38 6 2016 6 JournalofNingxiaMedicalUniversity 629 :1674-6309(2016)06-0629-05 A FSH P C 4 1, 567 1, 89 2, :( 1, ; 1,, 1, 5-1,2 (1. [ 'S[ / br, 750004; 2. R6W X, 750004) 34:5 89 : ( ; `\ JM(folicle-stimulatinyhormore,FSH)RE X \,7_ \ 89 ;: B 6 3. ICR \ 67 %8(FCG) 4U %89 : 8(B-VCG) FSH 89 : 8(FSH-VCG) E X (fetal bovineserum,fbs) 89 : 8(FBS-VCG) FBS FSH 89 : 8(FBS+FSH-VCG)5 8, \ O `9O \, 8 `9=TU /TUBcl- 2 Bax 2 78 FBS+FSH-VCG8 \ 6 B-VCG8(P =0.012),FBS+FSH- VCG8S FBS-VCG(P =0.751)S FSH-VCG(P =0.693)8 KLMNO (P>0.05);R FCG 8,89 : D8\ 8 TU Bax2 (P <0 05), TU Bcl-22 (P <0.05);R B-VCG8,FBS-VCG FSH-VCGS FBS+FSH-VCG8 Bcl-2TU2 (P <0.05),BaxTU2 (P <0.05); FBS+FSH-VCG8,Bcl-2/Bax 1,Bcl-2 2 _M,R B-VCG8 KL NO (P =0.034),FBS+FSH-VCG8R FCG8 Bcl-22 79 \ 889 : E X S FSH ; Z \ 8 ;:, \ 6,\3 ; \ 1:;:89 : ; \ ; \ 6 ;E X ;FSHBcl-2/Bax -:R588.6 <:A DOI:10.16050/j.cnki.isn1674-6309.2016.06.006 ), \ 8 5: = : ; \ 8 ;,7FGP/ *)*. B b G 3 /- \ : 89 : 7_,89 ;: N/ ;: OS 5`9,3(< \ 8 W3 89^, 5 5, N/ ; : O ES 7.,3I \ =Zb :, : \ 8\ 8, Zb @ 7 A )> < 7 : \ \ 8, 89 :? `\ JM(folicle-stimulatinghormone, FSH), 3Z \ 6 [1], b ^,X * X TU=R =>?:2015-12-23 @AB5: (81260110) CDE : (1989-),, -, PQ, b! CD: ",ST,Q, br b / VW, @A. O 7., \ \ 89 :? / FSH RX, b ^ H \ ]FR Sb,A ;: ( ; FSH E X (fetalbovineserum,fbs) : \, ^ H_ 89 ;:,= \ :,7 \ 8 89 : B $ 1 6 1.1 3 SPFT ICR., D B, `B,C 24 0 5,12h % /12hDE % ^ 1.2 NO -O 8 `\ JM(r-hFSH)(? ) J ;E X (FetalBovine Serum,FBS) Hyclone ; X UTU (AlbuminBovineV,BSA) ;Bax Bcl-2S β-actin S\ N
630 K 38L O 1.3 `9 1.3.1 NO a 1 (:D-MEM/F-12 R 9 M R,, ph7 7.2~7.4, ;2V (: 12%E X ; FSH;389 (7 EG5.5-3089 (;4 :(:6 7 0 5 mol L -1 0.25mol L -1 = 0.125mol L -1 ( O,6 ; FSHSE X V ( 89 ( O (= :( 1.3.2 68 +, 67: 1 %8(freshcontrolgroup,FCG):7F 3 ICR. \ 8, 4, 24U %89 : 8(blank -vitrificationcontrolgroup,b-vcg): ( (a, ; E X S FSH 3 FSH 89 : 8(FSH-vitrificationcontrol group,fsh -VCG): ( ; 0.3IU FSH 4E X 89 : 8(FBS-vitri ficationcontrolgroup,fbs-vcg): ( ; 12% (V/V)E X 5 E X FSH 89 : 8(FBS+FSH-vitrification controlgroup,fbs+fsh-vcg): ( ; 12%(V/V)E X S 0.3IUFSH 1.3.3 89 : = : [2] 3 ICR. \ 8 V 1h, ; @ \( \ 5~8min 89 ( 3~ 5min; @ -196 (<: 3d, a :,1 \ (<, 37 - :( O, DN/ :( E \ 10min, 5min, ; V ( V 2h, F6 O 1.3.4 \ 8GH%+ = HE S;: \ 8 4% I 5" 16h; \ E XJT SGH K;a %+,%+L/ 5μm, M - Y (hematoxylin-eosin,he),? 6+,DT\ ^ 1.3.5 \ O [3] \ 8 %+, Y 5 %+ 1, 8 6 %+a HE, ; L %+45 5 2. $N,\ ^ (! <":\ ^7 \ =\ OA. B J,\ B ^ b N 3; @,/\ ^,\ 5, B JA 3 E 2 \,$ 7L ]F\ ), HE %+,O 5 $N \,, O ^ \,a O \ 6 1.3.6 8 \ 8 /T U Bax Bcl-2 GH%+a Bax Bcl-2 8 % + H S ;3% H 2 O 2 54 10min;O @ 25min; X 30min;6 Bax(1 200,PBS ),Bcl-2(1 200,PBS ) (,4 ;7F. \ <"NO,37,30min; DAB ; MM TU2-.!< ": 3P 8 &1 -.@A DPCtroler3.1.1.267 XN Q R% 1.3.7 Westernblot \ 8 /TU Bax Bcl-2 8 6 \ a TU, ; Westernblot a 1 \ 8TU, 3 BCATUN/ "N O a N/ ";2 TU,.;3a 12%6,5%N, ;4,S ;5,S* 180mA,2h;6,5%a 5 2h;7 5% BSA(TBSTa ), Bax Bcl-2 N/ 7 1:400,β-actin N/7 1 10000,4 b[,tbst,5min 3 ;8 1%BSA(TBSTa ) \ 7 1:8000, 5 2h,TBST,5min 3 ; 9E ;10TU ], QuantityOne 6 ], 1.4 NO`9 SPSS17.0NO a 6,] ±<"K( x±s)2,8j V 珋 M`K6,P 0.057KL NO 2 78 2.1 FSH=E X 89 : \ 8 \ 6 %& 8J \ 6 KL NO (F=12.244,P=0.001),Z FCG8 \ 6 VD8(P<0.05);FBS+FSH- VCG8 \ 6 B-VCG8(P = 0.012),FBS+FSH -VCG 8 S FBS-VCG 8 (P=0.751)S FSH-VCG8(P=0.693) K LMNO, 2 1
6^, E X FSH 89 : \ 631 1 FSH FBS Q ( x±s,\ 珋 =6) 68 \ /( /HP) \ /( /HP) \ 6 /% FCG 17.83±2.42 9.33±2.75 0.52±0.08 B-VCG 18.28±1.27 4.14±0.97 0.23±0.05 * FBS-VCG 16.28±1.70 4.56±0.51 0.28±0.01 * FSH-VCG 17.94±0.86 4.28±0.92 0.23±0.06 * FBS+FSH-VCG 17.06±1.67 5.33±1.53 0.31±0.06 * R FCG * P<0.05;R B-VCG P<0.05 2.2 \ 8 Bcl-2S BaxTU2 8 ] 8 ],Bcl-2S BaxTU S;:;\ DT\ \ B W-.2,ZR 8,: 8 Bcl-2TU-.2,BaxTU-.2, 1 2( 2) 2.3 \ 8 Bcl-2S BaxT U 2 Westernblot ] Westernblot ] :Bcl-2TUS BaxTU 2 8 J K L N O (F =13.459,P = 0.017;F = 12.536,P = 0 027),Z TU2] R 8 ] ;R FCG8,89 : D8\ 8 TU Bax2 (P < 0 05), TU Bcl-22 (P < 0 05);R B-VCG8,FBS-VCG FSH - VCGS FBS+FSH-VCG8 Bcl-2TU2 (P <0.05),BaxTU2 (P < 0.05); FBS+FSH-VCG8,Bcl-2/Bax 1,Bcl-22 _M,R B-VCG8 KL NO (P = 0.034),FBS+FSH- VCG8R FCG8 Bcl-22, 8, 3 1.FCG;2.FSH-VCG;3.FBS+FSH-VCG;4.FBS-VCG;5.B-VCG;R B-VCG P<0.05;R FCG # P<0.05 3 Bcl-2 Bax P H FF ^ (A)`!"# (B) 3 T9 \. b 6 V,4.A 23,7 [,\ 8 5 : J W 7 X Y [4] ) 3 5;:b \ 8 \,V*\ / ; : ` 7 \ ; : `9,. 5 O +,W DM SO \ 6W :TU, R ] ;,Z ] /R /, ] 5 Z ;: N/, 3 5-196 K \ ;:= : ; VWb AH 89 \ 8,, ;: 8) N O Z (sphingosine-1-phos Z>ZG, : \ 8),;: 237\ ]F, 3\ \ 5,\ A 8 [5] V*, ;: I,: ( @ ",Z 5 N/ =TU phate,s1p) [6-8] [DJM [9] VEGF [10]. L. O (necrostatin) [11] B (catalase) [12] (alphalipoicacid) [13] b H ;:S ; \ 8 :I: ( @ "O X UT U E X 6W,Z =
632 K 38L \,Z + M7 [2,14-15],FSH ] 6 \ JM,b `\ =, \,A ( ; E X S FSH= A \, \ Bcl-2 TU / 06 W,*B [16], Bcl-27 Bcl-2 TU,Bax7 Bcl-2 ` TU, 3 J ",/ Bax _M, Bcl-22,,@ 8 [17-18], Bcl-2 $R \ \ ^ [19], 7 89 ;: ;: ( ; FSH E X \, TU Bcl-2S` TU Bax \ 2, * >P` ;: ], \ 89 : " R %8, VD: 8 \ 6,E X FSH 89 : 8 V: 8 \ 6 ;R %8,D : 8` TU Bax2, R B-VCG 8,FBS-VCG FSH -VCG S FBS+FSH -VCG 8 BaxT U 2, Bcl-2TU2,6 V,Zb FBS S FSH E \ 5: S : \ a,a /, \ 8 I 0,\ 889 : ",E X S FSH ; Z \ 8 ;:, \ 6, Z\3 ; \ ;: ( ; FSH E X,73 23X 7 \ : B $ UV : [1], _`, ",. \ 89 : > PI FSH Cx432 %&[J]. K,2012,32(9):827-829. [2] _`.. [ [M]. :,2002. [3] WangYR,ChangQ,SunJ,etal.EfectsofHMGon revascularizationandfolicularsurvivalinautotrans plantmouseovariantisue[j].reproductivebiomedi cineonline,2012,24(6):646. [4] Bos-MikichA,MarquesL,RodriguesJL,etal.The useofamentalcontainerforvitrificationofmouse ovarises,asaclinicalgrademodelforhumanovarian tisuecryopreservation,afterdiferenttimesandtem peraturesoftransport[j].jasistreprod Genet, 2012,29(11):1267. [5] GaborVajta.Vitrificationoftheoocytesandembryos ofdomesticanimals[j].animalsreproductionsci ence,2000,60:357-364. [6] MengY,XuZ,WuF,etal.Sphingosine-1-phos phatesuppresescyclophosphamide induced folicle apoptosisinhumanfetalovarianxenograftsinnude mice[j].fertilsteril,2014,102(3):871-877. [7] TsaiYC,TzengCR,WangCW,etal.Antiapoptotica gentsphingosine-1-phosphateprotectsvitrifiedmu rineovariangrafts[j].reprodsci,2014,21(2): 236-243. [8] JeeBC,LeeJR,YoumH,etal.Efectofsphingosine- 1-phosphatesupplementationonfolicularintegrityof vitrified-warmedmouseovariangrafts[j].eurjob stetgynecolreprodbiol,2010,152(2):176-180. [9] HemadiM,AbolhasaniF,AkbariM,etal.Melatonin promotesthecumulus-oocytecomplexesqualityof vitrified-thawedmurineovaries;withincreasedmean numberoffoliclessurvivalandovarysizefolowing heterotopic transplantation[j]. EurJPharmacol, 2009,618(1-3):84-90. [10] ShikanovA,ZhangZ,XuM,etal.Fibrinencapsula tionandvascularendothelialgrowthfactordelivery promotesovariangraftsurvivalinmice[j].tisue EngPartA,2011,17(23-24):3095-3104. [11] LeeJR,YoumHW,KimSK,etal.Efectofnecrosta tinonmouseovariancryopreservationandtransplan tation[j].eurjobstetgynecolreprodbiol,2014, 178:16-20. [12] CarvalhoAA,FaustinoLR,SilvaCM,etal.Catalase additiontovitrificationsolutionsmaintainsgoatovari anpreantralfoliclesstability[j].resvetsci,2014, 97(1):140-147. [13] HatamiS,ZavarehS,SalehniaM,etal.Theimpactof alphalipoicacid on developmentalcompetenceof mousevitrifiedpre-antralfoliclesincomparisonto thoseisolatedfrom vitrifiedovaries[j].iranjre prodmed,2014,12(1):57-64. [14] Vab,c,,. :O 8 β1 M=8 %& [J]. /,2007,9(3):307-309. [15] 9Y, W,. \ 8;:. 3 _La,2005,16(1):16-19. [16] OlaMS,NawazM,AhsanH.RoleofBcl-2family
6^, E X FSH 89 : \ 633 proteinsandcasepasesintheregulationofapoptosis [J].MolCelBiochem,2011,35(1):41-58. [17] WestphalD,DewsonG,CzabotarPE,etal.Molecular biologyofbaxandbakactivationandaction[j].bio chimbiophysacta,2011,1813(4):521-531. [18] EdlichF,BanerjeeS,SuzukiM,etal.Bcl-xLretro translocatesbaxfromthemitochondriaintothecyto sol[j].cel,2011,145(1):104-116. [19] a, b. [ _ "^\ S Bcl-2/BaxTU2[J]. /, 2004,20(10):1908-1911. (!:C67) TheProtectionEfectoftheJointAddingofFetalBovineSerum andfsh intheprocesofmouseovaryvitrification SUQin 1, PEIChengbin 1, ZHANGShuya 2, LUOXiaoqiang 1, XURongrong 1, WANGYanrong 1, PEIXiuying 1,2 (1.KeyLaboratoryofFertilityPreservationandMaintenance,MinistryofEducation, KeyLaboratoryofReproductioninNingxia,Yinchuan750004; 2.Dept.ofBiological ChemistryandMolecularBiology,NingxiaMed.University,Yinchuan750004) Abstract:Objective ToobservetheprotectionforovarybytheJointaddingoffetalbovineserum andfsh intheprocesofmouseovaryvitrification,whichprovidesascientificbasisforoptimizingtheovariesvitrifica tioncryopreservationtechnology.methods 3-week-oldICRfemalemiceweredividedintofreshcontrol group(fcg),blank-vitrificationcontrolgroup(b-vcg),fsh-vitrificationcontrolgroup(fsh-vcg), FBS-vitrificationcontrolgroup(FBS-VCG)andFBS+FSH-vitrificationcontrolgroup(FBS+FSH- VCG).Thepercentageofnormalfolicleswereobservedthroughmorphologyand,theexpresionofBcl-2, Baxindiferentgroupsofovaryweredetectedbyimmunhistochemicaltechnique.Theefectontheexpresion ofbcl-2,baxproteinaboutdiferentgroupofvitrificationweredeterminedbyaddingjointlyfetalbovineser umandfsh.results Normalfolicles,apoptosisproteinBaxandantiapoptosisproteinBcl-2weresignifi cantlydiferentbetweengroups.significantdiferencesofthebcl-2proteinandbaxproteinexpresionamong groupswasfound(f=13.459,p=0.017andf=12.536,p=0.027),andtwokindsofproteinexpres sionresultswerebasicalyidenticalwithimmunohistochemicalresults.comparedwithfcggroup,apoptosis proteinsofeachgroupinthevitrifiedcryopreservationovariantisuebaxexpresionweresignificantlyhigher (P <0.05),andtheexpresionofantiapoptoticproteinsBcl-2werereducedsignificantly(P<0.05). ComparedwithB-VCG,theBcl-2proteinexpresionwashighersignificantly(P<0.05),andBaxpro teinexpresionwassignificantlylower(p<0.05)infbs-vcg,fsh-vcgandfbs+fsh-vcggroup. IntheFBS+FSH-VCGgroup,theBcl-2/Baxratiowassignificantlygreaterthan1whiletheBcl-2ex presionwasdominant.conclusion Addingjointlyfetalbovineserum andfsh intotheprotectionfluidof cryopreservedovarianfunctioncanimprovethepercentageofnormalovarianfoliclesandminimizethedamage ofcryopreservedovarian. Keywords:vitrification;mouseovary;thepercentageofnormalfolicles;fetalbovineserum;folicle-stim ulatinghormone;bcl-2/bax