JournalofZhengzhouUniversity(MedicalSciences) Sept Vol.52 No T,[ ) B DCFH DA ROS,AnnexinV FITC/PIF 4 L Z D ; >?,E Westernblot ; c U V Bax

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1 doi: /j.issn TCDD "#! ROS(7,,,,,, ( # &' (, 5.,0"P &' # (,),1970* 11+,,-.,01,"# :,E mail:zly@zzu.edu.cn :;X ;; c UV; Y R114 $: 2,3,7,8 = :(TCDD)B YX ; c UV<=@ ROS CD %&: H TCDD( nmol/L) YX 24h,E MTT 9 :

2 JournalofZhengzhouUniversity(MedicalSciences) Sept Vol.52 No T,[ ) B DCFH DA ROS,AnnexinV FITC/PIF 4 L Z D ; >?,E Westernblot ; c U V Bax Bcl 2 Caspase 3 < = Z [ ' : TCDDH 9, 9: TS ROS. (P<0.05), Bax Caspase 3UVcB<=.9, Bcl 2UVcB<=. (P<0.05) ' :TCDD YX ; EfectsofTCDD onexpresionsofapoptosisrelatedproteinsandros contentinleydigcelsofrats YINJun,TANXuemei,ZHANGXiuli,JIMengmeng,TAOYuchang,LIUManman,YUZengli DepartmentofNutritionandFoodHygiene,ColegeofPublicHealth,ZhengzhouUniversity,Zhengzhou Keywords TCDD;leydigcel;apoptosisrelatedprotein;rat Abstract Aim:ToexploretheefectsofTCDDonexpressionsofapoptosisrelatedproteinsandROScontentinleydig celsofrats.methods:leydigcelsfrom ratsweretreatedwithdiferentdosesoftcdd(0,1,5,10,50nmol/l)for24 hours,andtheproliferationinhibitionrateofleydigcelswasdeterminedbymttassay,roscontentinleydigcelswas determinedbydcfh DAmethod,apoptosiswasobservedbyconfocalmicroscopycombinedAnnexinV FITC/PIstaining, andtheexpressionsofbax,caspase 3andBcl 2proteinsweredetectedbyWesternblot.Results:Withtheincreaseofthe doseoftcdd,theproliferationinhibitionrateandroscontentshowedarisingtrend(p<0.05),theexpressionsofbax andcaspase 3increasedwhereasthatofBcl 2proteindecreased(P<0.05).Conclusion:TCDDcouldsignificantlyinduce apoptosisofratleydigcels. *,J,:?_c TJ * $, c : [ \F4, 210 L4,A " Y 2,3,7,8 = : (TCDD) [1] ]a4 ^_ W ` KW@a, TCDD, [2] " # [3 6] <,TCDD J b, B O c?_w@,: S c %,, :? _ B TCDDX Y,TCDD Y#$X Z[W@ 3VMS A,:?_ J [7],Q ^ $ X 2 6 b, X 2% ~4% [8] X X LMS2 X 19 c3bm, 3 M,,: S, Z [ [9] a, ( W c W@, TCDDF B ' YX 9: CD,W@AB ; c U V Bax Caspase 3 Bcl 2 < = S D (ROS) CD, TCDD,: Q,YN c 1 %& 1.1 X$ _ SD Y,8~10, 4 [ :SCXK( ) ], P 1, 2 SPF_ 4 (QM J#(_ ) 1.2 ( ) * Q ( 3 23 ()),JY SCZ2 SDS PAGEUV ( ),Z [ * ( 4, 4 ()), O MZ (3 ^), C TCSSP5 Z D(4 C ()) DMEM/F 12' 3 Hy clone(),!" # $ ' R ( ), (DMSO) c Ⅳ 3 Sigma() ROS G H, 4 ( ),AnnexinV FITC/PIF ; GH F,4!./ (),ECL G H 3 Genview( ) 2 Y Bax Caspase 3 Bcl 2 β actin( =) 2 2 B D 4 S 2 IgG 238,4! () 1.3 L L= I Y &, 2 75% = 5 min, ^ P X D ^ PBS', b' S#6 IX b $ D

560 &' ( ) 2017* 9+ 52 5 c DMEM/F 12',34 Q 10~15min(90r/min), %& ^#$' U`, ` 5min $,1000r/min 5min,a $, ^#$' K, D 2min,100 I K M Percol2 (70% 58% 30% 5%),1600r/min 20min, F 4, 3 (30% ~58% ) 6, 2 10%!

3 560 &' ( ) 2017* c DMEM/F 12',34 Q 10~15min(90r/min), %& ^#$' U`, ` 5min $,1000r/min 5min,a $, ^#$' K, D 2min,100 I K M Percol2 (70% 58% 30% 5%),1600r/min 20min, F 4, 3 (30% ~58% ) 6, 2 10%!"#$ ', ' S 6 I, %CO 2 ' ' MY ml -1, 96 I,2: 0(B ) nmol/LTCDD F,A H -D 6 [,24h 1.4 -U &'( ) # $ % F 24h,A 20μLMTT5 (5g/L) ' 4h,ab ',A 150μLDMSO 37 Q 20min, 4 M(A) 9: T =1-( b A- A)/(B A- A) 100% &' ROS #$% F 24h ; $, nmol/L DCFH DAK,D ' R S 30min, PBS 3 W 2b X DCFH DA PBS 96 I,T 488nm 525nm, &'*+$% F 24h ;, 1 AnnexinV BindingSolution M VMY ml -1, 100μL, 5μL FITCS 5μLPI,,PQ 0> 15min, 400μLBindingSolution,, ;, Z D &' Bax Caspase 3 Bcl 2-.#$% F 24h ;, UV,BCA U VV M A b 30μgU V SDS PAGE 2,220V 1.5h 50g/L " 37 2h,TBST 4, 2 4 RS J,TBST 4, 2 37 RS 2h,TBST 4, ECL, O M Z, E ImageJ cb SPSS17.0 _ $ 2, E \2,b, \5E LSD t, \ 5E Dunnet t3, Z α= ' 2.1 TCDD "# < 1 F H 9, 9 : T TCDD ROS(7" # < 1, TCDDH 9, ROS. 1 5= A ROS(7?@ c(tcdd)/(nmol/l) n 9: T /% ROS ± ± ± ± ± ± ± ± ±3.01 F P <0.001 <0.001 : 0nmol/LB b,p< TCDD! " # AnnexinV FITC/PIF D, 1 F H 9, ; ;. 9", TCDD YX ; 1~5: nmol/LTCDDb 1 5=! *

JournalofZhengzhouUniversity(MedicalSciences) Sept. 2017 Vol.52 No.5 561 2.4 TCDD! "# 2S < 2, TCDD F H 9, YX Bax Caspase 3 UVcB<=., Bcl 2U V c B < =. 1~5:0 1 5 10 50nmol/LTCDDb 2 5=!

4 JournalofZhengzhouUniversity(MedicalSciences) Sept Vol.52 No TCDD! "# 2S < 2, TCDD F H 9, YX Bax Caspase 3 UVcB<=., Bcl 2U V c B < =. 1~5: nmol/LTCDDb 2 5=! 2 5=! c(tcdd)/(nmol/l) n Bax Caspase 3 Bcl ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.03 F P <0.001 <0.001 <0.001 : 0nmol/LB b,p< C,, 9:S ; C [ C,W a K, S ; ( Q ), ( 2 4 " " ),,4 ( ), 9: R, ; C "# [10] [,K 8 ; : < S& R ROS "D QR D, ; % [11] " # [12] [,ROS < S Q, <, 0 ROS,M, M, a Y ROS= <; Bcl 2 Bax Bcl 2UV M,Bcl 2Y ; UV,BaxY ; UV, ( c = CSA ; 3 W, = <; [13] Caspase 3 ; 3,A ; [14 15] <, ; U V, A < < Caspase, _ 4 0 >, ; +, Bcl 2 ; U V C <=, Caspase^ TCDD Y \ F 4,A B, :? _ CD.R / [16] "#, [ F H 9, YX Bax Caspase 3UV<= 9, Bcl 2< =. "#, H TCDDF 24hBX 9:^ CD,Q F H 9, 9: T 9,; 9, TCDD X ; " #, TCDDF H 9,X ROS, _ Bax Caspase 3 UV<=,Bcl 2U V < = " #,TCDD Q X < ;,TCDDQ Y X 9: A;,A Q ; < DE [1] $,&, b. : " # [J].,C,2006,1(2):105 [2] KERKVLIETNI.TCDD:anenvironmentalimmunotoxicant revealsanovelpathwayofimmunoregulation:a30 yearod yssey[j].toxicolpathol,2012,40(2):138 [3],,V,.TCDDB SD Y O CD[J]. K-,2004,17(6):370 [4] IKUTA T,NAMIKIT,FUJI KURIYAMA Y,etal.AhR proteintrafickingandfunctionintheskin[j].biochem Pharmacol,2009,77(4):588 [5] COLLINSLL,WILLIAMSON MA,THOMPSON BD,etal. 2,3,7,8 tetracholorodibenzo p dioxin exposure disrupts granule neuron precursormaturation in the developing mousecerebelum[j].toxicolsci,2008,103(1):125 [6],,,.2,3,7,8 = : (TC DD)[ F \M *! 1 %, : 4c U V < = [J]., C, 2010,5(3):334 [7] BEYTUR A,CIFTCIO,AYDIN M,etal.Protocatechuic acidpreventsreproductivedamagecausedby2,3,7,8 tet rachlorodibenzo p dioxin(tcdd)inmalerats[j].andro logia,2012,44(suppl1):454 [8] VELDHUISJD.Recentneuroendocrinefacetsofmalere productiveaging[j].expgerontol,2000,35(9/10):1281 [9] CHENH,HARDYMP,ZIRKINBR.Age relateddecreases inleydigceltestosteroneproductionarenotrestoredby exposuretolh invitro[j].endocrinology,2002,143(5):

5 1637 [10]$,,,. ; " # [J].,2007,17(5):297 [11]MARTINDALE JL,HOLBROOKNJ.Celularresponseto oxidativestress:signalingforsuicideandsurvival[j].j CelPhysiol,2002,192(1):1 [12] [.MC LR Y X ; < Caspase <"#[D].&':&',2015. [13]SHARPE JC,ARNOULTD,YOULE RJ.Controlofmito chondrialpermeabilitybybcl 2familymembers[J].Bio chimbiophysacta,2004,1644(2/3):107 [14],,,.α B Y < D >J S; CD[J]. : # ( ),2016,37(1):54 [15] ",1. Y > 3 b Bcl 2 BaxS Caspase 9UV < = [J].& ' ( ),2016,51(2):183 [16], R,&,.[ B Leydig caspase 9 caspase 3U V < = C D [J].,2014,34(1):131 ( ;/ 01 )

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