c-Jun-N-Terminal Kinase Drives Cyclin D1Expression and Proliferation During Liver Regeneration Robert F.Schwabe,1,2Cynthia A.Bradham,1,2Tetsuya Uehara,1,2Etsuro Hatano,1,2Brydon L.Bennett,3
Robert Schoonhoven,4and David A.Brenner1,2
The c-Jun-N-terminal kinase(JNK)pathway is strongly activated after partial hepatectomy
(PH),but its role in hepatocyte proliferation is not known.In this study,JNK activity was
blocked with the small molecule inhibitor JNK SP600125in vivo and in vitro as shown by
a reduction of c-Jun phosphorylation,AP-1DNA binding activity,and c-jun messenger
RNA(mRNA)expression.SP600125inhibited proliferating cell nuclear antigen(PCNA)
expression,cyclin D1mRNA and protein expression and reduced mitotic?gures after PH.
Survival was reduced signi?cantly3days after PH in SP600125-treated versus vehicle-
treated rats(3of11vs.8of9,P<.01).In epidermal growth factor(EGF)-treated primary
cultures of rat hepatocytes,SP600125decreased3H-thymidine uptake,cyclin D1mRNA
and protein expression,and inhibited the EGF-induced transcription of a cyclin D1pro-
moter-driven reporter gene.The defective regeneration and the decreased survival in
SP600125-treated rats did not result from a major increase in apoptosis as shown by normal
levels of caspase3activity and only slight increases in apoptotic?gures.In conclusion,our
data show that JNK drives G0to G1transition in hepatocytes and that cyclin D1is a
downstream target of the JNK pathway during liver regeneration.(H EPATOLOGY2003;37:
824-832.)
T wo-thirds partial hepatectomy(PH)of the liver results in the rapid and potent activation of mul-
tiple cellular signaling pathways inducing a pro-liferative response of hepatocytes,which allows the liver to regain its original size and cell mass within7to10days.1,2 Although c-Jun-N-terminal kinase(JNK)is strongly ac-tivated within minutes after PH,3,4the function of JNK activation in liver regeneration has not yet been deter-mined.The JNK/c-Jun pathway is a critical component of the proliferative response and induces G0to G1cell cycle progression in many cell types.5Fibroblasts derived from c-Jun null mice have an impaired proliferation and reduced activity of cyclin D1–dependent kinases.6Fibro-blasts derived from JNK1?/?JNK2?/?mice have a defect in proliferation that resembles the defect of c-Jun ?/??broblast and?broblasts with a mutated c-Jun.6-8
The cyclin D1gene has emerged as an important target for the JNK/c-Jun pathway in driving proliferation.The cyclin D1promoter contains an activating protein1 (AP-1)site and ectopic expression of either c-fos or c-Jun induces cyclin D1messenger RNA(mRNA),9which in turn is critical in driving G0to G1cell cycle progression in many cell types including hepatocytes.10During liver regeneration,the activation of JNK is preceded by the release of tumor necrosis factor?(TNF-?).Inhibition of TNF-?or inactivation of the TNF receptor(TNFR)1 lead to defects in liver regeneration and increased mortal-ity after PH and is associated with a reduced JNK activa-tion.3,11TNF-?does not act as a strong mitogen itself, but may prime hepatocytes for the proliferative action of growth factors such as epidermal growth factor(EGF), hepatocyte growth factor(HGF),and transforming growth factor?through mechanisms that potentially in-volve AP-1,STAT3,C/EBP?,and nuclear factor?B (NF-?B).2,12
In this study,we determined the role of JNK in hepa-tocyte proliferation during liver regeneration and in EGF-
Abbreviations:PH,partial hepatectomy;JNK,c-Jun-N-terminal kinase;AP-1, activating protein1;mRNA,messenger RNA;TNF,tumor necrosis factor;EGF, epidermal growth factor;HGF,hepatocyte growth factor;NF-?B,nuclear factor?B;PCNA,proliferating cell nuclear antigen;PCR,polymerase chain reaction;IL, interleukin;DMSO,dimethyl sulfoxide.
From the1Departments of Medicine,2Biochemistry and Biophysics,4Environ-mental Sciences and Engineering,University of North Carolina at Chapel Hill, Chapel Hill,NC;and3Signal Research Division,Celgene Corporation,San Diego,CA.
Received August8,2002;accepted January6,2003.
Supported in part by grants DK-34987and GM41804from the National Institutes of Health.
Address reprint requests to:David A.Brenner,M.D.,University of North Caro-lina,Department of Medicine,CB#7038,Chapel Hill,NC27599.E-mail: dab@https://www.doczj.com/doc/0d14097419.html,;fax:919-966-7468.
Copyright?2003by the American Association for the Study of Liver Diseases. 0270-9139/03/3704-0016$30.00/0
doi:10.1053/jhep.2003.50135
824
treated primary hepatocytes by blocking JNK with the small molecule inhibitor SP600125.13Inhibition of JNK resulted in a diminished proliferative response as shown by a decrease of proliferating cell nuclear antigen(PCNA) expression and mitotic?gures.Cyclin D1was a transcrip-tional target of JNK and may mediate its pro-proliferative effects in hepatocytes.
Materials and Methods
SP600125Treatment in Animals Undergoing PH. All animal studies were approved by the University of North Carolina Ethics Board.Male Sprague-Dawley rats (200-225g)were subcutaneously injected with either SP600125(a gift from Celgene Corporation,San Diego, CA)at a dose of6mg/kg or the corresponding volume(6 mL/kg)of PPCES vehicle(30%PEG-400/20%polypro-pylene glycol/15%Cremophor EL/5%ethanol/30%sa-line).One hour later,animals were anesthetized with ketamine and two-thirds PH was performed according to Anderson and Higgins as previously described.14Animals were killed at various time points after PH and the rem-nant liver was snap frozen or?xed in4%paraformalde-hyde.For all24-and60-hour PH and survival experiments,treatment groups(i.e.,vehicle or SP600125 treated)consisted of at least4rats.
Hepatocyte Isolation and SP600125Treatment of Cultured Hepatocytes.Hepatocytes were isolated from male Sprague-Dawley rats by collagen perfusion as de-scribed previously.15Hepatocytes were plated in6-well plates(0.5?106cells/well)coated with rat type I collagen in Waymouth’s medium containing10%fetal bovine se-rum,0.1mmol/L insulin,and0.1mmol/L dexametha-sone in a humidi?ed atmosphere at37°C and5%CO2. After3hours,the culture was washed with phosphate-buffered saline and changed to hormonally de?ned medium containing0.1mmol/L insulin,2mmol/L L-glutamine,5mg/mL transferrin,1nmol/L selenium, and10nmol/L free fatty acids in RPMI basal medium. For cyclin D1expression and3H-thymidine incorpora-tion studies,hepatocytes were plated in Williams E media containing5mmol/L pyruvate and0.1mmol/L insulin. Two hours before EGF(20ng/mL)treatment,hepato-cytes were pretreated with SP600125at a concentration of20?mol/L or0.1%dimethyl sulfoxide(DMSO). Western Blot Analysis.Snap frozen liver samples or cultured hepatocytes were lysed as previously described.16 Acrylamide gels were loaded with50to100?g protein. Protein transfer onto nitrocellulose membranes was checked by Ponceau S.Blots were incubated with either anti–cyclin D1antibody(Santa Cruz Biotechnology, Santa Cruz,CA)anti–phospho-c-Jun(Santa Cruz Bio-technology),anti–phospho-STAT3(Cell Signaling,Bev-erly,MA),or?-phospho Erk antibody(New England Biolabs,Beverly,MA)at a dilution of1:1,000for1to3 hours followed by incubation with goat-anti-mouse sec-ondary antibody at1:1,000for1hour and chemilumi-nescent detection.For the con?rmation of equal loading, blots were reprobed with anti-tubulin primary antibody (Oncogene Science,Cambridge,MA)or anti-actin pri-mary antibody(ICN,Costa Mesa,CA).
Northern Blot Analysis.Twenty micrograms of RNA was separated by gel electrophoresis on2.2mol/L formaldehyde1%agarose gels,transferred to nylon mem-branes,and hybridized with32P-labeled probes for c-jun as previously described.17
Reverse-Transcription Polymerase Chain Reaction. RNA was prepared from frozen liver tissue by the Trizol method.One microgram of RNA was reverse transcribed as previously described.16One microliter of the reverse-transcription reaction was subjected to polymerase chain reaction(PCR)for either cyclin D1,?-actin,or interleu-kin6(IL-6).Cyclin D1was ampli?ed for28cycles using 5?-GCG AAG TGG AGA CCA TCC G-3?sense and 5?-GTC CAC ATC TCG GAC GTC G-3?antisense primer at a concentration of1?mol/L in10mmol/L Tris (pH9.2),35mmol/L MgCl2,75mmol/L KCl,and100?g/mL bovine serum albumin.IL-6was ampli?ed for40 cycles in10mmol/L Tris(pH8.3),50mmol/L KCl,and 1.5mmol/L MgCl2using previously described primers.18
?-actin was ampli?ed for28cycles as previously de-scribed.16
Electrophoretic Mobility Shift Assay.Nuclear ex-tracts were prepared as described.16Five micrograms of nuclear protein were incubated with100pg of32P labeled probe containing the AP-1consensus site in buffer con-taining10mmol/L HEPES pH7.8,2mmol/L MgCl2,50 mmol/L KCl,1mmol/L dithiothreitol,0.1mmol/L ethylenediaminetetraacetic acid,20%glycerol,single-stranded oligonucleotide(25?g/mL),and poly dI/dC (25?g/mL)for15minutes on ice. Immunohistochemistry.Liver tissue was?xed in4% paraformaldehyde for20hours.PCNA expression was detected by immunostaining using monoclonal anti-PCNA primary antibody(DAKO,Carpinteria,CA)at a concentration of50?g/mL for10minutes,the DAKO Envision system(DAKO),and3.3-diaminobenzidine substrate as previously described.19PCNA expression was quanti?ed using Bioquant TCW98software(Biometrics, Nashville,TN)and an Olympus microscope(Melville, NY).
3H-Thymidine Incorporation Assay.Two hours af-ter plating,hepatocytes were pretreated with20?mol/L SP600125or DMSO for2hours and then treated with
HEPATOLOGY,Vol.37,No.4,2003SCHWABE ET AL.825
20ng/mL EGF(R&D Systems,Minneapolis,MN).Two ?Ci3H-thymidine were added18hours before subject-ing the cells to precipitation with10%trichloroacetic acid.Cells were lysed in0.2N NaOH and counted in a scintillation counter(Beckman-Coulter,Fullerton,CA). TNF-?Enzyme-Linked Immunosorbent Assay. Whole-liver tissue was lysed in25mmol/L HEPES(pH 7.4)containing0.1%CHAPS(Sigma),5mmol/L MgCl2,1.3mmol/L ethylenediaminetetraacetic acid,1 mmol/L EGTA,protease and phosphatase inhibitors. Cleared lysates were measured by enzyme-linked immu-nosorbent assay(R&D Systems)at a1:5dilution accord-ing to the manufacturer’s description and adjusted to total protein concentration.
Detection of Apoptosis.Apoptosis was quanti?ed by counting nuclei with an apoptotic morphology as previ-ously described.14Caspase3–like activity was determined by incubating whole-cell extracts with amino-4-tri?u-oromethyl coumarin-DEVD as previously described.16 Reporter Gene Assays.To assess the effects of SP600125on cyclin D1transcription,hepatocytes were transfected with either?964CD1Luc,or ?964mtCD1luc.9For transfection,2.5?g of plasmid DNA,0.5?g of Renilla-luc Tk plasmid,and2.5?L transfect F1reagent(Targeting Systems,San Diego,CA) were added to the cells in OptiMEM media for2hours. After12hours,the cells were treated with EGF for16 hours.Luciferase activity was determined on a2010lu-minometer(Analytical Luminescence,San Diego,CA) and adjusted to the internal Renilla-luc control. Statistics.A paired Student’s t test was performed us-ing Microsoft Excel(Redmond,WA)and a P level less than.01was considered statistically signi?cant.For sur-vival studies after PH,the Wilcoxon test was performed and a P value of.01was used as criterion of statistical signi?cance.
Results
SP600125Inhibits JNK Activity After PH and in
Cultured Hepatocytes.To analyze the effects of SP600125on JNK activity,rats were treated with SP600125or vehicle1hour before PH and killed1hour after PH,which corresponded to the peak of JNK activity after PH.4Because SP600125acts as a reversible inhibitor of JNK and may be washed out in JNK activity assays,we analyzed signaling events downstream of JNK activation. SP600125speci?cally inhibited c-Jun phosphorylation (Fig.1A)and c-Jun mRNA induction and blunted the induction of AP-1DNA binding after PH(Fig.1B),but did not affect signal transducer and activator of(STAT)3 phosphorylation(Fig.1A).In cultured hepatocytes,EGF rapidly induced the phosphorylation of c-Jun,which was almost completely blocked by SP600125(Fig.1C). SP600125speci?cally blocked JNK and did not in?uence TNF-?–induced I?B degradation,EGF-induced Akt ac-tivation(unpublished data),and EGF-induced Erk phos-phorylation(Fig.1C),which is consistent with previous studies in other cell types.13,20To exclude that the effects of SP600125after PH were caused mainly by blocking the release of the cytokines that are required for normal liver regeneration,we determined the effects of SP600125 on IL-6and TNF-?after PH.Hepatic levels of IL-6 mRNA and TNF-?protein were not in?uenced
by Fig.1.SP600125inhibits JNK activity in the regenerating liver and in cultured hepatocytes.(A)The effect of SP600125on JNK activity after PH was determined by Western blot analysis of the JNK target c-Jun in vehicle-and SP600125-treated rats(upper panel).To show the speci-?city of SP600125,STAT3phosphorylation was analyzed after PH in vehicle-and SP600125-treated rats(lower panel).(B)The induction of c-jun mRNA and AP-1DNA binding activity was examined by Northern blot and electrophoretic mobility shift assays,respectively,1hour after 70%PH in untreated,vehicle-treated,and SP600125-treated rats.(C) Hepatocytes were pretreated with SP600125(20?mol/L)for2hours followed by EGF(20nmol/L)treatment for20minutes.Phosphorylation of c-Jun and Erk were determined by Western blot analysis.(D)IL-6 mRNA was determined by reverse-transcription PCR in livers from vehicle-or SP600125-treated rats before or after PH and compared with levels of ?-actin.RNA from Rat1?broblasts served as a positive control for IL-6.
(E)TNF-?levels from whole-liver extracts from vehicle-or SP600125-treated rats were determined by enzyme-linked immunosorbent assays before PH,30minutes,and90minutes after PH.
826SCHWABE ET AL.HEPATOLOGY,April2003
SP600125(Fig.1D and E).The decrease of intrahepatic TNF-?seen after PH most likely was caused by the re-lease of preformed TNF-?and loss of intracellular stor-ages.
SP600125Inhibits Proliferation After PH.To ad-dress the speci ?c role of JNK in liver regeneration,we analyzed the proliferative response after PH in the pres-ence or absence of SP600125.As expected,vehicle-treated rats showed a strong increase in the number of PCNA-positive nuclei 24hours after PH (12.3%?7%,n ?4)compared with rats before PH (0.15%?0.12%,Fig.2A).In contrast,SP6-treated rats showed a marked decrease in the number of PCNA-positive nuclei (1.14%?0.22%,n ?4)that signi ?cantly differed from vehicle-treated rats (Student ’s t test,P value ?.01)and was nearly as low as those of livers before PH.After 60hours there was a further increase in PCNA expression in vehicle-treated animals (31.7%?2.9%)and at this time point,SP600125-treated animals also showed a signi ?cant per-centage of PCNA-positive nuclei (21.0%?7.1%)that was reduced only slightly in comparison with the vehicle control (Fig.2A).Mitotic ?gures were low in both treat-ment groups 24hours after PH.Sixty hours after PH there was a high percentage of mitotic hepatocytes in ve-hicle-treated animals,but almost no mitotic ?gures in SP600125-treated hepatocytes (Fig.2B).These ?ndings suggest that JNK exerts an important role in promoting G0exit and cell cycle progression in hepatocytes after PH.SP600125Inhibits Cyclin D1Expression After PH.To further investigate the functional links between JNK activation and proliferation,we analyzed the ex-pression of the G1cell cycle regulator cyclin D1that contains an AP-1site in its promoter.9Cyclin D1ex-pression is suf ?cient to drive hepatocytes to enter the cell cycle.10In vehicle-treated animals,cyclin D1pro-tein expression was strongly up-regulated 24hours af-ter PH.In contrast,SP600125-pretreated animals showed a marked reduction in cyclin D1protein ex-pression after 24hours (Fig.3A).To determine whether JNK exerted its effect on cyclin D1through a transcriptionally regulated mechanism as suggested by the AP-1site in its promoter,we analyzed cyclin D1mRNA levels by reverse-transcription PCR.Cyclin D1mRNA was induced 24hours after PH in vehicle-treated animals (Fig.3B).This induction was reduced in SP600125-treated animals in comparison with vehi-cle treated animals (Fig.3B),but to a slightly lesser degree than cyclin D1protein levels,whereas ?-actin mRNA levels were not affected,suggesting that JNK controls cyclin D1expression mainly through a tran-scriptionally regulated
pathway.
Fig.2.SP600125inhibits PCNA expression and mitosis in the regen-erating liver.(A)PCNA expression in vehicle-and SP600125-treated rats was examined 24and 60hours af-ter PH by immunohistochemistry as described in the Materials and Methods section and quanti?ed us-ing image analysis software.Statis-tical signi?cance was determined by the t test.(B)Mitotic ?gures are indicated by arrows in hematoxylin-eosin–stained section 60hours after PH.Mitotic ?gures were counted in 20?elds of animals killed after 24and 60hours after PH.
HEPATOLOGY,Vol.37,No.4,2003SCHWABE ET AL.827
SP600125Inhibits Hepatocyte Proliferation and Cyclin D1Expression in Cultured Hepatocytes.The complete mitogen EGF signi ?cantly increases c-Jun phosphorylation in cultured primary rat hepatocytes (Fig.1C)and drives cell cycle progression in cultured hepato-cytes,thereby mimicking some of the pro-proliferative signaling occurring during PH.4,21To determine whether JNK is critical for EGF-induced proliferation in cultured hepatocytes,we examined the effect of SP600125on 3H-thymidine incorporation and cyclin D1expression.EGF induced a 2-fold increase in 3H-thymidine incorporation after 24hours,and a 6-fold increase after 48hours (Fig.4A).When preincubated with SP600125,3H-thymidine uptake decreased by 48%(24-hour time point,P ?.01)and 67%(48-hour time point,P ?.01)in the EGF-treated hepatocytes and also was lower in the absence of EGF.To test whether cyclin D1transcription was inhib-ited by SP600125in vitro ,we transfected hepatocytes with a reporter gene containing the ?964cyclin D1pro-moter linked to ?re ?y luciferase (?964CD1Luc)or the same reporter gene with a mutated AP-1site at ?954(?964mtCD1Luc).9EGF induced the expression of this reporter gene 2-fold (Fig.4B).Pretreatment with SP600125reduced the transcription of this reporter gene in EGF-treated hepatocytes by 40%but did not affect the internal control Tk-driven renilla luciferase.The reduced luciferase activity was comparable with the level observed
in cells transfected with ?964mtCD1Luc,which lacks the AP-1binding site in its promoter (Fig.4B).To further con ?rm this data,we analyzed cyclin D1mRNA and protein expression in EGF-treated hepatocytes in the presence or absence of SP600125.Both cyclin D1mRNA and protein levels were induced after 48hours of EGF treatment,and pretreatment with SP600125strongly re-duced the levels of cyclin D1mRNA and protein (Fig.4C and D).
Inhibition of JNK Decreases Survival After PH.Previous studies have shown that the absence of critical signaling molecules such as c-Jun or TNF-receptor 1in-hibits liver regeneration and decreases survival after PH and is associated with increased microvesicular steato-sis.11,22In the present study,JNK inhibition
decreased
Fig.3.SP600125inhibits cyclin D1expression after PH.(A)Cyclin D1protein expression was determined by Western blot analysis in SP600125-or vehicle-treated rats 24hours after PH.Cyclin D1expres-sion was quanti?ed using NIH Image software (Bethesda,MD)and normalized to a band from the Coomassie stained blot.(B)Cyclin D1mRNA and ?-actin mRNA were detected by reverse-transcription PCR in either SP600125-or vehicle-treated rats 24hours after PH.Cyclin D1and ?-actin bands were quanti?ed by densitometry.Shown is the average of the ratio between cyclin D1and ?-actin mRNA in each treatment
group.
Fig.4.SP600125inhibits 3H-thymidine incorporation and cyclin D1transcription in cultured hepatocytes.(A)3H-thymidine incorporation was measured in hepatocytes in the presence or absence of EGF (20nmol/L)in either SP600125-treated (20?mol/L)or DMSO-treated (0.1%)hepa-tocytes after 24and 48hours of treatment.Statistical signi?cance was determined by the t test.(B)Cyclin D1–dependent transcription was determined by reporter gene assay.Hepatocytes were transfected with ?964CD1luc or ?964mutCD1luc and treated with EGF (20nmol/L)after SP600125(20?mol/L)or DMSO (0.1%)pretreatment as de-scribed in the Materials and Methods section.Statistical signi?cance was determined by the t test.(C)Cyclin D1and ?-actin mRNA levels were determined by PCR 48hours after EGF (20nmol/L)stimulation in the presence or absence of SP600125(20?mol/L).(D)Cyclin D1and actin protein levels were determined by Western blot analysis 48hours after EGF (20nmol/L)stimulation in the presence or absence of SP600125(20?mol/L).
828SCHWABE ET AL.HEPATOLOGY,April 2003
survival 3days after PH (Fig.5A,vehicle 89%vs.SP60012527%,P ?.01,Wilcoxon test)and SP600125-treated animals had elevated transaminase levels 60hours after PH (Fig.5B,P ?.01).SP600125-treated animals showed a high degree of microvesicular steatosis 2to 3days after PH (Fig.5C).However,vehicle-treated animals also showed microvesicular steatosis,albeit to a lesser de-gree.To determine whether JNK blockade induced an increase in mortality due to higher rates of apoptosis as shown in animals with defective NF-?B and inducible nitric oxide synthase in previous studies,14,23we quanti-?ed apoptotic ?gures in hematoxylin-eosin –stained liver sections of SP600125-and vehicle-treated animals.In ad-dition,caspase 3–like activity was measured in extracts from these livers.In both vehicle-and SP600125-treated animals,the numbers of apoptotic hepatocytes were low.Twenty-four hours after PH,apoptotic ?gures were ele-vated slightly in SP600125-treated animals,but at all other time points the number of apoptotic ?gures was
similar (Fig.5D).These ?ndings were con ?rmed by the low caspase 3–like activity after PH in both SP600125-and vehicle-treated animals after 24and 60hours,whereas caspase 3–like activity was highly elevated in hepatocytes treated with the agonistic Jo2antibody (Fig.5E).Thus,the reduced hepatocyte proliferation in SP600125-treated animals after PH did not appear to be caused by an increase in hepatocyte apoptosis.
Discussion
In the adult liver,hepatocytes are long-lived and rarely undergo proliferation,yet they retain a remarkable ability to proliferate.1This allows the liver to regain its original size within 7to 10days after 70%PH and quickly restore function.This regenerative response is initiated by a series of signaling events that allow the hepatocyte to enter the cell cycle and undergo several rounds of proliferation.JNK activation is one of the earliest signals to be
detected
Fig. 5.JNK inhibition increases mortality after PH.(A)Rats were pre-treated with SP600125or vehicle 1hour before PH.Survival was moni-tored over the following 3days and is shown as a Kaplan-Meier graph.Statistical signi?cance was deter-mined by the Wilcoxon test.(B)Rats were pretreated with SP600125or vehicle 1hour before PH.The levels of aspartate and alanine amino-transferases were determined 60hours after PH.Statistical signi?-cance was determined by the t test.(C)Sections from SP600125-or ve-hicle-treated animals 60hours after PH were stained with hematoxylin-eosin.(D)Rats were pretreated with SP600125or vehicle 1hour before PH and livers were harvested 24or 60hours after PH.Cells with typical apoptotic morphology were counted in 20?200?elds in 2different animals per treatment group.The number of apoptotic cells was di-vided by the total number of paren-chymal cells and is expressed as percentage.(E)Caspase 3activity was determined by incubating cell extracts from livers harvested 24and 60hours after PH with the caspase 3substrate AFC-DEVD for 2hours.Mouse hepatocytes treated with the agonistic Jo2antibody served as positive control.Caspase 3activity is shown as AFC release (pmol)per ?g protein extract after 2hours of incubation.
HEPATOLOGY,Vol.37,No.4,2003SCHWABE ET AL.829
after PH,but the function of JNK in liver regeneration remains largely unknown.4Several agonists may induce JNK during liver regeneration:(1)TNF-?is an impor-tant mediator of early-phase JNK activation after PH as shown by the reduced JNK activation and AP-1binding activity in rats injected with antibodies to TNF-?and mice with inactivated TNFR1.3,11TNF-?acts as a comi-togen that primes hepatocytes for the pro-proliferative effects of hepatic mitogens including EGF and HGF through the activation of NF-?B,AP-1,STAT3,and the release of IL-6,but alone is incapable to induce hepato-cyte proliferation.3,24,25However,the relative role of these pathways in driving proliferation after PH is unclear and some factors such as NF-?B may only play a minor role.26 (2)EGF and HGF are believed to play a key role in liver regeneration and both induce JNK activation and AP-1–dependent gene transcription in cultured hepatocytes.4,27 The merging of2crucial pro-proliferative signaling cas-cades(i.e.,cytokines and growth factors)at the level of JNK points toward a role for JNK in driving proliferation during liver regeneration.This hypothesis is supported by studies showing JNKs to be an important regulator of cell proliferation in other cell types.8,28JNKs phosphorylate the N-terminal domain of c-Jun,increase its transactiva-tion,and thereby up-regulate AP-1–dependent transcrip-tion.Additionally,JNKs phosphorylate the transcription factors ATF2and JunD.These factors homodimerize and heterodimerize with other AP-1components to induce AP-1–dependent transcription and the up-regulation of many genes that are critically involved in cell proliferation and survival.5
JNK Activity Is Required for Hepatocyte Prolifera-tion.The liver contains JNK1and JNK2,which are be-lieved to ful?ll similar functions,but it does not contain the neuronal form JNK3.Because JNK1?/?/JNK2?/?mice are not viable,29,30our study used a pharmacologic approach to study the role of JNKs in liver regeneration. The small molecule JNK inhibitor SP600125blocks both JNK1and JNK2in a highly speci?c manner13and ef?-ciently inhibited JNK activity in the liver and cultured hepatocytes in our study.Blocking JNK activity with SP600125inhibited the normal proliferative response af-ter PH,indicating that JNKs are essential components of the cellular machinery that drives hepatocytes to enter the cell cycle after PH.SP600125-treated animals showed a blocked G1/S transition,as apparent by the decreased expression of PCNA and by the reduction of the levels of cyclin D1,which is predominantly expressed during G1. SP600125additionally reduced mitotic?gures after PH and3H-thymidine incorporation in cultured hepatocytes. At later time points,SP600125-treated animals had sim-ilar expression levels of PCNA,indicating the absence of early-phase JNK activity still allows hepatocytes to enter the cell cycle,albeit in a delayed manner.
JNK Activation Drives Cyclin D1Expression.Sev-eral of the AP-1regulated genes are critical regulators of cell growth,among them cyclin D1and PCNA.9,31The importance of cyclin D1as a critical downstream target of JNK is suggested by the impaired transcription of cyclin D1and the reduced activity of cyclin D1–dependent ki-nases in c-jun?/?mouse embryonic?broblasts.6,32In hepatocytes,cyclin D1plays a crucial role in driving pro-liferation as recently shown by a study in which cyclin D1 overexpression per se was suf?cient to promote hepato-cyte replication.10Moreover,it has been shown that cy-clin D1is the most prominently up-regulated type D cyclin after PH and in response to mitogenic stimuli and plays a critical role in the driving cells through the G1 restriction point.33Our study found a strong reduction of cyclin D1protein and mRNA expression in SP600125-treated animals after PH.Similar results were obtained in cultured hepatocytes in which cyclin D1mRNA and pro-tein levels as well as the transcription of a cyclin D1–promoter-driven reporter gene were decreased by SP600125in EGF-treated hepatocytes.Cyclin D1ex-pression in the liver may be regulated at the posttranscrip-tional level.34,35In our study,however,cyclin D1was regulated mainly at the transcriptional level through a JNK-dependent pathway,suggesting that cyclin D1is an important mediator of pro-proliferative effects of JNK.In conjunction with results from previous studies,our data indicates that the growth factor?TNF-?3JNK3 AP-13cyclin D1pathway is crucial in driving prolifer-ation during liver regeneration.
JNK Blockade Decreases Survival After PH.Inac-tivation of important signaling pathways such as TNF-?and IL-6impairs liver regeneration and decreases survival after PH.11,25In our study,SP600125signi?cantly re-duced survival3days after PH.SP600125-treated ani-mals showed a higher degree of microvesicular steatosis similar to mice with inactivated TNFR1or c-jun.11,22 However,vehicle-treated animals,also showed increased microvesicular steatosis in our study,presumably due to components in the drug vehicle that may exacerbate the low degree of microvesicular steatosis occurring normally after PH.11Animals that survived day3after PH showed a slightly delayed but ultimately normal liver regeneration with a slightly decreased liver-to-body-weight ratio at day 3and a normal liver-to-body-weight ratio at day5(data not shown).These data are consistent with other studies in which the blockade of single signaling pathways caused a signi?cant delay of liver regeneration and increased mortality,but an ultimately normal regeneration in sur-viving animals,most likely due to compensatory effects of
830SCHWABE ET AL.HEPATOLOGY,April2003
other pathways.25This hypothesis is supported addition-ally by our?nding that the expression of PCNA was al-most as high in SP600125-as in vehicle-treated animals 60hours after PH.
JNK Blockade Does Not Signi?cantly Increase Apoptosis After PH.Previous studies have shown that some signaling cascades after PH such as NF-?B,induc-ible nitric oxide sythase,and Akt function to protect hepa-tocytes from undergoing apoptosis.14,23,36JNK is involved in the regulation of cell death in many cell types and may have both pro-or antiapoptotic effects.37JNK activity and c-Jun are required for normal liver develop-ment and appear to protect hepatocytes from apoptosis during development as seen in mice lacking either the JNK kinase SEK1or c-Jun.38-42Therefore,we wanted to exclude the possibility that the decreased survival and the decreased proliferation in SP600125-treated animals after PH was caused by an increased rate of apoptosis.Caspase 3–like activity was similar in the treatment groups and apoptotic bodies were only slightly more frequent in SP600125-treated animals24hours after PH,but not at other time points.These?ndings are further supported by data from our laboratory that JNK has proapoptotic ef-fects in TNF-?–induced apoptosis of hepatocytes43and reperfusion injury.44
The Role of c-Jun Phosphorylation in Liver Regen-eration.The AP-1component c-Jun is a major contrib-utor of AP-1activity in quiescent liver and after PH.4The occurrence of proliferative defects in both JNK1?/?/ JNK2?/?MEFs and c-jun?/?MEFs indicate that c-Jun serves as a major JNK target in proliferation.6,8,32 However,proliferative defects in c-jun?/?MEFs are more severe than in MEFs containing a c-Jun with mu-tated phosphorylation sites63and73,7and N-terminal phosphorylation of c-Jun is dispensable for liver develop-ment and hepatocyte proliferation after PH.22,45These results indicate that c-Jun has functions independent of its N-terminal phosphorylation.Our study indicates that JNK activity is required for hepatocyte proliferation after PH and in culture and seems to contradict previous stud-ies.22,45However,JNKs regulate AP-1–dependent tran-scription through the phosphorylation of several targets including c-Jun,ATF2,and JunD,indicating that inhi-bition of c-Jun phosphorylation through the use of a mu-tated c-Jun in previous studies may have been compensated for by other JNK targets.Evidence support-ing this hypothesis comes from a recent study showing that the knock-in of a second JunB allele can reverse de-velopmental hepatic abnormalities and embryonic lethal-ity of c-Jun?/?mice and up-regulate c-Jun targets including cyclin D1,46indicating that Jun family mem-bers,and potentially other AP-1components,have a higher degree of redundancy than previously appreciated. However,it appears that the presence of c-Jun in the AP-1 complex is absolutely required for cell cycle progression and protection from apoptosis after PH as seen in the c-jun?/?mice.22The striking difference between the complete absence of c-Jun and deletion of its N-terminal phosphorylation sites indicate that unphosphorylated c-Jun may contribute to AP-1–dependent transcription after PH.22Potential explanations are that(1)het-erodimerization of other AP-1components with c-Jun induces AP-1transcription independent of the phosphor-ylation status of c-Jun and/or that(2)JNK targets such as JunD,which lack a JNK docking site,use a JNK docking site containing partners(including mutated c-Jun)for JNK recruitment and their subsequent phosphorylation resulting in increased AP-1–dependent transcription.47 Future studies have to determine whether JNK has other substrates than c-Jun to drive hepatocyte proliferation. References
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Harbin Institute of Technology 机械原理课程设计说明书 课程名称：机械原理 设计题目：产品包装生产线（方案1） 院系：机电学院 班级： 设计者： 学号： 指导教师： 设计时间：
一、绪论 机械原理课程设计是在我们学习了机械原理之后的实践项目，通过老师和书本的传授，我们了解了机构的结构，掌握了机构的简化方式与运动规律，理论知识需要与实践相结合，这便是课程设计的重要性。我们每个人都需要独立完成一个简单机构的设计，计算各机构的尺寸，同时还需要编写符合规范的设计说明书，正确绘制相关图纸。 通过这个项目，我们应学会如何收集与分析资料，如何正确阅读与书写说明书，如何利用现代化的设备辅助工作。这种真正动手动脑的设计有效的增强我们对该课程的理解与领会，同时培养了我们的创新能力，为以后机械设计课程打下了坚实的基础。 二、设计题目 产品包装生产线使用功能描述 图中所示，输送线1上为小包装产品，其尺寸为长?宽?高=600?200?200，小包装产品送至A处达到2包时，被送到下一个工位进行包装。原动机转速为1430rpm，每分钟向下一工位可以分别输送14，22，30件小包装产品。 产品包装生产线（方案一）功能简图 三、设计机械系统运动循环图 由设计题目可以看出，推动产品在输送线1上运动的是执行构件1，在A处把产品推到下一工位的是执行构件2，这两个执行构件的运动协调关系如图所示。 ?1?1 执行构件一 执行构件二 ?01?02 运动循环图
图中?1 是执行构件1的工作周期，?01 是执行构件2的工作周期，?02是执行构件2的动作周期。因此，执行构件1是做连续往复运动，执行构件2是间歇运动，执行构件2的工作周期?01 是执行构件1的工作周期T1的2倍。执行构件2的动作周期?02则只有执行构件1的工作周期T1的二分之一左右。 四、 设计机械系统运动功能系统图 根据分析，驱动执行构件1工作的执行机构应该具有的运动功能如图所示。运动功能单元把一个连续的单向传动转换为连续的往复运动，主动件每转动一周，从动件（执行构件1）往复运动一次，主动件转速分别为14,22,30rpm 14,22,30rpm 执行机构1的运动功能 由于电动机的转速为1430rpm ，为了在执行机构1的主动件上分别得到14、22、30rpm 的转速，则由电动机到执行机构1之间的总传动比i z 有3种，分别为 i z1= 141430 =102.14 i z2=221430=65.00 i z3=30 1430=47.67 总传动比由定传动比i c 和变传动比i v 两部分构成，即 i z1=i c i v1 i z2=i c i v2 i z3=i c i v3 3种总传动比中i z1最大，i z3最小。由于定传动比i c 是常数，因此，3种变传动比中i v1最大，i v3最小。为满足最大传动比不超过4，选择i v1 =4 。 定传动比为 i c = v1 z1i i =4102.14=25.54 变传动比为 i v2= c z2i i =54.2565=2.55 i v3= c z3i i =54 .2547.67=1.87 传动系统的有级变速功能单元如图所示。 i=4,2.55,1.87 有级变速运动功能单元
1、函数 ()12 ++=x x x f 与函数()11 3--=x x x g 相同． 错误 ∵当两个函数的定义域和函数关系相同时，则这两个函数是相同的。 ∴()12 ++=x x x f 与()11 3--=x x x g 函数关系相同，但定义域不同，所以()x f 与 ()x g 是不同的函数。 2、如果()M x f >（M 为一个常数），则()x f 为无穷大． 错误 根据无穷大的定义，此题是错误的。 3、如果数列有界，则极限存在． 错误 如：数列()n n x 1-=是有界数列，但极限不存在 4、a a n n =∞ →lim ，a a n n =∞ →lim ． 错误 如：数列()n n a 1-=，1)1(lim =-∞ →n n ，但n n )1(lim -∞ →不存在。 5、如果()A x f x =∞ →lim ，则()α+=A x f （当∞→x 时，α为无穷小）． 正确 根据函数、极限值、无穷小量的关系，此题是正确的。 6、如果α～β，则()α=β-αo ． 正确 ∵1lim =α β ，是 ∴01lim lim =?? ? ??-=-αβαβα，即βα-是α的高阶无穷小量。 7、当0→x 时，x cos 1-与2x 是同阶无穷小． 正确 ∵2122sin 412lim 2sin 2lim cos 1lim 2 02 2020=????? ? ????==-→→→x x x x x x x x x 8、 01 sin lim lim 1sin lim 000=?=→→→x x x x x x x ． 错误 ∵x x 1 sin lim 0→不存在，∴不可利用两个函数乘积求极限的法则计算。 9、 e x x x =?? ? ??+→11lim 0 ． 错误 ∵e x x x =?? ? ??+∞ →11lim 10、点0=x 是函数x x y =的无穷间断点． 错误 =-→x x x 00lim 1lim 00-=--→x x x ，=+→x x x 00lim 1lim 00=+→x x x ∴点0=x 是函数x x y =的第一类间断点． 11、函数()x f x 1 =必在闭区间[]b a ,内取得最大值、最小值．
3.2高速级齿轮传动的设计 3.2.1传动齿轮的设计要求 1)齿轮材料：软齿面齿轮传动 小齿轮：45号钢，调质处理，齿面硬度为240HBS； 大齿轮：45号钢，正火处理，齿面硬度为200HBS。 2)轴向力指向轴的非伸出端； 3)每年300日，每班8小时，两班制 4)齿宽系数； 5)螺旋角； 6)中心距取整，分度圆直径精确计算(保留小数点后两位)。 3.2.2选择齿轮类型，精度等级及齿数 1)参考表10.6，取通用减速器精度等级为7级精度 2)取小齿轮齿数为，齿数比，即大齿轮齿数 ,取； 3)选择斜齿圆柱齿轮，取压力角°； 4)初选螺旋角. 3.2.3按齿面接触疲劳强度设计 1.计算小齿轮的分度圆直径，即 ≥ 1)确定公式中的各参数值 a)试选载荷系数=1.3 b)计算小齿轮传递的转矩
=9.55*?=9.55**4.496/1450(N?mm)=2.96*N?mm c)取齿宽系数=1.0 d)由图10.20查得区域系数=2.433； e)由表10.5查得材料的弹性影响系数=189.8 f)计算接触疲劳强度用重合度系数 =arctan(tan/tan)=arctan(tan20/tan14)=20.562° =arccos =arccos[24*cos20.562/(24+2*1*cos14)]=29.974 =arccos = 22.963 = =[24*(tan29.974-tan22.963)+115*(tan22.963-tan20.562)]/2 =1.474 ==1*24*tan14/=1.905 = g)螺旋角系数===0.985 h)计算接触疲劳许用应力 由图10.25c,d查得小齿轮和大齿轮的接触疲劳极限分别为 =500MPa，=375MPa 应力循环次数分别为 =60=60*1450*1*(2*8*300*8)=3.341*
高中数学·· 教师版 page 1 of 9 函数的相关概念与映射 __________________________________________________________________________________ __________________________________________________________________________________ 1、 通过丰富实例，进一步体会函数是描述变量之间的依赖关系的重要数学模型； 2、 学习用集合与对应的语言来刻画函数，体会对应关系在刻画函数概念中的作用； 3、 了解构成函数的要素，会求一些简单函数的定义域和值域. 一、映射的概念： 设A 、B 是两个非空的集合，如果按某个确定的对应关系f ，对于集合A 中的任意一个元素，在集合B 中都有唯一确定的元素和它对应，那么这样的对应（包括集合A 、B ，以及对应关系f ）叫做集合A 到集合B 的映射，记作：:f A B →。 二、像与原像的概念： 给定一个集合A 到集合B 的映射，且,a A b B ∈∈，如果元素a 和元素b 对应，那么我们把元素b 叫做元素a 的像，元素a 叫做元素b 的原像。 特别提醒：1、对于映射:f A →B 来说，则应注意理解以下四点： （1）集合A 中每一个元素，在集合B 中必有唯一的象；（2）集合A 中不同元素，在集合B 中可以有相同的象；（3）集合A 中的元素与集合B 中的元素的对应关系，可以是：“一对一”、“多对一”，但不能是“一对多”。（4）允许集合B 中的元素没有象； 2、集合A 、B 及对应法则f 是确定的，是一个系统； 3、对应法则f 有“方向性”。即强调从集合A 到集合B 的对应，它与从B 到A 的对应关系一般是不同的； 三、映射： 一般地，设A ，B 是两个非空的集合，:f A →B 是集合A 到集合B 的映射，如果在这个映射
机械原理课程设计大作业 ——齿轮传动系统20 课程名称：机械原理课程设计 设计题目：齿轮传动系统分析 院系：机电工程学院 班级： 15 设计者： 学号： 115 指导教师：陈 设计时间： 2017年6月
1、设计题目 1.1机构运动简图 2、传动比的分配计算 电动机转速min /970r n =，输出转速min /3001r n =，n /3502mi r n =， min /4003r n =，带传动的最大传动比5.2m ax =p i ,滑移齿轮传动的最大传动比 4m ax =v i ，定轴齿轮传动的最大传动比4max =d i 。 根据传动系统的原始参数可知，传动系统的总传动比为： 333.3230970 011=== n n i 714.2735 970 022=== n n i 250.2440 970 033=== n n i
传动系统的总传动比由带传动、滑移齿轮传动和定轴齿轮传动三部分实现。设带传动的传动比为5.2m ax =p i ，滑移齿轮的传动比为321v v v i i i 、、，定轴齿轮传动的传动比为f i ，则总传动比 f v p i i i i 1m ax 1= f v p i i i i 2m ax 2= f v p i i i i 3max 3= 令 4max 3==v v i i 则可得定轴齿轮传动部分的传动比为 425.24 *5.2250 .24max max 3=== v p f i i i i 滑移齿轮传动的传动比为 333.5425 .2*5.2333 .32max 11== = f p v i i i i 571.4425 .2*5.2714 .27max 22== = f p v i i i i 设定轴齿轮传动由3对齿轮传动组成，则每对齿轮的传动比为 4343.1425.2max 3 3=≤== =d f d i i i 3、齿轮齿数的确定 根据滑移齿轮变速传动系统中对齿轮齿数的要求，可大致选择齿轮5、6、7、8、9和10为角度变位齿轮，其齿数：42,8,41，9,40,101098765======z z z z z z ；它们的齿 顶高系数1=* a h ， 径向间隙系数25.0=* c ，分度圆压力角020=α，实际中心距mm a 50' =。 根据定轴齿轮变速传动系统中对齿轮齿数的要求，可大致选择齿轮11、12、13和14 为高度变位齿轮，其齿数：21,1314121311====z z z z 。它们的齿顶高系数1=* a h ，径向 间隙系数25.0=* c ，分度圆压力角020=α,实际中心距mm a 51'=。圆锥齿轮15和16 选择为标准齿轮29,171615==z z ，齿顶高系数1=*a h ，径向间隙系数25.0=* c ，分度 圆压力角为020=α（等于啮合角'α）。 4、齿轮变速传动中每对齿轮几何尺寸及重合度的计算 4.1 滑移齿轮5和齿轮6
第一章 函数、极限与连续 由于社会和科学发展的需要，到了17世纪，对物体运动的研究成为自然科学的中心问题.与之相适应，数学在经历了两千多年的发展之后进入了一个被称为“高等数学时期”的新时代，这一时代集中的特点是超越了希腊数学传统的观点，认识到“数”的研究比“形”更重要，以积极的态度开展对“无限”的研究，由常量数学发展为变量数学，微积分的创立更是这一时期最突出的成就之一.微积分研究的基本对象是定义在实数集上的函数. 极限是研究函数的一种基本方法，而连续性则是函数的一种重要属性.因此，本章内容是整个微积分学的基础.本章将简要地介绍高等数学的一些基本概念，其中重点介绍极限的概念、性质和运算性质，以及与极限概念密切相关的，并且在微积分运算中起重要作用的无穷小量的概念和性质.此外，还给出了两个极其重要的极限.随后，运用极限的概念引入函数的连续性概念，它是客观世界中广泛存在的连续变化这一现象的数学描述. 第一节 变量与函数 一、变量及其变化范围的常用表示法 在自然现象或工程技术中，常常会遇到各种各样的量.有一种量，在考察过程中是不断变化的，可以取得各种不同的数值，我们把这一类量叫做变量；另一类量在考察过程中保持不变，它取同样的数值，我们把这一类量叫做常量.变量的变化有跳跃性的，如自然数由小到大变化、数列的变化等，而更多的则是在某个范围内变化，即该变量的取值可以是某个范围内的任何一个数.变量取值范围常用区间来表示.满足不等式a x b ≤≤的实数的全体组成的集合叫做闭区间，记为,a b ????，即 ,{|}a b x a x b =≤≤????； 满足不等式a x b <<的实数的全体组成的集合叫做开区间，记为(,)a b ，即 (,){|}a b x a x b =<<； 满足不等式a x b <≤(或a x b ≤<)的实数的全体组成的集合叫做左(右)开右(左)闭区间，记为 (,a b ?? (或),a b ??)，即 (,{|}a b x a x b =<≤?? (或),{|}a b x a x b =≤
优秀设计 单级圆柱齿轮减速器的高速级齿轮传动设计
目录 一、传动方案的拟定及电动机的选择 (2) 二、V带选择 (4) 三．高速级齿轮传动设计 (6) 四、轴的设计计算 (9) 五、滚动轴承的选择及计算 (13) 六、键联接的选择及校核计算 (14) 七、联轴器的选择 (14) 八、减速器附件的选择 (14) 九、润滑与密封 (15) 十、设计小结 (16) 十一、参考资料目录 (16)
数据如下: 已知带式输送滚筒直径320mm ，转矩T=130 N ·m ，带速 V=1.6m/s ，传动装置总效率为?=82%。 一、拟定传动方案 由已知条件计算驱动滚筒的转速n ω,即 5.953206 .1100060100060≈??=?= π πυωD n r/min 一般选用同步转速为1000r/min 或1500r/min 的电动机作为原动机，因此传动装置传动比约为10或15。根据总传动比数值，初步拟定出以二级传动为主的多种传动方案。 2.选择电动机 1）电动机类型和结构型式 按工作要求和工作条件，选用一般用途的Y （IP44）系列三相异步电动机。它为卧式封闭结构。 2）电动机容量 （1）滚筒输出功率P w kw n T 3.19550 5.951309550P =?=?= ωω （2）电动机输出功率P kw d 59.1% 823 .1P P == = η ω 根据传动装置总效率及查表2-4得：V 带传动?1=0.945；滚动轴承?2 =0.98；圆柱齿轮传动 ?3 =0.97；弹性联轴器?4 =0.99；滚筒轴滑动轴承?5 =0.94。 （3）电动机额定功率P ed 由表20-1选取电动机额定功率P ed =2.2kw 。
基于MATLAB的齿轮传动系统优化设计 摘要：某高速重载齿轮进行了优化设计，在分析齿轮在各工况下的弯曲强度后，根据齿轮的优化设计原则，选择齿轮体积最小为优化设计原则，对传动齿轮中的小齿轮进行了优化设计，设计模数、齿数、齿宽系数、螺旋角为变量，根据各参数的设计要求来确定约束条件，同时根据齿根弯曲疲劳强度和齿面接触疲劳强度进行条件约束，最后用MATLAB进行编程计算，最后得出优化后的结果，该结果满足要求。本文的研究对机械系统的优化设计具有指导意义和工程应用价值。关键词：齿轮；优化设计；MATLAB； 0引言 优化设计是近年发展起来的一门新的学科，也是一项新技术，在工程设计的各个领域都已经得到了更为广泛的应用。通过实际的应用过程表明：工程设计中采用优化设计这种新的科学设计方法，不仅使得在解决复杂问题时，能够从众多纷繁复杂的设计方案中找到尽可能完善的或者最适合的设计方案，而且，采用这种方法还能够提高设计效率和设计质量，使其的经济和社会效益都非常明显。优化设计的理论基础是数学规划，采用的工具是计算机。 优化设计具有一般的设计方法所不具备的一些特点。优化设计能够使各种设计参数自动向更优的方向进行调整，直到找到一个尽可能完善的或最适合的设计方案。一般的设计方法只是依靠设计人员的经验来找到最佳方案，这样不足以保证设计参数一定能够向更优方向调整，也不能够保证一定能找到最适合的设计方案。优化设计的手段是采用计算机，在很短的时间内就可以分析一个设计方案，并判断方案的优劣、是否可行，因此就能够从大量的方案中选出更加适合的设计方案，这是常规设计所不能比的。 1 机械系统优化设计方法概述 许多机械工程设计都需要进行优化。优化过程可以分为三个部分：综合与分析、评价、改变参数三部分组成。其中，综合与分析部分的主要功能是建立产品设计参数与设计性能、设计要求之间的关系，这也就是一个建立数学模型的过程。评价部分就是对该产品的性能和设计要求进行分析，这就相当于是评价目标函数是否得到改善或者达到最优，也就是检验数学模型中的约束条件是否全部得到满足。改变参数部分就是选择优化方法，使得目标函数（数学模型）得到解，同时根据这种优化方法来改变设计参数。 在许多机械工程设计问题中，优化设计的目标是多种多样的，按照所追求的目标的多少，目标函数可以分为单目标函数和多目标函数。以多级齿轮传动系统设计过程为例，要求在满足规定的传动比和给定最小齿轮、大齿轮直径的条件下，追求系统的转动惯量最小，箱体的体积最小，各级传动中心距和最小，承载能力最高，寿命最长等，这就是一个多目标函数。目标函数作为评价方案中的一个很重要的标准，它不一定有明显的物理意义、量纲，它只是代表设计指标的一个值。所以，目标函数的建立是否正确是优化设计中很重要的一项工作，它既要反映用户的需求，又要敏感地、直接地反映设计变量的变化，对优化设计的质量及计算难易程度都有一定的影响。表2.1给出了常用优化设计中的可供选择的优化目标。 优化设计问题的前提是选择优化设计方法，选用哪个方法好，这就主要是由优化设计方法的特性和实际设计问题的具体情况来决定。一般来讲，评价一种优
3 高速级齿轮设计 3.1 选定齿轮类型，精度等级，材料及齿数 3.1.1 压力角 选定直齿圆柱齿轮，属于一般用途的齿轮传动，压力角取20°。 3.1.2 精度选择 带式输送机为一般工作机器（通用减速器），参考表10-6[2]，选用7级精度。 3.1.3 材料选择 由表10-1[2]，选择小齿轮材料为40Cr （调质），齿面硬度280HBS ，大齿轮材料为45号钢（调质），齿面硬度为240HBS 。硬度差为40HBS 。 3.1.4 齿数选择 闭式齿轮传动，试选小齿轮齿数z 1=20，大齿轮齿数z 2为： 21=z u z ? （3-1） 式中：z 1 ——小齿轮齿数； u ——Ⅰ轴与Ⅱ轴之间的传动比。 故由式3-1，得大齿轮齿数z 2： 2=4.8320=96.6z ? 取z 2=97。 3.2按齿面接触疲劳强度设计 3.2.1 试算小齿轮分度圆直径 小齿轮分度圆直径d 1t 可由下式近似计算： [] 2 131 21 Ht H E d H K T Z Z Z u d m u m ε φσ?? +=?? ? ??? （3-2） （1）确定公式中的各参数值 ①试选K Ht =1.3。
②小齿轮传递的转矩T 1为： 6 19.5510 I I P T N mm n =?? （3-3） 式中：P Ⅰ ——Ⅰ轴的输入功率，单位：kW ； n Ⅰ ——Ⅰ轴的转速，单位：r/min 。 故由式3-3，得小齿轮传递的转矩T 1： 6 411 9.5510 2.38110T P N mm N mm n =??=?? ③因为小齿轮相对支承非对称布置，所以由表10-7[2]，可查得齿宽系数Φd =1。 ④由图10-20[2]，可查得区域系数Z H =2.5。 ⑤由表10-5[2]，可查得材料的弹性影响系数Z E =189.8MPa 1/2。 ⑥接触疲劳强度用重合度系数Z ?为： 3 4α εε-= Z （3-4） 式中：?α——端面重合度，按下式计算： 11* 122* 21122cos arccos[]2cos arccos[]2(tan tan )(tan tan ) 2a a a a a a z z h z z h z z αα αα αααααεπ =+=+-+-= （3-5） 式中：z 1 ——小齿轮齿数； z 2 ——大齿轮齿数； h a * ——齿顶高系数； α ——压力角，单位：°。 故由式3-4、3-5，得接触疲劳强度用重合度系数Z ?：
考研数学高数公式:函数与极限 第一章:函数与极限 第一节:函数 函数属于初等数学的预备知识,在高数的学习中起到铺垫作用,直接考察的内容比较少,但是如果这章节有所缺陷对以后的学习都会有所影响。 基础阶段: 1.理解函数的概念,能在实际问题的背景下建立函数关系; 2.掌握并会计算函数的定义域、值域和解析式; 3.了解并会判断函数的有界性、单调性、周期性、奇偶性等性质; 4.理解复合函数和反函数的概念,并会应用它们解决相关的问题; 强化阶段: 1.了解函数的不同表现形式:显式表示,隐式表示,参数式,分段表示; 2.掌握基本初等函数的性质及其图形,了解初等函数的概念。 冲刺阶段: 1.综合应用函数解决相关的问题; 2.掌握特殊形式的函数(含极限的函数,导函数,变上限积分,并会讨论它们的相关性质。 第二节:极限
极限可以说是高等数学的基础,极限的计算也是高等数学中最基本的运算。在考试大纲中明确要求考生熟练掌握的基本技能之一。虽在考试中站的分值不大。但是在其他的试题中得到广泛应用。因此这部分学习直接营销到整个学科的复习结果 基础阶段 1.了解极限的概念及其主要的性质。 2.会计算一些简单的极限。 3.了解无穷大量与无穷小量的关系,了解无穷小量的比较方法,记住常见的等价无穷小量。 强化阶段: 1.理解极限的概念,理解函数左右极限的概念及其与极限的关系(数一数二/了解数列 极限和函数极限的概念(数三; ▲2.掌握计算极限的常用方法及理论(极限的性质,极限的四则运算法则,极限存在的两个准则,两个重要极限,等价无穷小替换,洛必达法则,泰勒公式; 3.会解决与极限的计算相关的问题(确定极限中的参数; 4.理解无穷大量和无穷小量的概念及相互关系,会进行无穷小量的比较,记住常见的等价无穷小量并能在计算极限时加以应用(数一数二/理解无穷小量的概念,会进行无穷小量的比较,记住常见的等价无穷小量并能在计算极限时加以应用,了解无穷大量的概念及其与无穷小量的关系(数三。 冲刺阶段: 深入理解极限理论在微积分中的中心地位,理解高等数学中其它运算(求导,求积分与极限之间的关系,建立完整的理论体系。
课时授课计划 课次序号：01 一、课题：§1.1 映射与函数 二、课型：新授课 三、目的要求：1.了解集合与映射的有关概念； 2.理解函数的概念，了解函数的四种特性； 3.理解复合函数的概念，了解反函数的概念； 4.熟悉基本初等函数的性质及其图形； 5.会建立简单实际问题的函数关系式． 四、教学重点：函数的概念，函数的各种性态. 教学难点：反函数、复合函数、分段函数的理解. 五、教学方法及手段：启发式教学，传统教学与多媒体教学相结合． 六、参考资料：1.《高等数学释疑解难》，工科数学课程教学指导委员会编， 高等教育出版社； 2.《高等数学教与学参考》，张宏志主编，西北工业大学出版社． 七、作业：习题1–1 3（1），6（4）（7），9（1） 八、授课记录： 九、授课效果分析：
第一章函数与极限 第一节映射与函数 高等数学研究的主要对象是函数. 为了准确而深刻地理解函数概念，集合与映射的知识是不可缺少的. 本节将简要复习回顾集合、映射的一些基本概念，在此基础上重点介绍函数概念与相关知识. 一、集合 1. 集合的概念 集合是数学中的一个最基本的概念．一般地，我们将具有某种确定性质的事物的全体叫做一个集合，简称集．组成集合的事物称为该集合的元素．例如，某大学一年级学生的全体组成一个集合，其中的每一个学生为该集合的一个元素；自然数的全体组成自然数集合，每个自然数是它的元素，等等． 通常我们用大写的英文字母A，B，C，…表示集合；用小写的英文字母a，b，c，…表示集合的元素．若a是集合A的元素，则称a属于A，记作a∈A；否则称a不属于A，记作 a?A（或a∈A）． 含有有限个元素的集合称为有限集；不含任何元素的集合称为空集，用?表示；不是有限集也不是空集的集合称为无限集．例如，某大学一年级学生的全体组成的集合是有限集； 全体实数组成的集合是无限集；方程2x+1=0的实根组成的集合是空集． 集合的表示方法：一种是列举法，即将集合的元素一一列举出来，写在一个花括号内．例如，所有正整数组成的集合可以表示为N={1，2，…，n，…}．另一种表示方法是指明集合元素所具有的性质，即将具有性质p（x）的元素x所组成的集合A记作 A ={x｜x具有性质p（x）}． 例如，正整数集N也可表示成N={n｜n =1，2，3，…}； 又如A={（x，y）｜2x+2y=1，x，y为实数}表示xOy平面单位圆周上点的集合． 2. 集合的运算 设A，B是两个集合，若A的每个元素都是B的元素，则称A是B的子集，记作A?B （或B?A）；若A?B，且有元素a∈b，但a?A，则说A是B的真子集，记作A?B．对任何集A，规定??A．若A ?B，且B?A，则称集A与B相等，记作A=B．由属于A或属于B的所有元素组成的集称为A与B的并集，记作A∪B，即 A∪B={x｜x∈A或x∈B}． 由同时属于A与B的元素组成的集称为A与B的交集，记作A∩B，即 A∩B={x｜x∈A且x∈B}． 由属于A但不属于B的元素组成的集称为A与B的差集，记作A\B，即 A\B={x｜x∈A但x?B}． 如图1-1所示阴影部分．
机械设计课程设计 设计题目：偏心齿轮传动的快速优化设计学校： 专业：机械设计与制造2012级秋 姓名： 指导老师： 完成设计时间：
目录 摘要 (2) 绪论 (3) 1 偏心齿轮简介化原理 (4) 2 偏心齿轮快速优化设计 (5) 2.1 偏心齿轮传动设计计算公式推导 (5) 2.2 偏心齿轮优化设计模型的建立 (6) 2.3偏心齿轮优化设计的程序实现 (8) 2.4偏心齿轮优化设计示例 (9) 结论 (10) 参考文献 (11)
摘要 偏心齿轮虽然在制造上与普通渐开线齿轮无异,却属于变传动比的非圆齿轮传动,设计计算十分复杂。本文将优化设计概念引入非圆齿轮设计,使非圆齿轮设计方法从传统的基于分析的设计发展为基于综合的设计,避免了带有较大盲目性的参数试凑和反复校验过程, 提高了非圆齿轮传动设计的科学性和一次成功率。 关键词：偏心齿轮非圆齿轮优化目标规划
绪论 齿轮机构是应用最为广泛的机械传动机构, 具有传递功率大、效率高、传动准确可靠、寿命长、结构紧凑等优点。通常所说的齿轮传动是指传动比为常数的齿轮传动, 其主要功能是传递匀速运动和恒定的动力(功率), 而非圆齿轮则更多地作为运动控制元件使用, 广泛应用于轻工、纺织、烟草、食品等机械中[1~ 5 ], 在机构创新设计中具有重要作用。 非圆齿轮传动20世纪30年代就已出现, 20世纪50年代原苏联学者李特文在文献[1]中首次建立了非圆齿轮传动的系统理论, 20世纪70年代起这项技术被介绍到国内, 并开始进行系统研究, 但至今应用有限, 甚至在我国机械专业的本科生教材中都未包含这部分内容。其重要原因在于, 非圆齿轮设计计算复杂, 制造也很困难。进入20世纪70年代以后, 由于计算机技术和数控技术的发展和广泛应用, 使制约非圆齿轮应用的两大难点都有了得以克服的可能, 因而掀起了新的一轮非圆齿轮研究及应用热潮, 国外甚至有人将其称为非圆齿轮的“再发明( Rediscovering)”, 不仅开展非圆齿轮传动的研究, 而且开展了非圆带、链传动的研究, 形成一个内容丰富的非匀速比传动研究领域[ 4 ]。由于齿轮数控技术的发展, 非圆齿轮的制造已不再困难, 但是, 非圆齿轮设计计算复杂这一难点尚未得到根本克服, 具体表现在以下两点。 1)现有文献中给出的某些计算公式作为分析计算工具无疑是正确的, 但是如果将其用于设计计算, 则缺乏可操作性, 例如, 文献[ 4 ]中给出的偏心齿轮计算公式以瞬时啮合角作为基本变量, 要求计算时首先设定α值, 其“缺点是α角的设定范围不易掌握, 而且几何中心距的变化情况、特别是它的最小值l min不能直接求出”。[ 4 ] 2）现有文献中给出的设计方法( 包括计算机辅助设计方法) 均属于基于分析的设计方法, 即, 给定一组参数, 得到分析计算(校核计算)结果, 如发现不妥, 则修改给定参数, 再作分析与校核, 具有较大的盲目性。 本文将优化设计概念引入非圆齿轮设计, 使非圆齿轮设计方法从传统的基于分析的设计发展为基于综合的设计, 避免了带有较大盲目性的参数试凑和反复校验过程, 提高了非圆齿轮传动设计的科学性和一次成功率, 力求从根本上扭转由于非圆齿轮设计计算复杂困难而限制其广泛应用的局面。
目 录 一、传动方案的拟定及电动机的选择 (2) 二、V 带选择 (4) 三．高速级齿轮传动设计 (6) 四、轴的设计计算 (9) 五、滚动轴承的选择及计算 (13) 六、键联接的选择及校核计算 (14) 七、联轴器的选择 (14) 八、减速器附件的选择 (14) 九、润滑与密封 (15) 十、设计小结 (16) 十一、参考资料目录 (16)
数据如下: 已知带式输送滚筒直径 320mm，转矩 T=130 N·m，带速 V=1.6m/s，传动装 置总效率为 ?=82%。 一、拟定传动方案 由已知条件计算驱动滚筒的转速 n ω,即 5 . 95 320 6 . 1 1000 60 1000 60 ? ′ ′ = ′ = p p u w D n r/min 一般选用同步转速为 1000r/min 或 1500r/min 的电动机作为原动机，因此 传动装置传动比约为 10 或 15。根据总传动比数值，初步拟定出以二级传动为 主的多种传动方案。 2.选择电动机 1）电动机类型和结构型式 按工作要求和工作条件，选用一般用途的 Y（IP44）系列三相异步电动机。 它为卧式封闭结构。 2）电动机容量 （1）滚筒输出功率 P w kw n T 3 . 1 9550 5 . 95 130 9550 P = ′ = × = w w （2）电动机输出功率 P kw d 59 . 1 % 82 3 . 1 P P = = = h w 根据传动装置总效率及查表 2-4 得： V 带传动 ?1=0.945； 滚动轴承 ?2 =0.98； 圆柱齿轮传动 ?3 =0.97；弹性联轴器 ?4 =0.99；滚筒轴滑动轴承 ?5 =0.94。 （3）电动机额定功率 P ed 由表 20-1 选取电动机额定功率 P ed =2.2kw。
哈工大机械原理课程设计齿轮传动设计大作业20无错版
机械原理课程设计大作业 ——齿轮传动系统20 课程名称：机械原理课程设计 设计题目：齿轮传动系统分析 院系：机电工程学院 班级： 15 设计者： 学号： 115 指导教师：陈 设计时间： 2017年6月
1、设计题目 1.1机构运动简图 1 序号 电机转速（r/min ） 输出轴转速（r/min ） 带传动最大传动比 滑移齿轮传动 定轴齿轮传动 最大传动比 模数 圆柱齿轮 圆锥齿轮 一对齿 轮最大 传动比 模 数 一对齿轮最大传动比 模数 20 970 30 35 40 ≤2.5 ≤4 2 ≤4 3 ≤4 3 2、传动比的分配计算 电动机转速min /970r n =，输出转速min /3001r n =， n /3502mi r n =，min /4003r n =，带传动的最大传动比5.2m ax =p i ,滑移齿轮传动的最大传动比4m ax =v i ，定轴齿轮传动的最大传动比4max =d i 。 根据传动系统的原始参数可知，传动系统的总传动比为： 333.3230970 011=== n n i 714.2735 970 022=== n n i
250.2440 970 033=== n n i 传动系统的总传动比由带传动、滑移齿轮传动和定轴齿轮传动三部分实现。设带传动的传动比为5.2m ax =p i ，滑移齿轮的传动比为321v v v i i i 、、，定轴齿轮传动的传动比为f i ，则总传动比 f v p i i i i 1m ax 1= f v p i i i i 2m ax 2= f v p i i i i 3max 3= 令 4max 3==v v i i 则可得定轴齿轮传动部分的传动比为 425.24 *5.2250 .24max max 3=== v p f i i i i 滑移齿轮传动的传动比为 333.5425 .2*5.2333 .32max 11== = f p v i i i i 571.4425 .2*5.2714 .27max 22== = f p v i i i i 设定轴齿轮传动由3对齿轮传动组成，则每对齿轮的传动比为 4343.1425.2max 33 =≤===d f d i i i 3、齿轮齿数的确定 根据滑移齿轮变速传动系统中对齿轮齿数的要求，可大致选择齿轮5、6、7、8、9和10为角度变位齿轮，其齿数： 42,8,41，9,40,101098765======z z z z z z ；它们的齿顶高系数1=* a h ，径向间隙系数25.0=* c ，分度圆压力角0 20=α，实际中心距mm a 50'=。
多元函数的极限与连续习题 1. 用极限定义证明:14)23(lim 1 2=+→→y x y x 。 2. 讨论下列函数在（0,0）处的两个累次极限，并讨论在该点处的二重极限的存在性。 （1）y x y x y x f +-=),(； (2) y x y x y x f 1s i n 1s i n )(),(+=； (3) y x y x y x f ++=23 3),(； (4) x y y x f 1 s i n ),(=。 3. 求极限 （1）2 20 ) (lim 22 y x x y x y +→→； （2）1 1lim 2 2 220 0-+++→→y x y x y x ； （3）2 20 01 sin )(lim y x y x y x ++→→； （4）22220 0) sin(lim y x y x y x ++→→。 4. 试证明函数?? ???=≠+=0 0)1ln(),(x y x x xy y x f 在其定义域上是连续的。
1. 用极限定义证明:14)23(lim 2 1 2=+→→y x y x 。 因为1,2→→y x ，不妨设0|1|,0|2|<-<-y x ， 有54|2||42||2|<+-≤+-=+x x x ， |22123||1423|2 2 -+-=-+y x y x |1|2|2|15|1|2|2||2|3-+-<-++-≤y x y x x |]1||2[|15-+-
函数与映射的概念知识梳理第 1 页 共 1 页 函数与映射的概念主要知识梳理 ●函数的基本概念： 1、函数的定义：设B A ,是非空的数集，如果按某个确定的对应关系f ，使对于集合A 中的任意一个元素x ，在集合B 中都有唯一确定的数)(x f 和它对应，则称B A f →:为从A 到B 的一个函数。 ①关键词：非空的数集、任意性、唯一性 ②作用：判断一个对应是否是函数 2、函数的三要素： 定义域A 、值域(?B)、对应法则f （定义域和对应法则最为关键） 作用：判断两函数是否是同一函数的依据（只要判断定义域和对应法则是否相同即可） ●函数的表示方法： 解析式法，列表法，图像法 ●分段函数与复合函数 分段函数：? ??∈∈=)()()()()(21D x x h D x x g x f ，复合函数：))((x g f y = ●映射的概念 1、定义：设设B A ,是非空集合，如果按某个确定的对应关系f ，使对于集合A 中的任意一个元素x ， 在集合B 中都有唯一确定的数)(x f 和它对应，则称B A f →:为从A 到B 的一个映射。 ①关键词：非空集合、任意性、唯一性 ②作用：判断一个对应是否是映射 2、映射的三要素： 原象集A 、象集(?B)、对应法则f 作用：判断两映射是否是同一映射的依据（只要判断原象集和对应法则是否相同即可） 3、函数是特殊的映射； ●反函数 1、概念; 设函数()y f x =的定义域为A ，值域为C ，由()y f x =求出()x y ?=.如果对于C 中 每个y 值，在A 中都有唯一的值和它对应，那么()x y ?=为以y 为自变量的函数，叫做()y f x =的反函数，记作1()y f x -=，（x C ∈） 2、存在反函数的条件：函数()y f x =在定义域内单调（一 一映射） 3、求反函数的一般步骤： （1）求原函数的值域； （2）反解，由()y f x =解出)(y x ?=； （3）写出反函数的解析式1()y f x -=（互换,x y ），并注明反函数的定义域（即原函数的值域）. 4、互为反函数的两个函数具有如下性质： （1）反函数的定义域、值域上分别是原函数的值域、定义域； （2）互为反函数的两个函数在各自的定义域内具有相同的单调性；它们的图象关于x y = 对称； （3）?=b a f )(a b f =-)(1 ●常见的思想方法 1、主要思想: ①数形结合:-------树形图 ②分类讨论：①按象的个数分类；②按原象个数分类； ③按对应关系（一对一、多对一，不能一对多）分类. 2、易错易混点 ①映射B A f →:与函数的定义).(x f y =-----A 中元素的任意性和B 中元素的唯一性? ②一个映射与某一对应的值. ③定义域与原象集以及与集合A 的关系. 值域与象集以及集合B 的关系. 3、主要题型: ①判断映射与函数； ②知原象、象、对应法则三者中的任意二个求余下一个； ③求映射与函数的个数.(注意分类讨论、注意和排列组合知识的综合应用)
一、 设 计题目 设计带式运输机中的齿轮传动：带式运输机的传动方案如下图所示，机器运行平稳、单向回转、成批生产，其他数据参见下方表格。 方案 电动机工作 功率P d /kW 电动机满载转速n m /(r/min) 工作机的转速n w /(r/min) 第一级传动比i 1 轴承座中心高H/mm 最短工作年限 工作环境 5.1.3 3 960 110 2 180 5年2班 室外、 有尘 二、 选择齿轮材料、热处理方式、精度等级 考虑到带式运输机为一般机械，且仅有一级齿轮减速传动，故大、小齿轮均选用40Cr 合金钢，调质处理，采用软齿面。大小齿面硬度为241~286HBW ，平均硬度264HBW 。 由要求，该齿轮传动按8级精度设计。 三、 初步计算传动主要尺寸 本装置的齿轮传动为采用软齿面开式传动，齿面磨损是其主要失效形式。其设计准则按齿根疲劳强度进行设计，并考虑磨损的影响将模数增大10%~15%。 齿根弯曲疲劳强度设计公式; m ≥√2KT 1?d z 12?Y F Y s Y ε[σ]F 3
式中Y F——齿形系数，反映了轮齿几何形状对齿根弯曲应力σF的影响。 Y s——应力修正系数，用以考虑齿根过度圆角处的应力集中和除弯曲应力以外的其它应力对齿根应力的影响。 Yε——重合度系数，是将全部载荷作用于齿顶时的齿根应力折算为载荷作用于单对齿啮合区上界点时的齿根应力系数。 [σ]F——许用齿根弯曲应力。 1.小齿轮传递的转矩 T1=9.55×106×P1 n1 p1=η1η2P d 根据参考文献[2]表9.1，取η1=0.96，η2=0.97。 由此 P1=η1η2P d=0.96×0.97×3=2.7936KW T1=9.55×106×P1 1 =9.55×106× 2.7936 960 2 =55581N?mm 2.齿数Z的初步确定 为了避免根切，选小齿轮z1=17，设计要求中齿轮传动比i=n1 n w =960/2 110 =4.3636，故 z2=i×z1=4.3636×17=74.1818，取z2=75。 此时的传动比误差为 ε=|i?i0 i |×100%=| 4.3636?75/17 4.3636 |×100%=1.1%<5% 满足误差要求，故可用。 3.载荷系数K的确定 由于v值未知，K v不能确定，故可初选载荷系数K t=1.1~1.8，本设计中初选K t=1.4。 4.齿宽系数?d的确定 根据参考文献[1]表8.6，齿轮在轴承上为悬臂布置，软齿面，选取齿宽系数?d=0.35。 5.齿形系数Y F和应力修正系数Y s的确定 根据参考文献[1]图8.19，Y F1=2.95，Y F2=2.25。 根据参考文献[2]图8.20，Y s1=1.52，Y s2=1.76。 6.重合度系数Yε的确定 对于标准外啮合直齿圆柱齿轮传动，端面重合度 εα=[1.88?3.2(1 z1 + 1 z2 )]=[1.88?3.2( 1 17 + 1 75 )]=1.6491 Yε=0.25+0.75 εα =0.25+ 0.75 1.6491 =0.72 7.许用弯曲应力[σ]F的确定