by PTKIs in a
dose-dependent manner with the following order of potency:
geldanamycin>herbimycin A>genestein>MDC. In contrast,
however, the HGF-induced tubulogenic cell differentiation was not inhibited
specifically by PTKIs. Conclusions: The finding that
PTKIs inhibited the mitogenic response but not the tubulogenic
differentiation induced by HGF indicated different intracellular signal
transduction pathways. We suggest that pp60 c-src
plays a key role in mediating the mitogenic and motogenic action of HGF,
whereas tubulogenic cell differentiation induced by HGF is transduced by a
pp60 c-srcindependent signalling pathway. 相似文献
8.
【摘要】 目的〓探讨姜黄素与PI3K/Akt抑制剂LY294002联合应用对前列腺癌PC-3细胞体外生长的影响。方法〓根据给药不同将实验分为对照组、姜黄素组、LY294002组和姜黄素组+LY294002联合组,分别加入等量的培养基、25 μmoL/L姜黄素、25 μmoL/L的LY294002和两种混合液。采用MTS法检测各组细胞的增殖情况、流式细胞仪术检测各组细胞凋亡情况、q-PCR测定各组细胞中NF-κB、P53及caspase-9的表达情况。结果〓姜黄素组、LY组和联合组的细胞增值率均较对照组明显降低,且联合组明显低于两个单独用药组(P<0.05)。姜黄素与LY294002联合作用前列腺癌PC-3细胞后,细胞总凋亡率较姜黄素及LY294002单独使用后明显增加(P值均<0.05)。联合用药组的NF-κB表达量明显低于两单独用药组(P<0.05),而P53和caspase-9的表达量均明显高于两单独用药组(P<0.05)。结论〓姜黄素与LY294002联合使用较两种药物单独使用更能有效抑制前列腺癌PC-3细胞的体外生长,作用机制可能是通过抑制NF-κB的表达从而抑制细胞增殖,并增加P53和caspase-9的表达从而促进细胞凋亡来实现的。 相似文献
9.
目的 探讨HGF是否上调SMMC-7721细胞抗凋亡蛋白Mcl-1的表达以及HGF是通过哪条信号途径调节MCl-1.方法 人肝癌细胞SMMC-7721进行实验;采用RT-PCR检测HGF受体c-Met在SMMC-7721细胞的表达情况;用Western blot方法 检测MAPK和P13K/AKT磷酸化以及Mcl-1蛋白的表达;用信号通路阻断剂处理SMMC-7721细胞,检测HGF是通过哪条信号途径调节Mcl-1.结果 人肝癌细胞SMMC-7721表达HGF受体C-Met;HGF能够激活MAPK、PI3K/AKT信号转导途径;HGF能够上调SMMC-7721细胞Mcl-1表达;进一步用特异的抑制剂阻断MAPK途径后,并不能改变HGF对Mcl-1的上调作用,而阻断PI3K/AKT途径后,HGF对Mcl-1的上调作用被抑制.结论 HGF对Mcl-1的上调作用是通过PI3K/AKT途径介导的. 相似文献
10.
We have previously shown that treatment with bone marrow stromal cells (BMSCs) augments the healing of fascial wounds in the rat. However, the biochemical mechanism by which BMSCs improve wound healing was not investigated. Growth factors have been shown to play a key role in repairing damaged tissue. In this study, we investigated whether BMSCs are capable of producing growth factors that play a critical role in healing of the damaged tissue. Growth factor expression in BMSCs stimulated with pro-inflammatory cytokines or wound superfusate was measured by RT-PCR and growth factor-specific quantitative sandwich enzyme-linked immunosorbent assay (ELISA). RT-PCR analysis demonstrated that BMSCs are capable of expressing transforming growth factor beta-1 (TGF-beta1), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) constitutively or upon stimulation with LPS, IL-1alpha, or TNF-alpha. Quantitative analysis of growth factor production by ELISA showed that BMSCs do not secrete TGF-beta1, EGF or VEGF in response to uninjured fascia tissue superfusate; however, production of these growth factors is significantly increased when cells were stimulated with wound tissue superfusate. The ability of wound to stimulate growth factor production in BMSCs could be detected as early as day 1 and lasted through day 7 after wounding. Thus, growth factor production by BMSCs in response to wound microenvionment suggests that BMSCs might augment wound healing through the responsive secretion of growth factors that enhance angiogenesis and promote wound repair. 相似文献
11.
Background Identification of a novel biomarker of subclinical lymph node metastasis (SLNM) in papillary thyroid microcarcinoma (PTMC) could provide important clues regarding SLNM in PTMC. We evaluated the significance of HGF and c-Met expression in surgically removed tumor tissue from PTMC patients as a predictive marker of SLNM. Methods We analyzed the immunohistochemical relationship between HGF and c-Met expression and SLNM in 113 surgically treated PTMC patients with clinically negative nodes presurgery. In addition, we explored whether HGF/c-Met pathway activation enhanced the in vitro migration and invasion of PTC cells. Results Positive immunohistochemical HGF and c-Met staining was found in 107 (95 %) and 103 (91 %) cases, respectively. The HGF staining distribution was as follows: no staining in 6 cases, weak staining in 43, moderate staining in 55, and strong staining in 9. Of the nine cases with strong HGF staining, eight (89 %) had SLNM. The c-Met staining distribution was as follows: no staining in 10 cases, weak staining in 39, moderate staining in 59, and strong staining in 5. Of the five cases with strong c-Met staining, three (60 %) had SLNM. The presence of SLNM was strongly correlated with HGF and c-Met expression in PTMC in a univariate analysis ( P < 0.05). HGF overexpression was also associated with SLNM in a multivariate analysis ( P < 0.05). Stimulation with exogenous HGF and constitutive activation of c-Met enhanced the migration and invasion of PTC cells in vitro by enhancing VEGF-A expression. Conclusions HGF/c-Met pathway activation is associated with SLNM of the central neck in PTMC. 相似文献
12.
Bone marrow-derived stromal cells (BMSCs) exhibit extraordinary degree of plasticity and growth factor repertoire for which they have been investigated for repair and regeneration of damaged tissues, but have not been adequately examined for wound healing. The ability of BMSCs to accelerate healing of surgically inflicted cutaneous and fascial wounds was tested in vivo in rats and in vitro using a fibroblast monolayer wound model. Intravenous treatment with BMSCs augmented healing of both cutaneous and fascial wounds as determined by an increase in the biomechanical strength of wounds. In vitro experiments showed that incorporation of BMSCs in fibroblast monolayers accelerates the closure of mechanically disrupted monolayers, which was attributed to the enhanced migration of fibroblasts onto the denuded surfaces. Furthermore, culture medium conditioned by activated BMSCs promoted the closure of defects in monolayers and enhanced the proliferation/growth and directional migration (chemotaxis) of fibroblasts. This study demonstrates that BMSCs significantly augment healing of cutaneous and fascial wounds in vivo at least in part through interaction with fibroblasts in which BMSCs promote growth and chemotaxis of fibroblasts. 相似文献
13.
目的 探讨黄芪多糖在体内外通过PI3K/AKT/mTOR信号通路对糖皮质激素诱导的骨质疏松症(glucocorticoid-induced osteoporosis,GIOP)的保护作用。方法 从分化的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BM-MSCs)中培养成骨细胞,分为PBS组、模型组、LY294002组、黄芪多糖组和LY294002+黄芪多糖干预组。通过CCK-8法和碱性磷酸酶(alkaline phosphatase,ALP)染色检测细胞增殖和分化。MDC染色观察自噬体的形成。Western blot检测Beclin-1、p62等信号通路及自噬相关因子的蛋白表达。大鼠分为对照组、模型组、LY294002组、黄芪多糖组和LY294002+黄芪多糖组。比较各组大鼠的骨密度、骨组织形态学参数、组织中通路和自噬相关因子的表达。结果 黄芪多糖促进成骨细胞的增殖和分化能力(P<0.05)。与模型组相比,黄芪多糖组PI3K/AKT/mTOR通路相关磷酸化蛋白的表达、成骨细胞的增殖分化能力、自噬体及自噬相关因子的表达均升高,但在LY294002组中发现了相反的结果(P<0.05)。在体内实验中,与模型组相比,GIOP大鼠通过黄芪多糖干预改善了骨密度和骨形态参数,并提高了软骨组织中自噬相关因子的表达,而LY294002干预则表现出相反的结果(P<0.05)。LY294002部分逆转了黄芪多糖对GIOP中成骨分化和骨形态参数的影响。结论 黄芪多糖通过PI3K/AKT/mTOR通路对GIOP发挥保护作用,可能与诱导自噬和促进成骨细胞增殖有关。 相似文献
14.
目的:探讨肝细胞生长因子(HGF)对人肝癌细胞增殖的作用及其机制。方法:人肝癌细胞株HepG2用于实验。通过BrdU细胞增殖酶联免疫实验观察HGF对细胞增殖的作用,采用Western印迹和RT-PCR法检测HGF对S期激酶结合蛋白2(Skp2)表达的影响。将Skp2真核细胞重组表达质粒转染HepG2细胞并获得稳定转染的过表达Skp2的HepG2细胞,观察HGF对该转染细胞增殖的影响。结果: HGF对HepG2细胞的增殖具有浓度依赖性和时间依赖性的抑制作用;HGF明显抑制HepG2细胞表达Skp2蛋白和mRNA。但HGF对过表达Skp2的HepG2细胞无增殖抑制作用。结论:HGF对HepG2细胞具有明显的增殖抑制作用,其作用机制与下调Skp2表达密切相关。 相似文献
15.
Osteoblast recruitment to the site of future bone formation is essential for skeletal development, bone remodeling and fracture healing. A number of factors associated with bone tissue have been reported to induce directional migration of osteoblasts but the mechanism remains to be clarified. In this study, to explore a major chemotactic factor(s) for osteoblasts, we examined the serum-free medium conditioned by MC3T3-E1 osteoblast-like cells for its ability to induce osteoblast migration. Employing sequential chromatography and tandem mass spectrometry analysis, we purified and identified IGF-I as a potent chemotactic factor from the conditioned medium. IGF-I induced cell migration of both MC3T3-E1 cells and primary mouse osteoblasts, and checkerboard analysis revealed that IGF-I markedly induced directional migration (chemotaxis) of osteoblasts. Neutralization of mouse IGF-I with monoclonal antibodies resulted in delayed osteoblast monolayer wound healing and cellular polarization but addition of human IGF-I reversed these effects. IGF-I also promoted cell spreading on fibronectin in an integrin beta1-dependent manner. IGF-I induced Akt and Rac activation and localized accumulation of phosphatidylinositol 3,4,5-triphosphate (PtdIns (3,4,5)P3) at the membrane in osteoblasts. The phosphatidyl inositol 3 kinase (PI3K) inhibitor LY294002 inhibited IGF-I-induced cell migration and wound healing. Together, the results suggest that IGF-I secreted from osteoblasts in the bone tissue is a potent chemotactic factor that may play a major role in recruitment of osteoblasts during bone formation. 相似文献
16.
Bone marrow stem cells participate in tissue repair processes and may have a role in wound healing. Diabetes is characterised by delayed and poor wound healing. We investigated the potential of bone marrow-derived mesenchymal stromal cells (BMSCs) to promote healing of fascial wounds in diabetic rats. After manifestation of streptozotocin (STZ)-induced diabetic state for 5 weeks in male adult Sprague-Dawley rats, healing of fascial wounds was severely compromised. Compromised wound healing in diabetic rats was characterised by excessive polymorphonuclear cell infiltration, lack of granulation tissue formation, deficit of collagen and growth factor [transforming growth factor (TGF-beta), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor PDGF-BB and keratinocyte growth factor (KGF)] expression in the wound tissue and significant decrease in biomechanical strength of wounds. Treatment with BMSC systemically or locally at the wound site improved the wound-breaking strength (WBS) of fascial wounds. The improvement in WBS was associated with an immediate and significant increase in collagen levels (types I-V) in the wound bed. In addition, treatment with BMSCs increased the expression of growth factors critical to proper repair and regeneration of the damaged tissue moderately (TGF-beta, KGF) to markedly (EGF, VEGF, PDGF-BB). These data suggest that cell therapy with BMSCs has the potential to augment healing of the diabetic wounds. 相似文献
17.
目的:研究p38丝裂原激活蛋白激酶(p38MAPK)和磷脂酰肌醇-3激酶(PI/3K)通路在表皮生长因子(EGF)诱导的激素非依赖性前列腺癌(hormone-refractory prostate cancer,HRPC)PC-3细胞环氧化酶2(cyclooxygen-ase-2,COX-2)表达上调中的作用。方法:MTT法检测EGF(0μg/L)、EGF(10μg/L)、EGF(10μg/L)+PI-3K阻断剂(LY294002,20μmol/L)、EGF(10μg/L)+p38MAPK阻断剂(SC203580,20μmol/L)处理后的细胞增殖情况。RT-PCR和Western印迹测定上述处理24h后PC-3细胞COX-2的表达变化,ELISA测定细胞培养液中前列腺素E2(PGE2)的变化。结果:LY294002和SC203580明显抑制EGF刺激后的PC-3细胞增殖(P<0.05)及EGF诱导的COX-2上调和PGE2生成(P<0.05)。结论:PI-3K通路和p38MAPK通路可能参与了EGF诱导的PC-3细胞COX-2的表达上调。 相似文献
19.
Background. Hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, plays a role as organotrophic factor for regeneration of various organs. HGF has an angiogenic activity and exhibits a potent antiapoptotic activity in several types of cells. Although HGF and the c-Met/HGF receptor are expressed in the heart, the role of HGF in the heart has remained unknown. Methods. After we analyzed changes in expression of endogenous HGF and c-Met mRNA levels in the rat left ventricle after myocardial infarction, the human HGF gene in hemagglutinating virus of Japan (HVJ)-liposome was transfected into the normal whole rat heart. Three days after transfection, the heart was subjected to global warm ischemia and subsequent reperfusion, followed by assessment of its cardiac functions. Results. Both HGF and c-Met/HGF receptor mRNAs were expressed in adult rat heart, and c-Met/HGF receptor mRNA was upregulated in response to myocardial infarction. HGF-transfected heart showed significant increase of human HGF protein level in the heart. Cardiac functions in terms of the left ventricular developed pressure, maximum dp/dt, and pressure rate product in hearts with HGF gene transfection were significantly superior to those in control hearts. In addition, leakage of creatine phosphokinase in the coronary artery effluent in hearts with HGF gene transfection was significantly lower than that in control hearts. Conclusions. These data indicated that both HGF and c-Met/HGF receptor mRNAs were upregulated in response to myocardial ischemic injury, and that HGF is likely to have a cytoprotective effect on cardiac tissue, presumably through the c-Met/HGF receptor. 相似文献
20.
This study investigated the effects of different frequencies of low intensity ultrasound on osteoblast migration using an in vitro scratch-wound healing assay. Mouse calvarial-derived MC3T3-E1 osteoblasts in culture were exposed to continuous 45?kHz ultrasound (25?mW/cm(2)) or pulsed 1?MHz ultrasound (250?mW/cm(2)) for 30?min followed by 2?days' culture. Ultrasound treatment with either kHz or MHz output similarly and significantly increased cell numbers after 2?days in culture compared with untreated control cultures. In the scratch-wound healing assay the presence of the cell proliferation inhibitor mitomycin C (MMC) did not influence scratch-wound closure in control cultures indicating that cell migration was responsible for the in vitro wound healing. Application of ultrasound significantly stimulated wound closure. MMC did not affect kHz-stimulated in vitro wound healing; however, MMC reduced in part the scratch-wound closure rate in MHz-treated cultures suggesting that enhanced cell proliferation as well as migration was involved in the healing promoted by MHz ultrasound. In conclusion, both continuous kHz and pulsed MHz ultrasound promoted osteoblastic migration; however, subtle differences were apparent in the manner the different ultrasound regimens enhanced in vitro scratch-wound healing. 相似文献
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