全 文 ：·1188· 中草药ChineseTraditionalandHerbalDrugs第36卷第8期2005年8月
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ApoptoticeffectoforidoninonNB4cellsanditsmechanism
LIUJia—junl，LIQia02，PANXiang—lin3，PENGJun3，WUXiang—yuanl，
LIMing—quanl，LINDong—junl，LINQu1，HUANGRen—weil
(1．DepartmentofH matology，TheThirdAffiliatedHospitalofSunYat—senU iversity，Guangzhou510630，China；
2．DepartmentofBiomedicalEngineering，MedicalCollegeofShandongUniversity，Jinan250012，China；
3．DepartmentofH matologyandOncology，QiluHospitalofShandongUniversity，Jinan250012，China)
Abstract：ObjectiveToinvestigatethem chanismsoforidonininducingapoptosisonacute
leukeamiaNB4cellsanditsmechanism．MethodsNB4cellsinculturemediuminvitroweregivenwith
differentconcentrations(8，16，24，and32／1mol／L)ofori onin．Theinhibitoryrateofthecellswasmea—
suredbyMTTassay，cellapoptoticratewasdetectedbyflowcytometry(FCM)，morphologyofap ptosis
wasobservedbyHoechst33258fluorescencestaining，DNAfragmentationw sassayedbyagarosegel
electrophoresis，caspase一3expres ionwasdetectedbyWesternblotting，andcaspase一3activitywasas—
sayedwithcolorimetricassaykitbeforeandafterapoptosisccurred．ResultsOridonin(over16Fmol／L)
couldinhibitthegrowthofNB4cellsandcauseapoptosissignificantly，thesuppressionwasb thinatime—
andose—dependentmanner．Markedchangesofapoptosisincludifigcondensationofehromatinndnuclear
fragmentationwereobservedveryclearlybyHoechst33258fluorescencestaininga dacharacteristic“lad—
der”ofDNAfragmentswaselicitedbyagarosegellectrophoresis；Westernblotanalysisr vealedthat
caspase一3wasactivatedbythelOSSofcaspase一3proenzyme(32kDa)andtheappearanceofits20kDasub—
unit，andthatalongwiththeapoptoticpro essca pase一3activitywasincreasedconcurrently．Conclusion
Oridonincan duceapoptosisinNB4cellsviactivationofcaspase一3．TheseresultswilIprovidelaborato一
收稿日期：2005一Ol一07
基金项目：国家自然科学基金资助项目(30300312)
作者简介：刘加军(1966)，山东省日照市人，主治医师，讲师，硕士生导师，山东大学博士，中山大学博士后，主要从事血液肿瘤的分子
生物学及细胞凋亡机制等研究。Tel：(020)85516867—2227
万方数据
中草药ChineseTraditionalandHerbalDrugs第36卷第8期2005年8月·1189·
ryevidencefortheclinical
Keywords：oridonin；
treatmentofacutel ukemiawithoridonin．
caspase一3；apoptosis；NB4cell
冬凌草甲素对白血病NB4细胞的诱导凋亡作用及其机制
刘加军1，李桥2，潘祥林3，彭军3，吴祥元1，李铭权1，林东军1，林曲1，黄仁魏1
(1．中山大学附属第三医院血液科，广东广州 510630；2．山东大学医学院生物医学工程教研室，
山东济南250012；3．山东大学齐鲁医院血液肿瘤中心，山东济南250012)
摘 要：目的探讨冬凌草甲素对白血病NB4细胞的诱导凋亡作用及其机制。方法以不同质量浓度(8、16、24
和32gmol／L)的冬凌草甲素作用于体外培养的NB4细胞。应用四氮唑蓝(MTT)比色法检测细胞生长抑制率，
流式细胞术(FCM)检测细胞凋亡率；采用Hoechst33258荧光染色法和琼脂糖凝胶电泳观察细胞凋亡；采用蛋白
印迹(Westernblot)和比色法测定细胞凋亡前后caspase一3表达水平及其活性的变化。结果冬凌草甲素(16
／Lmol／L以上浓度)对白血病NB4细胞具有显著的增殖抑制及诱导凋亡作用，并且呈现出一定的时间一效应和剂
量一效应关系。Hoechst33258荧光染色观察到典型的核浓缩、核碎裂等细胞凋亡的形态学变化，琼脂糖凝胶电泳观
察到细胞凋亡时的DNA“梯形”条带；Westernblot检测结果表明32kDa的caspase一3酶原被激活，出现20kDa
的亚单位活化片段，同时在细胞凋亡过程中，caspase一3活性显著增高。结论冬凌草甲素能够通过激活caspase一3
的表达而诱导NB4细胞发生凋亡，可为冬凌草甲素进一步应用于临床治疗急性自血病提供实验依据。
关键词：冬凌草甲素；caspase一3；细胞凋亡；NB4细胞
中图分类号：R286．91 文献标识码：A 文章编号：0253—2670(2005)08—1188一06
1 Introduction
Oridonin，aditerpenoidcompound，isextract—
edandpurifiedfromtraditionalChinesemedicinal
herb，Rabdosiarubescens(Hemsl．)HaraorIsodon
japonicus(Burm．f．)Hara[1’2|．Itisoneofthe
mostimportantraditionalChineseherbsu edin
clinicaltreatmentnowadays．Morethanahalfcen—
turyago，oridoninshowedavarietyofbiological
effectssuchasimmunoregulatoryandan i—-inflam—-
matoryfunctionsaswellasantiviralfunctions
especiallyintheupperrespiratorytractinfection．
Recentlaboratoryandclinicald tasuggestthat
oridoninisaveryeffectiveantitumorreagentwi h
profoundeffectsonanumberofmalignantdiseases
suchasprostate，breast，non—smallcelllung
cancers[3]．
Apoptosisisaformofcelldeathdefinedbya
characteristicsetofmorphologicalandbiochemical
changes，andm yrecents udieshavebegunto
identifytheroleofcaspasesinapoptoticdeathas—
peciallyincancercells[4|．Differentmembersof he
caspasefamilymediatepoptosisindifferentc ll
types，andevenwithina givencelltype，distinct
caspaseshavebeenfoundtomediatepoptosisde—
pendingupontheapoptotics mulusreceivedby
thecellsE5|．Somecaspases，suchascaspase一3and
caspase一9，aremo andmoreimportantinthecas—
apasesmediatedapoptosisandthevariationoftheir
activityiscorrelatedtoa largevarietyofcancer
poptosis[6|．
Thoughoridoninhasbeenprovedtobevery
effctivena varietyofmalignancies，manyofits
antitumormechanismshavenotbeendemonstrat—
ed．UptOdate，thereisnodetailedlaboratoryevi—
denceaboutthemechanismsoforidoninon
1eukemicNB4ells．Inordertoclarifysomeofits
anti—leukemicm chanisms，theapoptotlceffectof
variousconcentrationsoforidonin(8—32／lmol／L)
onNB4cellsinvitrowasinvestigatedin his
study，andthevariationofcaspase一3xpression
wasdetectedtoprovidelaboratoryevidenceofori—
doninfortheclinicaltreatmentofacutel ukemia．
2 Materialsandmethods
2．1 Reagents：Oridoninwasprese tedbyProf．
P nXiang—lin，andHoechst33258waspurchased
fromSigmaCompany，NB4cellstrainwaspur—
chasedfromShanghaiRuijinHospital，anti—cas—
pase一3antibodywaspurchasedfromPharmingen，
andcaspase一3detectingkitwaspurchasedfrom
PromigaCompany．
2．2 Cellculture：Human1e kemiacell1ineNB4
cellswereculturedinRPMI—-1640mediumsupple——
mentedwith10％heat—inactivatedcalfserum，and
100U／mLPenicillininahumidified5％incubator
万方数据
·1190· 中草药ChineseTraditionalandHerbalDrugs第36卷第8期2005年8月，
at37。C．Cellswerepassagedtwicew eklyand
routinelyexaminedformycoplasmacontamination．
2．3 Cellinhibitoryrate(MTTAssay)：Cellin—
hibitoryratewasassayedus!ngthemicroculture
tetrazoliummethod．Briefly，NB4ce lsinlogarith—
micgrowth—phasewerecollectedand2×105cells／
wellweredispensedwithin96一wellcultureplates
in100mLvolumes．Thendiff rentco centrations
oforidonin(8，16，24，and32ttmol／L)wereput
indifferentw lls．Everyoneof-theconcentrations
abovewasregardedasonetreatedgroupwhile
therewasnooridonini thecontrolgroup．Eachof
thetreatedorcontrolgroupscontainedsixparallel
wells．Cultureplatesw rethenincubatedfor0．
24，48，and72hpriortotheadditionoftetrazo—
liumreagent．MTTworkingsolutionwasprepared
asfollows：5mgMTT／mLphosphatebuffered
saline(PBS)wassterilebybeingfilteredwith
0．45／zmfilterunits．Eachoftheabovecultured
wellswasadded20ttLofMTTworkingsolution
andthenincubatedcontinuouslyfor4h．Allcul—
turemediumsupernatantwasremovedfromwells
afterachoftheplateswascentrifuged(1000r／
min，15min)andreplacedwith100ttLofDMSO．
Followingthoroughsolublization，theabsorbance
(Avalue)．ofeachwellwasmeasuredusinga mi—
crocultureplater aderat570nm．Cellinhibitory
ratewascalculatedaccordingtotheformulaasfol—
lows：inhibitoryra e一(Avalueofcontrolgroup—
Avalueoftreatedgroup)／Avalueofcontrol
group×100％．
2．4 Flowcytometrydetection：ForDNAcontent
analysis，cellstr atedwithdifferentconcentrations
oforidoninwereeollected，pelleted，washedwith
PBS，andresuspendedinPBScontaining20mg／L
PIand1 g／LribonucleaseA．2×106fixedceils
wereexaminedperexperimentalconditionbyflow
cytometry(FCM)，andpercentageofdegraded
DNAwasdeterminedbythenumberofcells
dispayingsubdiploid(sub—G1)DNAdividedbythe
totalnumberofcellsexamined．
2．5 Hoeehst33258staining：Themorphologyof
NB4cellswhenexposedtooridoninfordifferent
timewasobservedfirstlyunderinvertedmicro—
scope，thenHo chst33258 stainingwasusedto
observetheapoptoticmorphologyofNB4cellse —
peciallytreatedwithoridoninfor48h．Cellswere
fix dwith4％formaldehydeinPBSfor10 min，
stainedbyHoechst33258(10mg／L)forlh，and
thensubjectedtofluorescencemicroscopicanaly—
sis．Aftertreatmentwithoridonin(16，24，and32
tLmol／L)，themorphologicchangesincluding
reductioninthevolumeandnuclearch omatincon—
densationwereobserved．
2．6 DNAFragmentationassay：Apoptosiswas
confirmedbydetectionoffragmentationofchro—
mosomalDNAwiththeclassicDNAladder
method．Briefly。2×106cellswereimmersedincy—
tolysisbuffer(Tris—HCl1 mmol／L，pH8．0，
e etica id10mmol／L，pH8．0，proteinaseK
200mg／L，0．5％SDS)andincubatedfor3hat50
℃．DNAwasextractedwithphenol—chloroform，
precipitatedin1／10volumeofNaAc2mol／Land2
volumesof than01at一207Covernight。recovered
bycentrifugationat1 000×gfor30minat4℃，
andthenresuspendedinTEbuffer．RNaseAw
addedata concentrationof200mg／L，thenthe
treatedextractwasincubatedfor30minat37℃
andlectrophoresedona1．2％agarosegel．
2．7 Westernblotanalysis：ForWesternbloting，
2×106ceilswerewashedwithice—coldPBStwice
andlysedfor30minat4 C，thendebriswasre—
movedbycentrifugationfor15minat15000×gat
4℃，andequivalentamountsofproteinweres pa—
ratedby1o％SDS—PAGEandtransferredontoni—
rocellulosefilt r．Thefilterswerefirsttainedto
confirmuniformt ansferofa11samplesandthen
incubatedinblockingsolutionfor2hatroomtern—
perature．Thefilterswerereactedfirstlywith
monoclonalantibodyatadilutionof1：1000for2
h，followedby xtensivewasheswithPBStwice
andTBSTtwice．Filterswerethenincubatedwith
horseradishperoxidase——conjugatedsecondaryanti——
bodyofl：1000for1h。washedwithTBSTand
immunoreactiveproteinsw redetectedusingan
ECLWesternblottingdetectionsystem．
2．8 Caspase一3activityassay：Caspase-3activity
wasassayedwithanApoalertcaspase——3Colorimet．．
万方数据
中草药ChineseTraditionalandHerbalDrugs第36卷第8期2005年8月。1191‘
ricAssayKit(Calbiochem，SanDi go，CA，USA)
accordingtothemanufacturer
7
s instructions．
Briefly，controla d eatedc llswerewashedwith
coldPBSandlysedinlysisbuffercontaining5O
mmol／LHEPES，pH7．4，100mmol／LNaCl，
0．1％CHAPS，1mmol／Ldithiothreitol(DTT)，
and0．1mmol／LEDTAfor10minandthencen—
trifugedat10000×gfor20rainbeforecollecting
thesupernatants．Fiftymicrogramsofeachlysate
wereaddedtotheassaybuffer(5Ommol／LHEP—
ES，pH7．4，100mmol／LNaCl，0．1％CHAPS，
1mmol／LDTT，0．1mmol／LEDTA，and10％
glycer01)tomakea totalvolumeof90肛Lwhich
wasthenincubatedfor10minat37‘C．Tenmi—
crolitersofcolorimetricsubstrateforcaspase．．3(fi．．
nalconcentration，200pmol／L)wereaddedtothe
mixtures．A405wasrecordedforachsamplesafter
incubationfor2hat37‘C．
2．9 Statisticalan ysis：Allexperimentsw re
performedintriplicateandtheresultswereex—
pressedasmean±SD．Statisticalanalysiswere
performedwitht-testusingSAS6．12software．
3 Results
3．1 Cellinhibitoryratecausedbyoridonin：Ori—
doninbelow8弘mol／Lhadlittleinhibitoryrateon
NB4cells。butitcouldinhibittheproliferationof
NB4cellssignificantlyata higherconcentration
(between16—32pmol／Loridonin)，especiallyth
concentrationof32F,mol／L．Thein ibitoryrateof
oridoninbetween24—32b￡_mol／Lismuchhigher
thanthatof10werconcentrationsoforidonin
(P<0．01)(Fig．1)．
100
80
0
0 24
Fig．1Cellinhibitory
3．2 Cellapoptoticrate
48
r／h
ratecaused
detected
byoridonin
byFCM：Ori
donin(over16>mol／L)couldind ceapoptosis
whenculturedwithNB4cellsafter24—72h，the
apoptoticratewasveryhighandover50％when
cellsculturedwithoridoninfor48h(oridonin>24
tlmol／L)(Fig．2)
—-．●一0um01．L。1
100
60
40
20
0
0 24 48 72
，／h
Fig．2Cellapoptoticratecausedbyoridonin
3．3 Morphologyofcellapoptosis：Apoptotic
cellsgraduallyincreasedinbothdose—andtime—
dependentmannerswhenexposedtooridonin
(over16／xmol／L)．Afterthecellsexposedtoori—
doninfor48h，markedmorphologicalchangesof
cellapoptosisincludingco densationofchromatin
andnuclearf agmentationwerefoundusing
Hoechst33258staining(Fig3)．Oridoninbelow16
>mol／LhadlittleapoptoticeffectonNB4cells
(Fig．3一A)，butapoptoticcellswerethengradually
increasedlongwiththenhancementofeo cen—
tration(Fig．3一Band—C)，theapoptoticcellswere
between40％一50％whenthNB4exposedto32
／lmol／Loridoninfor48h，andnoobviousapopto—
siswasfoundinthecontrolgroup(Omol／Lori—
donin)(Fig．3一D)
A——16pmol／LoridoninB—。24弘mol／Loridonin
C—-32弘mol／LoridoninD——control
Fig．3ApoptoticcellsobservedbyHoechst
33258staining(200×)aftercells
exposedtOoridoninfor48h
3．4 DNAFragmentation：TheintegrityofDNA
wasassessedbyagarosegellectrophoresis．Incu—
bationofNB4cellswithoridonin16—32／lmol／L
摹＼骂霉。口3口。口《
摹～等叠奇旦Iq—lIuI
万方数据
。1192。 中草药ChineseTraditionalandHerbalDrugs第36卷第8期2005年8月
2 3 4 5 6 for48helicitedacharac—
teristic“ladder”ofDNA
tragmentsrepresentmg
integermultiplesofthe
internucleosomalDNA
length(about180一200
l_contr012—5一treatedgroupsbp)(Fig·4)·
of8，16，24，and32“mol／L3．5 Westernblotanaly—
oridonin6-DNAMarkersis：Westernblotanalysis
Fig·4DNAfragmenta—revealedthatcaspase一3
tionofcellsex— wasactivatedbvthe10ss
posedto ifferemofcaspase一3proenzyme
。on。。nt。ation8
of(32～kDa)andtheappear一
盯m蚰蛔‰¨鼬ance。fits20_kDas：iunitnl工LkU■■Lo厶V—AI，d3UUU儿lI
1 2 3 4 afterthecellsexposedto
-32渤16—32“m。1／L。ridonin
-20kD8
for72h(Fig．5)．
1-control2 4-treatedgroups
3·6 Variationofcas—
of16，24，and32“mol／Lpase-3activity：Caspase—
oridonin 3 activityofNB4cells
Fig·5Westernblotana—wereincreasedr mark—
lysisofcaspase一3
ablywhenexposedtoori—
doninfor24—72h。thehighertheoridonincon—
centration，themoreincreasedexpressionofcas—
pase一3activityofNB4cells，andcaspase一3activity
wasenhancedtoitspeakat72h(Fig．6)．
04
O3
O2
0l
O
0 lamol·L
8 pmol·L
r-
’ V ’
0 24 48 72
r／h
Fig．6Caspase一3activitycausedbyoridonin
4 Discussion
TherearemanycompoundsextractedfromR．
rubescensandoridoniniso eofitsmosteffective
derivatives，whichisrecentlyprovedtohaveactiv—
ityagainstanumberofcancercells，andthemain
antitumoringredient，oridonin，chemicallybelongs
toent—kauranedit rpenoid．Recentdatahave
shownthatoridoninca inhibitthegrowthof
manycancercellsandinducingapoptosismaybe
oneofitsantitumormechanisms，TUNELassay
andcellcycleanalysishowedthatoridoninin—
ducedapoptosisandGo／G1cellcyclearrestedin
somecancercellssuchasinprostatecancerc ll．
Furthermore，detailedreferencesindicatedthat
oridonininhibitedthproliferationofcancercells
via poptosisandcellcyclearrestedwithp53play—
ingacentralo einseveralc ncertypeswhichex—
pressedthewild—typep53gene．Oridoninmaybea
novel，adjunctivetherapyforalargevarietyofma—
lignanciesE2’3|．
Ourpreviousstudies[7]demonstratedthatori—
doninhadconsiderableanti—proliferationeffectso l
someleukemiccellsinvitroanddown—regulating
thetelomeraseactivityofleukemiccellsmaybeits
importantanti—leukemicm chanism．Inthisstudy，
thatoridonincouldinhibittheproliferationandin—
duceapoptosisnleukemicNB4ellsinvitroina
time—anddose—dependentman erhasbeenfound．
Oridoninshoweda significantapoptosisinducing
effectsa thedosesrangingfrom16to32／1mol／L．
Markedmorphologicalchangesofcellapoptosis
wereobservedveryclearlybyHoechst33258stain—
ingaswellaselectronicmicroscopy，andDNA
fragmentationw sobservedbyagarosegellec—
trophoresisafterthecellsexposedtooridoninfor
48h；Westernblottingshowedcleavageofthecas—
pase一3zymogenprotein(32kDa)withtheappea—
ranceofits20一kDasubunit；alongwiththeapop—
toticprocessca pase一3activitywasincreasedre—
markably．Theapoptosisinducedbyoridoninona——
cuteleukemicNB4ellsvia ctivationofcaspase一3
couldbeconcluded．
Amongallofthecaspases，caspase3isoneof
themostimportanteffectororexecutor，andcas—
pase3 activityis hecommoneffectorofthemost
oftheapoptoticpathways[63．Onceactivat d，cas—
pase3iscapableofcleavingmanyimportantcellu—
larsubstrates。suchasICAD(inhibitorofcaspase—
activatedDNase)，poly(ADP—ribose)polymerase
(PARP，aDNArepairenzyme)，actin，fodrin，
andlamin．Manyl boratorydatahaveprovedthat
一§3鲁言S
n．Q％导≈Q
万方数据
中草药ChineseTraditionalandHerbalDrugs第36卷第8期2005年8月·1193·
activecaspase一3cancausemembraneblebbing，
disassemblyofthecellstructureandDNAfrag—
mentation，whicheventuallyleadtoeelldeath．
Someinitiatorcaspases，suchascaspase9，canac—
tivateprocaspase3，whichthencancleavethecel—
lularsubstratesne dedfortheorchestrationof
apoptosisandforma“wheelofdeath’，[8_10|．Recent
datahaveshownthatapoptosis，especiallythcas—
pase—mediatedcelld ath，playsanimportantrole
inthetiology，pathogenesis，andtherapyofava—
rietyofhematologicalmalignanciessuchasacute
leukemia，andcytotoxiceffectsofmostanti—
leukemiadrugsarebasedoninductionofapopto—
sis[11|．A11thesetudiesndicatethatinductionof
apoptosismaybeaindexfornewantitumordrug
selectionandanimportantmethodofassesement
fortheclinicaleffectivenessofmanyanti—leukemia
drugs[12|．
Insummary，theresultsdemonstratehatori—
donincaninduceapoptosisinNB4cellsviactiva—
tionofcaspase一3。Thisindicatesthatoridoninmay
beanimportantpotentialanti—leukemiareagents．
Acknowledgments：Wethankhemembersof
ourlaboratoriesfortheirnsightandechnicalsup-
port．
References：
[1]WangN，XieGX，ShixH，ela1．Studyonextractingand
separatingoforidonininRabdosiarubescens[J]．ChinTradit
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雷公藤甲素对胶原诱导的关节炎大鼠关节局部热休克蛋白
和主要组织相容性复合体I类分子表达的影响
涂胜豪，胡永红，曾克勤，张明敏，赖先阳，张玮琛
(华中科技大学同济医学院附属同济医院中西医结合科，湖北武汉430030)
摘要：目的观察雷公藤甲素对胶原诱导的关节炎大鼠关节局部热休克蛋白(HSPs)和主要组织相容性复合体
I类分子(MHC—I)表达的影响。方法建立胶原诱导的关节炎大鼠模型，雷公藤甲素按40卜g／kgim给药，运用
逆转录聚合酶链反应(RT—PCR)和免疫酶组织化学染色的方法，观察大鼠关节滑膜细胞和软骨细胞HSP60、
HSP70和MHC—I类分子的表达情况。结果与正常对照组相比，模型组大鼠关节滑膜细胞及软骨细胞HSP60、
HSP70和MHC—I类分子的表达均显著增高(P<0．05)，与模型组相比，雷公藤甲素可以下调关节炎大鼠关节局
部HSP60、HSP70和MHC—I类分子的表达(P<0．05)。结论降低关节炎大鼠关节局部软骨细胞和滑膜细胞异
常表达的HSPs与MHC—I类分子的表达，可能是雷公藤甲素治疗类风湿关节炎(RA)的作用机制之一。
收稿日期：2004—12—29
基金项目：国家自然科学基金资助项目(30070961)
作者简介：涂胜豪(1965一)，男，湖北武汉人，硕士，华中科技大学同济医学院附属同济医院副教授，硕士生导师，主要从事中西医结合治
疗风湿病研究。Tel：(027)83663379E—mail：shtu@tjh．tjmu．edu．cn
万方数据
冬凌草甲素对白血病NB4细胞的诱导凋亡作用及其机制
作者： 刘加军， 李桥， 潘祥林， 彭军， 吴祥元， 李铭权， 林东军， 林曲， 黄仁魏， LIU
Jia-jun， LI Qiao， PAN Xiang-lin， PENG Jun， WU Xiang-yuan， LI Ming-quan，
LIN Dong-jun， LIN Qu， HUANG Ren-wei
作者单位： 刘加军,吴祥元,李铭权,林东军,林曲,黄仁魏,LIU Jia-jun,WU Xiang-yuan,LI Ming-
quan,LIN Dong-jun,LIN Qu,HUANG Ren-wei(中山大学附属第三医院,血液科,广东,广州
,510630)， 李桥,LI Qiao(山东大学医学院,生物医学工程教研室,山东,济南,250012)， 潘
祥林,彭军,PAN Xiang-lin,PENG Jun(山东大学齐鲁医院,血液肿瘤中心,山东,济南,250012)
刊名： 中草药
英文刊名： CHINESE TRADITIONAL AND HERBAL DRUGS
年，卷(期)： 2005,36(8)
被引用次数： 7次
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