* Guihaia Jan.201333(1):112-117 htp://journal.gxzw.gxib.cn DOI:10.3969/j.isn.1000-3142.2013.01.020. HelaS3 [J]. 201333(1):112-117 ShiZSYuDYPengQetal.PotentialmechanismofCameliachrysanthaseeds-inducedapoptosisinhumancervicalcancerHelaS3cels[J].Guihaia 201333(1):112-117 HelaS3 * ( 116622 ) : HelaS3 HelaS3 Western Bax Bcl-2 Bid : HelaS3 Bax Bcl-2 Bid Caspase-8Caspase-3 BaxCaspase-3Caspase-8 Bcl-2Bid HelaS3 : ; ;HelaS3 ; :R285.5 :A :1000-3142(2013)01-0112-06 PotentialmechanismofCameliachrysantha seeds-inducedapoptosisinhuman cervicalcancerhelas3cels SHIZhen-SongYU Da-YongPENG QianSHILi-Ying * FENGBao-MinWANG Yong-Qi (Instituteof Materia MedicaDalian UniversityDalian116622China) Abstract:ToinvestigatetheefectoftheseedsofCameliachrysanthaonapoptosisinhumancervicalcancerHelaS3 cels.human HelaS3celsweretreatedwiththesolutionofBuOHfractionfromtheEtOHextractfromtheseedsof C.chrysanthaatdiferentconcentrations.Theapoptosiswasdetectedbyflowcytometry(FCM)apoptosisassociated proteinsweredeterminedbywesternblot.aftertreatmentwiththesolutionofbuohfractionfromtheetohex- tractoftheseedsofc.chrysanthaonhuman HelaS3celsasignificantenhancementofapoptosiswasobservedthe expressionofbaxwasenhancedtheexpressionofbcl-2andbiddecreasedandthedecreaseofmitochondrialmem- branepotential(mmp)andtheactivationofcaspase-8andcaspase-3wereobserved.thebuohfractionfromthe EtOHextractoftheseedsofC.chrysanthawastheactiveanticancerfractionwhichpromotedhuman HelaS3cels apoptosispossiblybyactivatingbaxmmpcaspase-8andcaspase-3andinhibitingbcl-2andbid. Keywords:Cameliachrysantha;humancervicalcancer;HelaS3cels;apoptosis * :2012-08-20 :2012-10-25 : (81002621) : (1986-) (E-mail)szs19860202@163.com * : (E-mail)shiliying99074@163.com
1 : HelaS3 113 570nm ( 2005; 2006) 1.4DNA Selins-Cohen DNA (Selinsetal.1987;Hopciaetal.1996;Yuet (al.2007) (10 mmol/l Tris1 1994; 2006) mmol/ledta0.2% TritonX-100pH7.5) (13000g10min) 4 12.5% (CCEB) HelaS3 (TCA) 5% 90 20 min DNA DNA HelaS3 1 1.1 (Cameliachrysantha) 5 10 20 40 80 160μg/mL (Theaceae) (Camelia) (sec- (DMSO) 24h tionchrysantha Chang) 20 60 4h 20μL MTT (5mg/mL) (Phosphatidylserine) 100 (van Heerdeetal.1995) g 95% 3 3h 37 6h4 PBS 1200r/min 3 min AnnexinV-FITC 105cels/mL5μLFITC Annexin V 5μLPI 4 DMSO 10min (EpicsXLBeck- 1.2 man-coultermiamifl) HelaS3 HumanSciences 1.6 ResearchResourceBank( (tetram- ) 10% ethylrhodamine methylestertmrm)(molecular DMEM 37 5% CO 2 ProbesEugeneOR) (Mitochon- driamembranepotentialmmp) 1.3 MMP (MTT) 37 6h PBS HelaS3 96 1200r/min 3 min 10nmol/L 5 10 3 DMEM 37 5% CO 2 24h 24h PBS 200μLDMSO DNA 1.5 (PI) (FITC) V(Annexin V) (ImmunotechMarseileFrance) TMRM 1mL1% PBS 15min ( 488nm 575nm) (Yuetal. 6 20072008) MMP 0.1~12
114 33 MMP 2 Bax Bid β -actin HRP (horserad- 1.7 caspase-3 Caspase8 Caspase8 ECL (Amersham BiosciencesBuchingham- Ac-IETD-pNA pna(p- nitroaniline) VilberLourmatBio-Profile BIO-ID Caspase8 pna 405nm (Yuetal.20072008) PBS 1.9 Ac-IETD-pNA37 1h ± t (Polarstar) ( 380 P<0.05 nm 430~460 nm)(yuetal. 2007) CaspGLOWTM Caspase-3 (MBLNagoyaJapan) caspase-3 FITC caspase-3 2.1CCEBHelaS3 DEVD (FITC-DEVD-FMK) (insitu) 5 10 20 40 80 160μg/mL (Yuetal.20072008) FITC- (CCEB) DEVD-FMK HelaS3 MTT caspase-3 FITC HelaS3 CCEB HelaS3 IC 50 capase-3 102.20μg/mL 1 37 6h (1 10 6 cels/ml) 300μL 1 μl FITC-DEVD-FMK37 0.5h 3000r/min 5 min 0.5 ml 500μL FL-1 1.8 Western PBS 50μL (1 mol/ltris-hcl5 mol/l NaCl1% Noni- detp-40(v/v)1% 0.05% SDS1mmol/ 1 CCEBHelaS3 L )10 6 Fig.1 EfectsofCCEBoncelproliferation in HelaS3cels 20min 4 12000r/min 10minBio-Rad 2.2CCEB HelaS3 30μL 5μLSDS (62.5mmol/LTris- CCEBHelaS3 DNA HCl20% SDS5% 2-10% 90 120 150 μg/ml CCEB 0.001% ) 5 minsds- PAGE HelaS3 ishperoxidase) IgGs shireuk) X 3 2 CCEB HelaS3 HelaS3 DNA 2 DNA Annexin V-FITC/PI -Bcl-2 -Bax -Bid (SantaCruzBio- technologyinc.santacruzca) -β-actin CCEB HelaS3 (Sigma) Western Bcl- 120μg/mL HelaS3
1 : HelaS3 115 20.6% 150μg/mL HelaS3 36.3% 3:A Caspase Z-VAD-FMK(CaspaseInhibitorZ- VAD-FMK) Caspase (Celpermeablepancaspaseinhibitor) Caspase 3:B Caspase CCEB CCED HelaS3 Caspase 2 CCEBHelaS3 DNA Fig.2 EfectsofCCEBonDNAfragmentation in HelaS3cels 2.3CCEBcaspase CCEB HelaS3 caspase CCEB HelaS3 MMP caspase-8caspase-3 90 120 150μg/mL CCEB HelaS3 TMRM MMP 4 CCEB HelaS3 MMP CCEB 100 μg/ml 42.4% CCEBHelaS3 caspase-8caspase- 3 90 120 150 μg/ml CCEB HelaS3 caspase-8 caspase-3 5 6 CCEB HelaS3 caspase 3 CCEBHelaS3 Fig.3 EfectsofCCEBonapoptosisin HelaS3cels 4 CCEBHelaS3 MMP Fig.4 EfectsofCCEBon MMPin HelaS3cels 2.4CCEB Bcl-2 HelaS3 c CCEB HelaS3 24hBax Western CCEB Bcl-2Bid 7
116 33 (FADD TRADD) Caspase810 Caspase Caspase3 67 Caspase ;2 : DNA C (Apaf-1) Caspase9 Caspase9 3 Caspase CCEB 5 CCEBHelaS3 caspase-8 HelaS3 MMP caspase-8 Fig.5 EfectsofCCEBoncaspase-8 caspase-3 Caspase activityin HelaS3cels CCEB HelaS3 6 CCEBHelaS3 caspase-3 Fig.6 EfectsofCCEBoncaspase-3 activityin HelaS3cels 3 ( 7 CCEB Fig.7 EfectsofCCEBonproteinexpression relatedtoapoptosis ) Bcl-2 Bcl-2 CCEB Bcl-2 HelaS3 PT DNA Bax ANT VDAC HelaS3 PT Bcl-2 CCEB HelaS3 Bcl-xL Bax ANT Bax ANT VDAC (Harrisetal.2000) (Green2000;Wang2001):1 : Bax TNFR ( TNFRl TNFR2 C Fas DR4DR5) Bcl-2 Bax C
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