2011 5 31 5 Basic& ClinicalMedicine May2011 Vol.31 No.5 :1001 6325(2011)05 0546 05 VEGFsiRNA%&' HL60 ( )*+,-./ 1, 2, 3 (01 12;2 3 45;3 6789:;, 510632)!": # VEGFsiRNA%&' HL60 <=*+ ( ),-./ $% 7>?@AB VEGFsiRNA,?C HL60,DE CCK8 %,,RT PCRFGHI 9JKL (VEGF)mRNA,MNO,ELISAF3 VEGF ',MNO,E P Q,R S &' VEGFsiRNATUVW HL60 ( ),-./,VEGFsiRNA ( )XE Y HL60,,Z 82 9%(F=121 63,P<0 01) VEGFsiRNA [\ VEGFmRNA 121 (F=17 17,P<0 01) ] VEGF '(F=3290 49,P<0 01),MNO ^ VEGFsiRNA_`ab ( )c, RSO & VEGFsiRNA,% %& HL60 ( ),-./ : GHI9JKL;siRNA; ( );*+-./ () :R730 53 *+,-:A VEGFsiRNAenhancessensitivityofleukemiacelstoAra C JINGChun xia 1,ZHANGHuan 2,YANGGuang 3 (1 Dept.ofEpidemiology;2 InstituteofHematology;3 Dept.ofParasitology,MedicalColege,JinanUniversity,Guangzhou510632,China) Abstract:Objective ToexploretheefectsofVEGFsiRNAonchemosensitivityofleukemiacelstoAra C.Meth ods VEGFsiRNAwasconstructedandthentransferedtoHL60cels.TheproliferationwasdetectedbyCCK8 method.thelevelofvegfmrnawasdetectedbyrt PCR.TheexpresionlevelofVEGFproteinwasdetermined byelisa.thecelapoptosiswasdetectedbyflowcytometry.results VEGFsiRNAincreasedthesensitivityof leukemiacelstoara C,VEGFsiRNAplusAra CsignificantlydecresedthesurvivalofHL60celsinwhichthein hibitionratewas82 9percentages(F=121 63,P<0 01).VEGFsiRNAdecreasedsignificantlyexpresionof VEGFmRNA 121 (F=17 17,P<0 01)andVEGFprotein(F=3290 49,P<0 01).However,VEGFsiRNA didn tpromotethelevelofapoptosisinducedbyara C.Conclusion VEGFsiRNAincreasesthesensitivityofleu kemiacelstoara Cbyinhibitingproliferation. Keywords:VEGF;siRNA;Ara C;drugsensitivity ' ;, /, W, b,,,<= Z =,<= /,,,9J] *+, ', */ <=,K [1] Z K 4 :2010-09-07 :2011-01-17 :!"(2001 Z 037 01);#$" (021195) (correspondingauthor):jcxphd@163.com
VEGFsiRNA%&' HL60 ( )*+,-./ 547,`,RNAi6, L, RNA(siRNA) `W, /,TU K [2] ' WMN VEGF 7 [3],K 4 7 >? @ A B, VEGFsiRNA, VEGFsiRNA <=*+XE VW' HL60 <=*+,-./ 1./0$% 1 1./ VEGFsiRNA7> (Ambion ),' HL60(* + ), ( )( & ),TaqDNAPolymerase (9 9+` ),Lipofectaming TM 2000 Opti MEMI(Invitrogen ),celcountingkit 8( < 45),6 VEGFELISA( 9+ ` ), RPMI 1640(Gibco ),! ("# * ), $ % (& ' 9+) 1 2 $% 1 2 1 VEGFsiRNA,AB: GenBank( @ : X62568)() VEGFmRNA*,+, sirna-. /01, VEGFmRNA, 386~ 406nt VEGFsiRNA 2!,34Z 5 AATATCTT TCTTTGGTCTGCA 3, 2!,34Z 5 AATGCAG ACCAAAGAAAGATA 3 sirna3459 9 + ` AB, E Ambion, sirna AB VEGFsiRNA 1 2 2 :HL60 DE 10%6 7$ %, RIPM 1640 ( 89:100U/mL ]100μg/mL! ), ;37 5% CO 2 ],H Z 4 : ', Ara C VEGFsiRNA ] Ara C+VEGF sirna <- 3 => 1 2 3 VEGFsiRNA ' HL60 *+-. /, :? HL60+5 10 5 /ml@ %,ABCDE 96> 4, >200μL, - 3 = >,D E 7 lipofectamine TM 2000? C VEGFsiRNA, @ Z50nmol/L,?C4h, 9 10%7$ %, 9, 1640, F 20h,?< = * + Ara C9 : G @ Z 25ng/mL, F 48h, >9 CCK8 10μL, F37 2h, 450nmHJ IJKL 1 2 4 RT PCRF VEGFmRNAMN: ]MNF,*+ E 48h KO, + TrizolRNAVV RNA?@] PCR % VEGFmRNA]G K GAPDHmRNA VEGFP1:5 GCCAGCACATAGGAG AGATGAGC 3 ;VEGFP2:5 TCACCGCCTCGGCTTG TCACATC 3 GAPDH P1:5 TCACCATCTTCCAGG AGCGAGA 3 ;GAPDH P2:5 GTGTCGCTGTTGAAG TCAGA 3 (UP+<59 9+ A B) % Q R Z:94 3min,94 30s 60 45s 72 50s,35 ST,72 5min %U+ 1 2%V (WX Y ( 0 5μg/mL < ) 1 2 5 ELISA VEGF '@ : ] MNF,*+ E48h, K %3 VEGF ' +6 VEGFELISAZ,E*# [\Q IJKL \]^_,? ),` VEGF,JI abz@ 1 2 6 P Q RS:? MNK c9 HL60,48h KO E PBS 2, RNA [ A 37 E 30min, < (PI, 50μg/mL)4 30min, P Q 1 2 7 E :U" < [4], ], 8 N,XAE* R (A+B) ], XAE* R (A+B),Q=R /R N = R (A+B) / R (A+B), R(A +B)=RA +RB-RA RB Q<0 85Z E,0 85 Q<1 15Z E 9, Q 1 15Z E 1 3 1 (.U< ±\] (x ±s)m DE SPSS13 0,DEF `. 2 &' 2 1 VEGFsiRNA2 HL60345678 9#:; VEGFsiRNA Ara CXE ` HL60,%, F,8 `Y / (F=121 63,P<0 01)(M 1) VEGFsiRNA Ara CXAE*, E<Z 9(Q=0 98)
548 Basic&ClinicalMedicine 2011 31(5) 1 VEGFsiRNA 2 HL6034 Ara C56789#:; Table1 ThesensitivityofAra Cimprovedby VEGFsiRNAinHL60cels(n=3) group Avalue(x ±s) inhibitionrate(%) control 1 316±0 012 0 Ara C 0 270±0 013 79 48 2 3 VEGF sirna Ara C HL603 4 VEGF= <#:; VEGFsiRNA <=*+ Ara CXE ` HL60 ', M N (P<0 01) Ara C+ VEGFsiRNA, 'MN Y \ VEGFsiRNA ] Ara C (P<0 01)( 2) VEGF±siRNA 0 998±0 182 24 16 VEGFsiRNA+Ara C 0 225±0 006 82 90 P<0 01comparedwithcontrol. 2 2 VEGF sirna Ara C HL603 4 VEGFmRNA <#:; VEGFsiRNA HL60 VEGFmRNA 121,MN(P<0 01)( 1), VEGFmRNA 121 MN,Ara C+VEGF sirna Ara C ] VEGFsiRNA, mrnamn <[\(P<0 01) 2 VEGFsiRNA Ara C HL6034 VEGF= <#:; Fig2 EfectsofVEGFsiRNAandAra Conthe VEGFproteinofleukemicHL60cels(n=3) P<0 01comparedwithcontrol 2 4 VEGFsiRNA Ara C 2 HL60 34>?#:; Ara C +VEGFsiRNA ] Ara C 7,RS, VEGFsiRNA ], RS ( 3) 3 @ 1 VEGFsiRNA Ara C2 HL6034 VEGFmRNA <#:; Fig1 EfectsofVEGFsiRNAandAra Conthe VEGFmRNAofleukemicHL60cels M DNAMarker;1 Ara C+VEGFsiRNA;2 Ara C; 3 VEGFsiRNA;4 control P<0 01comparedwithcontrol ',,D ] `, 8 Seftel [5] 9 / /',Z 7 99/10 Roswal [6] 30 U6 ',Z 10 8/10 'Z 2 76/10, / 5, S 6 ( )] 8 ( ),^ 35 UC6S [7],< =Z,^ /,*+ ]67, /\, E P,<=c, 9 * *' <=, K [8] K, 7> ' *+-./,F '=,
VEGFsiRNA%&' HL60 ( )*+,-./ 549 3 VEGFsiRNA Ara C HL6034>?#:; Fig3 EfectsofVEGFsiRNAandAra ContheapoptosisofleukemicHL60cels(n=3) A control;b Ara C;C VEGFsiRNA;D Ara C+VEGFsiRNA 1997, / ;' (AML), MN VEGF7 [9],H VEGFMNO, W,` [10] 50nmol/LVEGFsiRNA L7 >?C' 48hTW / VEGF mrna] VEGF ',MN, ' #, VEGF ab' K562 HL60,% ],V 'c VEGF, = TBZ`, K,? VEGFsiRNA L <=*+XET E<=*+ 5 U9 */,! 4,50nmol/L, VEGFsiRNA %9' HL60<=*+ ( ),-./, VEGFsiRNA ( ),XAE*, EZ 9 VEGFsiRNA <=*+ ( )XE ` ' HL60 VEGF ',MNO RSF,,VEGFsiRNA_`ab<=*+ c,',rs, VEGF ' 1RS, ab,%e, U, 4 [11] VEGFsiRNA?, S,, Ara C E DNAAB, S, /*+,K? sirna Ara CXAE*T` /,%& ' <=*+,-./,\ */ K VEGFsiRNA <=*+ ( )XA ETU =, VC,[\<=*+ E,, U\<=*+ 7U9,
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