2017 3 37 3 Basic& ClinicalMedicine March2017 Vol.37 No.3 :1001 6325(2017)03 0341 05 研究论文,-./01 2343567)89:;/Ⅱ,,,,,,!,"#$,%&' ("# <= 9>? =@=A B,"# 400016)!": # CD,E -./ 2343567)89 89:;/Ⅱ(Ang Ⅱ) F $% GHIJ-./KL,E -./MANO PQRSTU 6 7V 8W(D6 D7V D8)XY 8,8 Z[\9]^ PKL_`ab435NO,c D8RS XY ELISA 8 )-./ / 5,real timepcr 23 Ang Ⅱ mrna, Westernblot23 Ang Ⅱ 6 &',-./NO)8 -./E ctu D6 D7V D8,(P<0 001) /E ctu D8 H (P<0 05), /E ctu D6 D8 H (P<0 01) X,,-./NO) 23XY) Ang Ⅱ mrna 01(P<0 05) &,-./01 43567) 23XY Ang Ⅱ,F 2343567) 89= :-./; 23435;Ang Ⅱ;89= () :11 2652/R *+,-:A Highinsulinincreasesexpresionof angiogenensisⅡ intheendometriumofearlypregnantmice CHENWen qi,zhangchen,wangying xiong,hejun lin,liuxue qing,chenxue mei, MUXin yi,dingyu bin,gaoru fei (LaboratoryoftheReproductiveBiology,SchoolofPublicHealthandManagement,ChongqingMedicalUniversity,Chongqing400016,China) Abstract:Objective ToinvestigatetheefectofhighlevelinsulinonthevascularmarkermoleculesAng Ⅱ inthe endometriumofearlypregnantmice.methods Thehighinsulinanimalmodelwasconstructedbysubcutaneousin jectionofinsulin.theuterinetisuesandserumfromd6,d7,andd8daysofpregnancywhichwerecolectedaf termatingwithnormalmalerats,andbloodglucosewassimultaneouslymonitored.inadditiontothemaleratswith vasectomizedmatingtoconstructartificialdecidualization.elisaforthedetectionofseruminsulin,progesterone, andestradiolchanges,realtimepcrtodetecttheang Ⅱ mrnaexpresioninendometrialtisue.westernblotfor thedetectionofang Ⅱ proteinexpresioninuterinetisue.results Insulininthehighinsulingroupofpregnancy D6,D7,andD8daysincreasedsignificantly(P<0 001),estrogenlevelsinpregnancyD8daysappearsignificant lydecreased(p<0 05)andprogesteronelevelsinpregnancyD6andD8daysappeartodeclinesignificantly(P< :2016 07 01 :2016 10 31 :! (81300486);"#$%&' (cstc2015jcyja10013);"#$( )* &'+ (20150104) (correspondingauthor):gao_ru_fei@163.com
342 Basic&ClinicalMedicine 2017 37(3) 0 01).TheexpresionofAng ⅡmRNAandproteinsignificantlyincreasedinthehighinsulinmodel(P<0 05).Conclu sions HighinsulincanincreasetheexpresionofAng Ⅱintheendometriumofpregnantratsduringtheprocesofpreg nancy.itissuggestedthatinsulinmaymaycontributetotheangiogenesisintheendometriumoftheuterus. Keywords:insulin;decidualization;Ang Ⅱ;angiogenesis,,-./8 ) 23 [1 2] 89= 23435 " 7, 8 I 23 A V M 89,, 8 [2] 89= / 89= ) ", )89:;/ Ⅱ(an giotensinⅡ,ang Ⅱ) 89=, ] 89,8=89 [3] 23435 7),,-./ Ang Ⅱ8 9=F, & CD,-./ 2343567)89= Ang Ⅱ F 1./0$% 1 1./ 1 1 1 MA: 60(6~8, 20~30g),V 10 (6~8,30~ 40g)["# MA),MA :SCXK(()20070001 ] 1 1 2 :-./( \-./IJ, ) ("#!?);Ang Ⅱ" # IgG ($%&' =A+ ); (#)*+ EDTA#)*+,-#".#V SDS PAGE /0P1($%) =A+ );BCA SDS PAGE 2 345 ( 5)(2678W=A+ ); RNA9: Trizol(Invintrogen );PrimeScript RTreagentkit SYBRPremixExTaq TM Ⅱ(TaKaRa );PCR A(26=` ) 1 2 $% 1 2 1 MA;>:,-./NOKL: [4]KL,-./ NO,-./N O(treated)X(control) KL< NO V_`ab435NO(AI) < NO: XV,-./X =(,4 1),>? @ A B C B D, B D E T U 1W (D1) ctu 6 7V 8W(D6 D7V D8)RS8 XY6,8 _`ab435no: XV,-. /X[\9]^ =,>? @ A B CBD, BD E D1, 4W(D4) 2 9 00, I J 100μL,Z 6 IJ c D8 2 9 00R:_`ab435 6 F 1 2 2 ELISA 8 -./ /E :T U D6 D7V D8G HIJK:8,:8QLc 4 MN)O 30min, c 4 2 1000r/min 15min, :2P8 L -80 Q R,STUV W(26 X=A+ ) 8 )-./ / E 1 2 3 8:TU D6 D7V D8G YO :8RS8,8G ACCU CHEKPerforma (Roche,Germany)8 1 2 4 23 Ang Ⅱ mrna :: XYL CS;> ),1 1mL Trizol Z [\CXY]^,1 200μL_`abc L 4 12000r/min 15min, :2,1, 3 15min,: EP9Q 9 ]^A,1 75%,4 7500r/min 10min,1 DEPCE, (Thermo ) RNA3(A 260 /A 280 1 7~2 1), 1000ng 3 RNA) 23,1 4μL5 PrimeSript RTMasterMix, 1 DEPCE\ 20μLc )6, :20μL,37,15min;85,5s;4,forever
,-./012343567)89:;/Ⅱ 343 1 1 PCR Table1 Primerssequenceforreal timepcr gene primersequence Ang Ⅱ forword:5 CCAGGCCCGTTGTTCTTGAT 3 reverse:5 GCAAACCATTCTCACAGGCAATA 3 β actin forword:5 ATATCGCTGCGCTGGTCGTC 3 reverse:5 AGGATGGCGTGAGGGAGAGC 3 G 15μL :1 SYBR7 5μL A 0 6μL DEPC ; > E 5 1 μl cdna 1 2μL 0 7 :95 30s;95 5s,60 34s, 40;95 15s,60 1min,95 15s 2 -ΔΔct W6 F 1 2 5 Westernblot 23 Ang Ⅱ : : 20~40gXYL, XY" 1 PMSF, c M2 [\XY]^,O 30min 5minab 1>,4 12000r/min 15min, : 2: 2, Q BCAW SDS PAGE/0P1P1 10% 0, Q 0 β actin 42ku Ang Ⅱ 60ku, 3\ PVDF3 2,5% c 37 N) 1h, # c 4 MN >? PVDF3:, PBST 3> > 5min,1,-#".# (1 1000) 1h,PBST 3 3>, > 5min, c/0f Q6 F /2 x±s E (n=3) 1 3 234(5 G SPSS13 0, ± (x±s) 2 &' 2 1 6789:; 9:; < =>;#?@,-./X8 -./ctu D6 D7V D8,(P<0 001), 88 E01(P< 0 05)( 1A,B),8/E ctu D6W (P<0 01)( 1C),8/E ctu D6 D8 H (P<0 05)( 1D) 2 2 6789:; ABCD Ang ⅡmRNA1E?@ X,,-./XcTU D6 D7V D8 23) Ang Ⅱ mrna01(p< 0 05),-./X_`ab435Q Ang Ⅱ mrna X 01(P<0 05)( 2) P<0 05comparedwithnormalcontrolgroup 1 9:; < =>;78# Fig1 Changesofinsulin,bloodglucose,estrogenandprogesterone
344 Basic&ClinicalMedicine 2017 37(3) P<0 05comparedwithcontrolgroup 2 Ang Ⅱ mrna#1e Fig2 ExpresionofAng Ⅱ mrna(x±s,n=3) 2 3 6789:; ABCD Ang Ⅱ 1E?@,-./X c X,Ang Ⅱ TU D6 D7V D823XY) 01(P<0 05) _`ab435no),,-./;>x Ang Ⅱ 01 (P< 0 05)( 3) 3 23 435 " bcw:, b 89= 2 3 4 3 5 " 7 \ = 7),89 " 8 I 23,,-./8 V) 23 b [1 2] 23 2 -./,c 2 [3], -./ #23, 23 X 0 [5] A V M 89,, 8 [2] 89= / 89= ) ", ) Ang Ⅱ 89=, ] 89,8=89 [6] 2 O 89 / 89:;/ (renin P<0 05comparedwithcontrolgroup 3 Ang Ⅱ #1E Fig3 ExpresionofAng Ⅱ protein(x±s,n=3) angiotensinsystem,ras) [7] RAS -. # = R 6 [8],-./8 01 Ang Ⅱ = 1 Ang Ⅱ [9],-./8,,8 RAS,Ang Ⅱ, [10 11] 23435 7),,-./ Ang Ⅱ 8 9 = F, & CD,-./ 234356 7)89= Ang Ⅱ F ],-./ 8 -./E, / E ctu D8 H, /E ctu D6 D8 H X,,-./NO) 23XY) Ang Ⅱ mrna 01 9,-./01 43567) 23XY Ang Ⅱ,F 234 3567) 89=
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