( )016 56 4 JournalofNanchangUniversity(MedicalSciences)016Vol.56No.4 1-1 a b a a a ( a. ; b. 330006) : -l(fibroblast-specificprotein-1fsp-l) Transwel MD-M-31 ESF; RT-qPCR MD-M-31 ESF FSP-1 mrn ; MD-M-31 ESF FSP-1 ;CelCounting Kit-8(CCK-8) MD-M-31 ; FSP-1 FSP-1 MD-M-31 ESF ESF FSP-1 MD-M-31 ;ESF+MD-M-31 MD-M-31 FSP-1 MD-M-31 FSP-1 ; MD-M-31 ESF FSP-1 FSP-1 : -1; ; : R737.9 ;R-33 : : 095-477 (016)04-0001-06 DOI:10.13764/j.cnki.ncdm.016.04.001 Up-RegulationandSignificanceofFSP-1inHuman FibroblastsCo-CulturedwithreastCancerCels LIU Huan a LIWen-lin b CHENShao-fen a WU Hong a SHIXiao-yu a (a.departmentof Histology and EmbryologyColegeof asic Medicine; b.jiangxi Key Laboratoryof MedicaliologyNanchang UniversityNanchang330006China) STRCT:Objective Toinvestigatethemolecularmechanismsofactionofstromalfibroblasts onbreastcancerandtostudytheexpressionoffibroblast-specificprotein-1(fsp-1)anditsim- pactonbreastcancergrowth.methods HumanbreastcancerMD-M-31celsandhumanen- dometrialstromalfibroblasts(esf)wereco-culturedintranswelsystemorculturedalone.the expressionoffsp-1mrn wasexaminedin MD-M-31celsandESFbyRT-qPCR.Theex- pressionoffsp-1proteinwasdetectedbyimmunofluorescenceflowcytometry.theproliferation ofmd-m-31celswastestedusingcelcounting Kit-8.TheexpressionofFSP-1inbreast cancertissueoftumor-burdenednudemicewasanalyzedusingimmunofluorescenceconfocallaser microscopyandtheefectoffsp-1ontumorvolumewasexamined.results Theexpressionof FSP-1wasup-regulatedin ESFco-cultured with MD-M-31celsandtheup-regulationof FSP-1promotedtheproliferationof MD-M-31cels.FSP-1 wasexpressedin MD-M-31 :016-04-4 : (919118) : (1985 ) : E-mail:shixiaoyu999@163.com
celsco-culturedwithesfbutnotin MD-M-31celsculturedalone.InadditionFSP-1washighly expressedinthecancertissuewithco-growthofmd-m-31celsandesfinnudemiceandthehigh expressionoffsp-1acceleratedtumorgrowth.conclusion Theco-culturewithhumanbreastcancer celscanup-regulatefsp-1expressionandpromotebreastcancergrowth. KEY WORDS:fibroblast-specificprotein-1;humanfibroblasts;breastcancer;co-culture (ESF+MD-M-31 )6 10 5-1 Transwel ; 1.5 10 5-1 [1] [-5] Transwel ESF MD-M-31 [6] ESF MD-M-31 ( ESF MD-M-31 ) 1.3 RT-qPCR 3 4 Trizol -l(fibroblast-specific ThermoScientific )qpcr IQ SYR GreenSupermix( io-rad )Transwel ( 0.4μm FisherScientifi ) ( ) )CelCounting Kit-8(CCK-8) ( eyotime ) (3 6 ) FSP-1 : 5 -CCCC- ( SCXK( )01-000) GCTCCGT-3 5 -TTCG- FSP-1( bcam )FSP-1 1. 4% ESF MD-M-31 30min0.1%TritonX-100 8 min5% 10% 100 U ml -1 100μg S 60 min 37 ml -1 DMEM-H hps 3 /IgG- 37 5%CO 80% ~90% FITC 60minPS 3 Transwel 6 500μLFCM bufer ESF MD-M-31 RN DEPC-H O protein1fsp-l) μlrn 60nm/ 80nm RN RN 60nm/80nm>.0 <.3 cdn : RN10μL( 0.7μg) 1μL 1 5 5μLRN 1μL(0U 1.1 μl -1 )mix-dntpμl(10 mm)mmlv 1μL 0μL 5 5 min37 CCC-ESF-1( ESF 60min70 5 min -0 ) cdn PCR :10 μl IQ MD-M-31( SYR GreenSupermix8μL cdn(10μmol) )DMEM-H ( LifeTech-Gibco 1 μl (10 μmol) 1 μl )RevertidfirststrandcDN ( (10μmoL) 0μL 50 3min95 3min;95 8s63 35s39 ;73 7min ; 64~94 PCR io-radcfx Manager GCGCCCCGT-3 1.4 3 4 PS
: -1 3 1.5 CCK-8 MD-M-31 31 ESF FSP-1 mrn ESF (P<5) 3 5 1 6 ESF FSP-1mRN 4 MD-M-31 CCK-8 ESF FSP-1 mrn ;ESF 0μLCCK-8-1 3 4 FSP-1 h 100μL 96 (P>5) 450nm (OD ) FSP-1 MD-M-31 = OD / ( 1):ESF+MD-M-31 OD 100% ESF FSP-1 1.6 3 ESF FSP-1 6 4 ESF FSP-1 3 :ESF+MD-M-31 MD-M-31 16 ESF MD-M-31 7hPS 100μL :V(mm 3 )=ab / [7] 30 10% 3 4 FSP-1 ( HE ) ESF+MD-M-31 MD-M-31 FSP-1 3 FSP-1 MD-M-31 FSP-1 1.7 (P<5) 4 MD-M-31 10% FSP-1 3 (P>5) FSP-1 : 1 M ( ):MD-M-31 FSP-1 (ph6.0) ESF+ MD-M-31 3 PS 3 1%S37 30 min 3 MD-M-31 FSP-1 4 4 MD-M-31 FSP-1 30minPS 3 37 60 minps 3 5 min 3 DPI 10 minps 3 ESF Image-ProPlus.4 1.8.0 SPSSStatistics17.0 1.6 x±s t P< 1. 5 0.8 0.4.1 MD-M-31 ESF FSP-1 RT-qPCR ( 1):ESF+MD-M- RT-qPCR ( : 10 6 MD-M-31 1 10 6 ESF ) FSP-1 1d 4 (a) (b). ESF 5mm MD-M-31 FSP-1 RT-qPCR ( ):MD-M-31 ESF FSP-1 mrn ESF+MD-M-31 3 4 t/d
4 3 4 ESF :RT-qPCR ESF FSP-1mRN ;: ESF FSP-1 P<5;P>5ESF+MD-M-31 vs 3 4 ESF+ MD-M-31 3 vs 4 ; P>5 1 MD-M-31 ESF FSP-1.3 MD-M-31 FSP-1 ESF+MD-M-31 MD-M-31 FSP-1 (P < 6 MD-M-31 5 4 C) (P<5) 5 3 1: ESF+MD-M-31 MD-M-31 0 ESF+MD-M-31 3 ESF+MD-M- MD-M-31 (P<5) 31 MD-M-31 3 FSP-1 (P<5) 3 MD-M-31.4 ESF+MD-M-31.4 FSP-1.0 HE : 1.6 1. ( 4) 0.8 : 0.4 FSP-1ESF+MD-M-31 FSP-1MD-M-31 3 4 MD-M-31 FSP-1 mrn t/d 3 4 MD-M-31 :RT-qPCR MD-M-31 FSP-1 mrn ;: MD-M-31 FSP-1 P<5P>5 ESF MD-M-31 FSP-1
刘 欢等 与乳腺癌细胞共培养的人成纤维细胞特异蛋白 １ 表达上调及意义 ５ 3.0 MD-M-31 组 ESF+MD-M-31 组.5.0 MD-M-31 细胞活力 对照组值设为 100% MD-M-31 细胞增殖 450 nm 1.5 1.0 0.5 1 3 4 时间 t/d 5 50 00 150 MD-M-31 组 ESF+MD-M-31 组 100 50 0 1 6 3 4 5 6 时间 t/d 分别在细胞 培 养 第 １ ６ 天 收 集 ＭＤＡ ＭＢ ２３１ 细 胞 Ａ ＣＣＫ ８ 细 胞 增 殖 实 验 结 果 Ｂ ＭＤＡ ＭＢ ２３１ 细 胞 增 殖 活 力 ０５ Ｐ ０ ０５ Ｐ ０ 图 ３ 与人成纤维细胞 ＥＳＦ 共培养促进人乳腺癌细胞 ＭＤＡ ＭＢ ２３１ 增殖 C FSP-1 免疫荧光强度.0 1.5 1.0 0.5 ES MD-M-31 组 ESF+MD-M-31 组 F +M D -M - 31 组 M D -M - 31 组 在 ＭＤＡ ＭＢ ２３１ 细胞和 ＥＳＦ 细胞接种于裸鼠的第 ３０ 天 将 荷 瘤 裸 鼠 颈 椎 脱 臼 处 死 Ａ ＨＥ 染 色 光 学 显微镜观察荷瘤裸鼠乳腺肿瘤组织 Ｂ 免疫荧光激光共聚焦显微镜观 察 荷 瘤 裸 鼠 乳 腺 肿 瘤 组 织 ＦＳＰ １ 的 表 达 Ｃ Ｉｍａｇｅ Ｐｒ ｏｐ ｌ ｕｓ软件分析荧光强度的结果 Ｐ ０ ０５ 图 ４ 荷瘤裸鼠乳腺癌细胞和成纤维细胞共生长的癌组织高表达 ＦＳＰ １ 表 １ 裸鼠移植瘤体积 组别 ｎ １０ｄ １２ｄ ｘ±ｓ Ｖ ｍｍ３ １４ｄ １６ｄ １８ｄ ２０ｄ ＭＤＡ ＭＢ ２３１ 组 １３１ ８９±７ １８ １５０ ９５±１０ １１ １６７ ７５±１３ ０１ ２２８ ０１±１５ １２ ２８３ １８±１６ ３５ ３４５ ０４±１８ １３ ＥＳＦ ＭＤＡ ＭＢ ２３１ 组 １３２ ５０±７ ２２ １５１ １３±９ １６ 组别 １６７ ９７±１２ ６６ ２２７ ８１±１４ ８４ ２８２ ７３±１６ ４５ ３７１ ６７±１９ ３２ ｎ ２２ｄ ２４ｄ ２６ｄ ２８ｄ ３０ｄ ＭＤＡ ＭＢ ２３１ 组 ４１０ ０６±２０ ５７ ４７３ ３４±２２ ５２ ５３６ ７９±２３ ８６ ６０２ ６６±２５ ０９ ６６４ ３８±２７ ５７ ＥＳＦ ＭＤＡ ＭＢ ２３１ 组 ４９３ ７８±２１ ４７ ５９２ ９６±２４ ８１ ７０７ ２３±２８ ０４ ８１９ ４４±３１ ８１ ９５５ ３５±３４ ８５ ０５ 与 ＭＤＡ ＭＢ ２３１ 组比较 Ｐ ０
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