12 (autophagy),imagej,,, 3 [1-4] (self-eating) Transwelinvasion, 20μmol L -1 LY A549 [5] PI3K/AKT 24h,PBS, [6-8],, ml -1 ( ) 600μL

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1 ( ) JournalofNanchangUniversity(MedicalSciences)2017,Vol.57No.5 11 LY294002, (, ) : LY PI3K/AKT μmol L -1 LY294002(LY ) A549,PBS CCK8 LY A549 ; Westernblot 2 A549 Beclin-1 LC3B(Ⅱ Ⅰ) PI3K p-akt AKT ; WoundhealingTranswelinvasion 2A549 LY A549, 24h,24h IC μmol L -1 LY Beclin-1 PI3K p-akt LC3B(Ⅱ/Ⅰ) (P<0.05) LY PI3K/AKT A549 : LY ; A549;PI3K/AKT ; ; : R392 ;R734.2 : A : (2017) DOI: /j.cnki.ncdm : LY294002SuppressesAutophagy,Proliferation,Migration andinvasionoflungcancercelsinvitro : (20151BAB215026) : (1990 ),,, :,, [email protected] WU Hong-shan,LIUZhi-li (Departmentof Orthopaedics,the FirstAffiliated Hospitalof Nanchang University,Nanchang330006,China) ABSTRACT:Objective ToinvestigatewhetherLY294002inhibitstheautophagy,proliferation, migrationandinvasionoflungcancercelsthroughpi3k/aktsignalingpathway.methods Hu- manlungcancera549celsweretreated withly294002atconcentrationsof10,15,20,25and 30μmol L -1.Controlcelsweretreated withpbs.theproliferationofcelswasdetectedby CCK8assay.TheexpressionofBeclin-1,LC3B(Ⅱ,Ⅰ),PI3K,p-AKTandAKTinA549celswas measuredby Western blotanalysis.the migrationandinvasion ofcels weredetermined by WoundhealingandTranswelinvasionassays,respectively.Results LY294002treatmentinhibi- tedtheproliferationofa549celsinadose-dependentmanner,andthe24-houric 50 was μmol L -1.Comparedwithcontrolgroup,theexpressionofBeclin-1,PI3Kandp-AKT,ratioof LC3B Ⅱ/Ⅰ,cel migrationandinvasionsignificantlydecreasedin LY294002treatmentgroup (P<0.05).Conclusion LY294002treatmentsuppressestheautophagy,proliferation,migration andinvasionofa549celsthroughinhibitingpi3k/aktsignalingpathway. KEY WORDS:LY294002;humanlungcancerA549cels;PI3K/AKTsignalingpathway; autophagy;migration;invasion

2 12 (autophagy),imagej,,, 3 [1-4] (self-eating) Transwelinvasion, 20μmol L -1 LY A549 [5] PI3K/AKT 24h,PBS, [6-8],, ml -1 ( ) 600μL wel -1 10%, LY A549, PI3K/AKT, 200μL wel h,, 800μL wel -1 95%, 30 min,,800μl 1 wel -1 PBS3,800μL 1.1 wel % 30min, A549, ; ddh 2O, DMEM( ) ( ), 3 (TransGen ) Celcultureinsert(8.0μm,ImageJ, PETtrck-etched membrane, Corning ) 3 Westernblot MarkerPVDF ( Westernblot Formentas ) (Abcam ) ( ) LY294002( abmole) ( )201710,57 5 RIPA, 10%SDS-PAGE,5% 1h, ( PI3KIgG,1 2000; p-aktigg,1 DMEM 15% ( 2000; AKTIgG,1 2000; Beclin-1 100kU L -1 ) A549, IgG,1 1000; LC3BIgG,1 3000; 37 5%CO 2 β-actinigg,1 2500)4,TBST CelCountingKit-8(CCK8) PVDF3, (,1 5000) 2h,TBST 3, 10min,ECL,, 3, ml , 100μL, 12h SPSS19.0 x±s,, μmol L -1,P<0.05 LY294002,CCK8,, 450nm 2 (OD), LY294002A Woundhealing CCK8 LY294002, A549, 24h (1) ml -1, 6 24hIC,,20μmol L μmol L -1 20μmol L -1 LY LY A549 (PBS 2.2 LY294002A549 ),24h 100μL, Transwel,LY294002,PBS 2 (95±7) -1, ; DMEM (130±16) -1 (P<0.05), 2 24h,

伍宏山等ＬＹ２９４００２抑制肺癌细胞自噬增殖侵袭和迁徙的体外研究１３２３ＬＹ２９４００２对Ａ５４９细胞迁移能力的影响Ｗｕｎｄｈａｎｇ

４ＬＹ２９４００２对Ａ５４９细胞自噬相关蛋白Ｂｎ１及ＬＣ３Ｂ Ⅱ Ⅰ 的影响Ｗｒｎｂ检测结果显示ＬＹ２９４００２处理的Ａ５４９细胞中

对Ａ５４９细胞增殖的影响ＡＡ５４９细胞侵袭数量ＢＡ５４９细胞２４ｈ穿膜细胞数图２Ｔｒａｎｗ侵袭实验ＡＡ５４９细胞迁移数Ｂ

3 伍宏山等ＬＹ２９４００２抑制肺癌细胞自噬增殖侵袭和迁徙的体外研究１３２３ＬＹ２９４００２对Ａ５４９细胞迁移能力的影响Ｗｕｎｄｈａｎｇ实验结果显示ＬＹ２９４００２处理组细胞２４ｈ迁徙率为２２４±２６显著低于对照组３９１±３２Ｐ００５见图３２４ＬＹ２９４００２对Ａ５４９细胞自噬相关蛋白Ｂｎ１及ＬＣ３Ｂ Ⅱ Ⅰ 的影响Ｗｒｎｂ检测结果显示ＬＹ２９４００２处理的Ａ５４９细胞中Ｂｎ１蛋白表达和ＬＣ３Ｂ Ⅱ Ⅰ 比率比对照组显著降低均Ｐ００５见图４ＬＹ２９４００２浓度Ｌ１ μｍ图１ＬＹ２９４００２对Ａ５４９细胞增殖的影响ＡＡ５４９细胞侵袭数量ＢＡ５４９细胞２４ｈ穿膜细胞数图２Ｔｒａｎｗ侵袭实验ＡＡ５４９细胞迁移数ＢＡ５４９细胞２４ｈ细胞迁移率图３划痕实验检测细胞的迁徙能力ＡＷｒｎｂ电泳图Ｂ各蛋白与 β ａｎ蛋白灰度值之比图４Ｗｒｎｂ检测蛋白的表达

14 2.5 LY294002A549 PI3K/AKTPI3K,p-AKT (P< 0.

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:TGF-β bfgf 23 KEY WORDS:transforminggrowthfactor-β ;basicfibroblastgrowthfactor; bonemarrow mesenchymalstemcels;eukaryoticexpressionvector; liposomet

:TGF-β bfgf 23 KEY WORDS:transforminggrowthfactor-β ;basicfibroblastgrowthfactor; bonemarrow mesenchymalstemcels;eukaryoticexpressionvector; liposomet 2 ( )208 58 JournalofNanchangUniversity(MedicalSciences)208,Vol.58No. TGF-β bfgf,, 2 (., 330006; 2., 5040) : -β (TGF-β ) (bfgf) (BMSCs), SD BMSCs Percol PCR TGF-β bfgf, pcdna3.(+), 3 BMSCs RT-PCR Westernblot