中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/32570

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(1)! ύ!୯!ᙴ!ᛰ!ε!Ꮲ!! ୷ᘵᙴᏢࣴ‫!܌ز‬ ᅺγᏢՏፕЎ! ! CCN3 ࿶җ FAK, PI3K, AKT Ϸ NF-͒B ၡ৩ቚуΓ ᜪ೬ମዦಒझ‫ޑ‬౽ՉૈΚ !. ! CCN3 increases motility of human chondrosarcoma cell via FAK, PI3K, AKT and NF-ȩB pathways ! ! ! !. ࡰ!Ꮴ!௲!௤!Ǻ᛿ࡘᔺ!!റ!γ!! ! ӅӕࡰᏤ௲௤Ǻ෯ඵܷ!!റ!γ! !!! ! ! ࣴ!‫!ز‬ғǺ!!ഋ ഋ ྷߙ!!!!!! !. ύ๮҇୯ΐΜΐԃϤД!.

(2) . Ҟᒵ Ҟᒵ ..................................................................................................................................ǿ ᕭቪჹྣ߄ ................................................................................................................... III ύЎᄔा ........................................................................................................................ V मЎᄔा ....................................................................................................................... VI ಃ΋ക ‫ׇ‬ፕ ΋ǵ ࣴ‫ز‬ङඳ ............................................................................................................... 1 Βǵ ࣴ‫ز‬Ҟ‫ ޑ‬............................................................................................................... 2 ಃΒക Ў᝘௖૸ ΋ǵ೬ମԺዦ (Chondrosacoma)…………………………………………………3 Βǵᙯ౽ (Metastasis)…………………………………………………….……….8 Οǵߎឦೈқሇન (Matrix metalloproteinaseǹMMPs) ………………………….11 Ѥǵ CCN3………………………………………………………………………….15 ϖǵಒझᗹ๱‫ڙ‬ᡏ (Integrin)……………………………………………………..22 ಃΟക ჴᡍ‫׷‬਑ᆶБ‫ݤ‬ ΋ǵ (΋) (Β) (Ο) Βǵ. ჴᡍ‫׷‬਑ ಒझ…………………………………………………………………....27 ၂Ꮚ…………………………………………………………………....27 ჴᡍሺᏔᆶ઻‫……………………………………………………׷‬....29 ჴᡍБ‫ݤ‬. (΋) (Β) (Ο) (Ѥ) (ϖ) (Ϥ) (Ύ) (Ζ). ಒझ୻Ꭶ…………………………………………………………......30 ԏ໣ಒझ……………………………………………………………..32 ೈқ፦‫ۓ‬ໆෳ‫……………………………………………………ۓ‬..33 ՋБᏀᗺ‫( ݤ‬Western blot analysis)………………………………..34 ಒझӸࢲ౗ϩ‫( ݋‬MTT assay)…………………………………......38 ಒझ౽Չϩ‫( ݋‬Migration assay)……………………………………....39 ಒझᙯࢉ‫…………………………………………………………ݤ‬..40 ӄໆ RNA ‫………………………………………………………ڗܜ‬40. (ΐ) (Μ). Complementary DNA, cDNA ϸᙯᒵբҔ……………………...…..41 RT-PCR……………………………………………………………....42. (Μ΋) (ΜΒ) (ΜΟ). Luciferase ࢲ‫܄‬ෳ‫…………………………………………………ۓ‬43 Gelatin Zymography………………………………………………44. ಍ीϩ‫…………………………………………………………݋‬......46 ಃѤക ่݀ ΋ǵCCN3 ཮ߦ຾Γᜪ೬ମԺዦಒझ‫ޑ‬౽୏‫……………………………………܄‬47 ΒǵCCN3 ཮೷ԋΓᜪ೬ମԺዦಒझϐ MMP-13 ‫ޑ‬εໆ߄౜…………………….47 ΟǵCCN3 ೸ၸ Įvȕ3 Ϸ Įvȕ5 integrin receptor ೷ԋΓᜪ೬ମԺዦಒझ౽Չ ........ 48. I .

(3) . ѤǵFAK ୖᆶӧΓᜪ೬ମԺዦಒझ౽Չύ ...............................................................49 ϖǵPI3K/Akt ୖᆶӧΓᜪ೬ମԺዦಒझ౽Չύ .......................................................50 ϤǵNF-kB ୖᆶӧҗ CCN3 ፓ௓ቚуΓᜪ೬ମԺዦಒझ౽Չύ ...........................51 ಃϖക ૸ፕ ..................................................................................................................53 ಃϤക ่ፕ…………………………………………………………………………..57 ୖԵЎ᝘ ........................................................................................................................58 კ ....................................................................................................................................63. II .

(4) . ᕭቪ߄ ͉-MEM. Minimum Essential Medium. BSA. Bovine Serum Albumin. cDNA. Complementary DNA. DMEM. Dulbecco’s Modified Eagle’s Medium. DMSO. Dimethyl sulfoxide. ECL. Enhanced chemiluminescence. EDTA. Ethylenediaminetetraacetic acid. FAK. Focal Adhesion Kinase. FBS. Fetal Bovine Serum. NaCl. Sodium chloride. NaF. Sodium fluoride. NaHCO3. Sodium bicarbonate. NF-͒B. Nuclear Factor kappa-B. PI3K. Phosphatidylinositol 3-kinase. PMSF. Phenylmethylsulfonyl fluoride. PVDF. Polyvinylidene fluoride. RIPA lysis buffer. Non-ionic detergent-containing buffer. RT-PCR. Reverse Transcriptase-Polymerase Chain III. .

(5) . Reaction SDS. Sodium dodecyl sulfate. siRNA. Small interference RNA. TEMED. Tetramethylethylenediamine. TPCK. Tosyl phenylalanyl chloromethyl ketone. . IV .

(6) . ύЎᄔा NOVࢂCCN family (Cyr61-CTGF-Nov family) ύ‫ޑ‬΋঩ǴѬΞёа ᆀࣁCCN3ǶCCNৎ௼ёа຾ՉӚᅿғ౛‫ޑ‬ፓ௓ӵǺಒझߕ๱ૈΚǵߦ຾ ಒझ౽୏Ϸ‫ڈ‬ᐟಒझቚғբҔ฻...ǶԶCCN3ёа೸ၸᆶಒझ߄य़‫ޑ‬ integrin่ӝ຾Զߦ຾ಒझຠߕ‫܈‬౽୏ૈΚǴฅԶCCN3ᆶ೬ମԺዦ‫ޑ‬౽Չ ٠όܴਟǴӧҁჴᡍύ‫ॺך‬ว౜CCN3ёаቚуΓᜪ೬ମԺዦಒझ‫ޑ‬౽Չ ‫ک‬matrix metalloproteinase (MMP)-13‫߄ޑ‬౜Ƕќ΋Бय़ว౜RGD཮‫ڋ׭‬җ CCN3‫܌‬ቚу‫ޑ‬ಒझ౽ՉϷMMP-13‫ޑ‬ౢғǴՠ๏ϒRAG peptideਔؒԖᡉ ๱‫ޑ‬ৡ౦Ƕௗ๱‫ॺך‬٬Ҕૻ৲໺ሀ‫ڋ׭‬ᏊFAK mutantǵFAK si-RNAǵ Ly294002ǵwortmanninǵ Akt inhibitorǵNF-͒B inhibitor (PDTCǵTPCK ϷNF-͒B inhibitor peptide)ǵp85 mutantǵAkt mutantǵIKKD mutantϷ IKKȕ mutantࡕǴ೿཮‫ڋ׭‬CCN3‫ڈ‬ᐟΓᜪ೬ମԺዦಒझ‫ޑ‬౽ՉૈΚᆶ MMP-13‫ޑ‬ౢғǶ๏ϒCCN3ࡕΨ཮ߦ຾FAKǵPI3KϷAkt‫ࢲޑ‬ϯǶ೭٤่ ݀ࡰрCCN3཮࿶ၸĮvȕ3ϷĮvȕ5 integrin receptorፓ௓Γᜪ೬ମԺዦಒझ ‫ޑ‬౽ՉૈΚᆶMMP-13‫߄ޑ‬౜Ǵࢂ೸ၸPI3K/Akt/NF-͒B೭చၡ৩Ƕ ᜢᗖӷǺCCN3ǵಒझᗹ๱‫ڙ‬ᡏǵΓᜪ೬ମዦಒझǵ౽ՉǵߎឦೈқሇનǶ. V .

(7) . Abstract Nephroblastoma overexpressed (Nov; CCN3), from the CCN gene family, which is involved in many cellular activities such as growth, differentiation, cell motility, adhesion and division. However, the effect of CCN3 on migration activity in human chandrosarcoma cells is mostly unknown. Here, we found that CCN3 increased the migration and expression of matrix metalloproteinase (MMP)-13 through the Dvȕ3 and Dvȕ5 integrin receptor in human chondrosarcoma cells (JJ012 cells). RGD peptide, Dvȕ3 and Dvȕ5 monoclonal antibody but not RAD peptide inhibitor inhibited the CCN3-induced increase migration and MMP-13 expression. Activations of focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI3K), Akt and NF-țB. pathways. after. CCN3. treatment. was. demonstrated,. and. CCN3-induced expression of MMP-13 and migration activity was inhibited by the specific inhibitor of PI3K, Akt and NF-țB cascades. Transfection of cells with FAK, p85, Akt, IKKĮ and IKKȕ mutant also reduced CCN3-induced cancer migration. Taken together, our results suggest that CCN3 acts through FAK/PI3K/Akt, which in turn activates NF-țB, resulting in the activation of MMP-13 and contributing to the migration of human chondrosarcoma cells.. VI .

(8) . Key worldǺCCN3ǵIntegrinǵChondrosarcoma cellsǵMigrationǵMMP-13. . VII .

(9) ಃ΋ക ‫ׇ‬ፕ ΋ǵġ. ࣴ‫ز‬ङඳ. ᕎੱࢂҞ߻Γᜪ଼நғ‫ޑڮ‬ᓐဦఠЋǴଯۚ୯ϣΜεԝΫচӢᄦ २Ǵ೬ମዦᗨฅࢂ΋ᅿϿ‫ޑـ‬ൾ‫܄‬ဍዦǴՠલЮԖਏ‫ޑ‬ᇶշᕍ‫ݤ‬Ъ೬ ମዦ‫ڀ‬Ԗߟҍ‫܈‬ᇻᆄᙯ౽‫ૈޑ‬Κ (Tan et al., 2009)Ǵ೭٤೿٬ளੰΓ Ⴃࡕ‫ޑ‬Ӹࢲ౗࣬྽ৡ (Chow, 2007)ǶՠҞ߻ჹ‫ܭ‬೬ମᕎ‫ޑ‬ԋӢаϷᙯ ౽‫ޑ‬ᐒ‫ڋ‬೿ᗋόࢂࡐమཱǴՠऩૈவύᕕှᕎੱᙯ౽ၸำ‫ޑ‬ϩηᐒ ‫ڋ‬Ǵᔈ၀ёаග‫ٮ‬҂ٰᜢ‫ܭ‬ٛ‫ݯ‬೬ମԺዦᙯ౽ϐԖਏБ‫ݤ‬Ǵ‫ࢂ܈‬྽ԋ ᖏ‫׉‬΢ບᘐᕎੱᙯ౽‫ޑ‬ϩη኱૶Ƕ ӧဍዦ‫׎‬ԋᆶᙯ౽‫ޑ‬ၸำύԖ೚ӭϩηᐒ‫ୖڋ‬ᆶӧ‫ځ‬ύǴ೭хࡴ ᜢ‫ܭ‬ϩှಒझѦ୷፦ (Extracellular matrix ; ECM ) ‫ޑ‬ೈқ䁙୷ӢǴ‫ٯ‬ ӵǺMMP (Matrix metalloproteinase) (Egeblad and Werb, 2002)ǵаϷಒ झᆶಒझ໔‫܈‬ᆶ໔፦ϐ໔࣬ϕբҔ‫୷ޑ‬ӢǴ‫ٯ‬ӵಒझᗹ๱‫ڙ‬ᡏ (Integrin)ǶMMPs ࢂ΋ဂ཮ᆶ Zn2+่ӝ‫ޑ‬ೈқ䁙Ǵૈ୼ϩှಒझѦ໔ ፦ٰᔅշ҅ӧ౽୏‫ޑ‬ಒझቚ຾ߟҍૈΚǴ೭ࢂဍዦᙯ౽ၸำύ‫܌‬Ѹा ‫ޑ‬ǴӧЎ᝘ύΨว౜ǴMMPs ‫߄ޑ‬౜ໆࢂဍዦᙯ౽ϷߟҍૈΚ‫ࡰޑ‬኱ (McCawley and Matrisian, 2000)Ƕ CCN family ‫ࢂޑ‬΋ᅿᆶ୷፦Ԗᜢ‫ޑ‬ϩ‫ޑࠠݜ‬ೈқ፦ǴѬॺୖᆶ ೚ӭಒझ‫ٯ୏ࢲޑ‬ӵғߏǵϩϯǵ౽୏ǵғӸ฻ (Chen and Lau, 2009;. 1.

(10) Perbal, 2004)ǶԶ Nephroblastoma overexpressed (Nov; CCN3)Ǵឦ‫ܭ‬ CCN family ύ‫ޑ‬΋঩ǴԖЎ᝘ࡰр CCN3 ཮ߦ຾Γᜪဍዦಒझ‫ޑ‬౽ ୏‫܄‬ᆶߟҍૈΚ (Benini et al., 2005; Sin et al., 2009)Ƕᖏ‫׉‬΢‫زࣴޑ‬ᡉ Ң CCN3 ᆶဍዦಒझ‫߄ޑ‬౜Ԗᜢ (Perbal et al., 2008; Sin et al., 2009)ǶCCN3 ёаբࣁ integrin ‫ޑ‬ଛᡏ (Ligand)Ǵ೸ၸᆶ integrin ‫ޑ‬ ่ӝ໒௴Πෞ‫ૻޑ‬৲໺ሀ೷ԋಒझ౽୏ǵ₢Ϋǵ໾αঅൺǵՈᆅཥғ ᆶߕ๱ૈΚ‫ׯ‬ᡂ฻฻... (Chen and Lau, 2009)ǶฅԶ CCN3 ჹ‫ܭ‬Γᜪ೬ ମԺዦಒझ‫ ޑ‬MMP ߄౜аϷ೷ԋಒझ౽୏‫ޑ܄‬ᐒ‫ڋ‬Ҟ߻ᗋόࢂࡐమ ཱǶ Βǵġ. ࣴ‫ز‬Ҟ‫ޑ‬. ҁࣴ‫ޑز‬ЬाҞ‫ޑ‬Ǻ (΋)ǵ ௖૸ CCN3 ࢂցёа೷ԋ chondrosarcoma cells (JJ012 cells) ‫ޑ‬౽୏‫܄‬Ƕ (Β)ǵ ௖૸ЬाҗՖᅿ MMPs ଺ࣁಒझߟҍૈΚ‫ޑ‬ፓ௓Ƕ (Ο)ǵ ௖૸ CCN3 ᇨᏤΓᜪ೬ମಒझዦ‫ૻޑ‬৲໺Ꮴ೼৩Ƕ. 2.

(11) ಃΒക Ў᝘௖૸ ΋ǵġ ೬ମԺዦ (Chondrosacoma) Γ ᡏ ‫ ޑ‬ମ ᓝ ԰ ಍ ࢂ җ Ǻ ମ (Bone) ǵ ೬ ମ (Cartilage) Ϸ ᜢ ࿯ (Articulation) Ο‫ޣ‬ಔԋǴ೸ၸᆶԼԺ‫ޑ‬ҬϕբҔ຾Զၲ‫ډ‬ၮ୏ǵЍ࡭ ‫ߥک‬ៈ‫ي‬ᡏ‫ޑ‬фૈǶମᓐᄬ೷ύԖΟᅿ੝ϯ‫ޑ‬ମಒझǴϩձࢂ osteocyteǵosteoblast аϷ osteoclastǶ೬ମϩѲӧᜢ࿯‫߄ޑ‬य़Ǵߥៈ ମᓐǵ‫ނڙה‬౛‫܄‬ᓸΚǵೱௗ೬ಔᙃ‫ॄک‬ၩ‫ي‬ᡏख़ໆ (Poole et al., 1993)Ƕ ೬ମࢂҗ೬ମ҆ಒझ (Chondroblast) ‫׎܌‬ԋ‫ޑ‬೬ମಒझᆶಒझ Ѧ୷፦‫܌‬ಔԋ‫ޑ‬Ǵ‫ځ‬ύкᅈ๱җೈқӭᗐϩηᆶӭᗐೈқϩηᆫӝ‫ނ‬ ‫ޑ‬ጤচᠼᆢ (Collagen fiber) ᆶቸ‫܄‬ᠼᆢ (Elastic fiber) ‫ޑ‬ᆫӝ‫܌ނ‬ ಔ ԋ Ǵ Ѭ ॺ ࢂ ᄬ ԋ ೬ ମ ୲ ฯ Զ ࢋ ೬ ‫ ޑ‬Ь ा Ӣ ન (Paulsson et al., 1987)Ƕ೬ମёаϩԋΟᅿǴЬा‫ޑ‬ৡձӧ‫୷ܭ‬፦‫ޑ‬ಔԋόӕǴϩձ ࣁ೸ܴ೬ମ (Hyaline cartilage )Ǵቸ‫܄‬೬ମ (Elastic cartilage)ǴаϷᠼ ᆢ೬ମ (Fibrocartilage)Ƕа೸ܴ೬ମ‫ޑ‬ϩѲനቶǴӵᜢ࿯ǵԻ೬ମǵ ‫֎ڥ‬ၰǴЬाࢂҗಃΒࠠ‫ޑ‬ጤচೈқ (Type ɛ collagen) ‫׎‬ԋǶԶ྽ ೬ମр౜ୢᚒਔǴёૈ཮೷ԋӭᅿ‫ޑ‬ମᓝ੯ੰ‫ٯ‬ӵମᓐว‫ػ‬ό‫ੱؼ‬ (Skeletal dysplasias)ǵϣғ‫܄‬೬ମዦ (Enchondromatosis)ǶԶ‫ځ‬ύϣғ ‫܄‬೬ମዦᗋԖёૈ཮ൾ‫܄‬ᙯϯࣁ೬ମԺዦ (Yu et al., 2003)ǶନԜϐѦ 3.

(12) ྽วᐨ‫ࢂ܈‬Ԗ‫ځ‬д‫ޑ‬ว‫ݹ‬ϸᔈਔǴ཮ߦ٬ྖጢϩ‫ݜ‬΋ᅿћ଺ MMPs ‫ނޑ‬፦ǴѬઇᚯጤচೈқᆶᗐೈқ຾Զઇᚯ೬ମಒझ‫ޑ‬ว‫ػ‬Ǵ೷ԋ೬ ମ഼ѨфૈǴΨ཮೷ԋ΋ೱՍ‫ޑ‬೬ମੰᡂ (Aigner et al., 2006)Ƕ྽ MMPs ϩ‫ݜ‬ό‫ى‬Ǵҭ཮ቹៜମᓐ‫ޑ‬ғߏ (Page-McCaw et al., 2007) (Fig. 1)Ƕ. 4.

(13) 5.

(14) Fig 1. Skeletal phenotypes of MMP mutants. (A) Long bones in mice and humans develop through the process of endochondral ossification, in which a cartilage template froms first and then is resorbed and replaced by mineralized bone. This process requires extensive matrix remodeling and invasion of new blood vessels. (B) Martix metalloproteinase-9 (MMP-9)- and MMP-13 null femurs display greatly expanded hypertrophic cartilages zones (HC; red line) and altered trabecular bone (TB; blue line). Despite this expansion, MMP-9 and MMP-13 null phenotypes eventually resolve, resulting in good bone formation. The MMP-9 MMP-13 double mutant has an even greater expansion of hypertrophic cartilage, and significantly and persistently shorter long bones. Images courtesy of D. Stickens, D. Behonick and N. Ortega, University of California, San Francisco, USA (Page-McCaw et al., 2007).. 6.

(15) . ೬ମԺዦࢂ΋ᅿҗ೬ମౢғ‫ޑ‬ൾ‫܄‬ဍዦǴᅿᜪεԿ΢ϩԋΟᅿǺ. ‫ࠠڂ‬ମԺዦ. (Classic chondrosarcoma) ǵ ѐ ϩ ϯ ࠠ ମ Ժ ዦ. (Dedifferentiated. chondrosarcoma). а Ϸ ໔ ယ ࠠ ೬ ମ Ժ ዦ. (Mesenchymal chondrosarcoma)Ƕ‫ځ‬ύ೬ମԺዦࢂಃΒঁനத‫ޑـ‬চว ‫܄‬ମဍዦǴ೯தр౜ӧԃइКၨε‫ޑ‬௼ဂǴԶ೬ମԺዦёૈр౜ӧ‫ي‬ ᡏ‫ޑ‬ҺՖ೽ϩǴεӭத‫ܭـ‬ମࣧǵԻମǵЋᖉ (ާମ)ǵުछମ‫ک‬ᆲ (߈ ᆄިମǴ⅂ମ)Ǵଽᅟ཮ӧૉ෎‫܈‬ᓐମϣว౜Ƕ җ‫ܭ‬೬ମԺዦჹ‫ܭ‬ϯᏢ‫ܫ܈‬ᕍ‫ݯޑ‬ᕍਏ݀٠όӳǴ‫܌‬аҞ߻аЋ ೌϪନࣁЬा‫ک‬നԋф‫ޑ‬ЋࢤǴՠࢂဍዦ࿶த೏ว౜ӧ΋٤όֹܰӄ Ϫନ‫ޑ‬ӦБǴӵମࣧ‫ߏ܈‬ମ߈ᆄǴ೭٤೿೷ԋЋೌ‫ݯ‬ᕍ‫ޑ‬ᜤᚒ‫ࢂ܈‬Ԗ ёૈౢғЋೌࡕ‫ࡕޑ‬ᒪੱǴу΢લЮԖਏ‫ޑ‬ᇶշᕍ‫ݤ‬Ǵ٬ள೬ମԺዦ ‫ੰޑ‬Γ‫ޑ‬ႣࡕૈΚৡǵᗋ཮ԖൺวϷᙯ౽‫ޑ‬ёૈ‫( ܄‬Fong et al., 2007)ǶԖ᠘‫ܭ‬ԜǴऩૈவύᕕှ೬ମԺዦᙯ౽ၸำ‫ޑ‬ϩηᐒ‫ڋ‬Ǵ‫܈‬ ೚ёаග‫ٮ‬҂ٰᜢ‫ܭ‬ٛ‫ݯ‬೬ମԺዦᙯ౽ϐԖਏБ‫ݤ‬Ƕ. 7.

(16) Βǵġ ᙯ౽ (Metastasis) ࣴ‫ز‬಍ीऊԭϩϐΐΜ‫ޑ‬ᕎੱੰ஻ԝΫচӢࢂᕎಒझ‫ޑ‬ᙯ౽ (Sporn, 1996)Ƕဍዦӧᙯ౽‫ޑ‬ၸำࣁǺ1ǵ ᕎಒझগᚆ (Detachment)Ǵ ᚆ໒চҁ‫ޑ‬ဍዦǶ2ǵ ௗ๱ᕎಒझ཮ϩ‫ݜ‬рϩှಒझ୷፦‫ޑ‬ሇન٬ள ᕎಒझ‫ڀ‬ԖߟҍૈΚ (Duffy, 1996)ǴջёΕߟ‫ډ‬Ոᆅ‫܈‬రЃ԰಍ύǶ ‫׳‬຾΋‫؁‬Ǵᕎಒझ཮ౢғ‫ٯ‬ӵǺVEGFǵPDGF ฻…ғߏӢη (Blood and Zetter, 1990)Ǵ٬ளՈᆅཥғǶӧՈᆅ‫܈‬రЃᆅύǴဍዦಒझӵ݀ ёа‫ܢ‬Ᏺխࣝϸᔈ൩཮ຠߕ‫ډ‬ϣҜಒझ‫܈‬΋٤ཥғ‫ޑ‬ՈᆅᏛ΢Ƕ3ǵ ऀၸՈᆅ‫܈‬రЃᆅ‫ډ‬Ҟ኱Ꮤ‫۔‬ғߏǴౢғཥ‫ޑ‬ဍዦ (Fig. 2)Ƕ ӧᕎಒझ‫ޑ‬ᙯ౽ၸำύǴᕎಒझ཮ౢғѤᜪϩှಒझѦ୷፦‫ޑ‬ሇ નǺ MMPsǵserine proteinase (Urokinase plasminogen activator, u-PA)ǵ aspartic proteinase (Pepsin)ǵcysteineǶ྽ύΞа MMPs ᆶ u-PA ‫ת‬ᄽઇ ᚯ୷ۭጢύख़ा‫فޑ‬ՅǴ‫܌‬а೭٤Нှೈқሇન‫܄ࢲޑ‬ᆶဍዦ‫ޑ‬ൾ‫܄‬ ำࡋϷଯࡋ‫ޑ‬ᙯ౽ૈΚ೿Ԗ࣬ᜢ‫( ܄‬Cockett et al., 1998)Ƕ. 8.

(17) . 9.

(18) Fig 2. A schematic of the metastatic process. A schematic of the metastatic process beginning with (a) an in situ cancer surrounded by an intact basement membrane. (b) Invasion requires reversible changes in cell–cell and cell–extracellular-matrix adherence, destruction of proteins in the matrix and stroma, and motility. Metastasizing cells can (c) enter via the lymphatics, or (d) directly enter the circulation. (e) Survival and arrest of tumour cells, and extravasation of the circulatory system follows. (f) Metastatic colonization of the distant site progresses through single cells, which might remain dormant for years, to occult micrometastases and (g) progressively growing, angiogenic metastases (Steeg, 2003).. 10.

(19) Οǵߎឦೈқሇન (Matrix metalloproteinaseǹMMPs) ӧဍዦಒझᙯ౽‫ޑ‬ၸำǴϩှဍዦ‫ڬ‬ൎಒझѦ୷፦‫ޑ‬ሇનӵ MMPs ࢂဍዦᙯ౽ၸำύ‫܌‬Ѹा‫( ޑ‬Hood and Cheresh, 2002)ǶҞ߻ς ࿶‫ޕ‬ၰ MMPs ёаቹៜखजว‫ػ‬ǵ໾αᘰӝǵว‫ݹ‬ϸᔈаϷᕎಒझᙯ ౽ (Curran and Murray, 1999; Nagase and Woessner, 1999)Ƕ MMPs ࢂ΋ᅿ֖ᎋ‫ߎޑ‬ឦೈқሇનǴ‫ځ‬ᄬ೷Ьा֖ԖΟঁ domainǺ߻ᴏ两୔ (Propeptide domain)ǵ໽ϯ୔ (Catalytic domain) а Ϸ‫ڙ‬፦բҔ୔ (Substrate domain)ǴMMPs ໒‫ࢂۈ‬аؒԖࢲ‫׎ޑ܄‬Ԅ (Proenzyme) ೏ញ‫ډܫ‬ಒझѦǴ྽ MMPs ᄬ೷‫ ޑ‬N ᆄ߻ᴏ两୔೏Нှ ‫ޑ‬ਔࡕω཮‫ڀ‬Ԗࢲ‫܄‬. (Knauper et al., 1996; Van Wart and. Birkedal-Hansen, 1990)ǶҞ߻ว౜‫ ޑ‬MMPs Ԗ 28 ᅿǴԶਥᏵ MMPs ‫ޑ‬ ่ᄬаϷ٩ྣ‫܌‬ϩှ‫ނޑ‬፦‫ޑ‬όӕεԿ΢ϩԋϖεᜪ (Fig. 3)Ǻ 1.. CollagenaseǺ хࡴ MMP-1ǵ-8ǵ-13ǵ-18ǴЬा‫ޑ‬фૈёаफ़ှಃ΋ǵ ΒǵΟǵϷΎࠠጤচǴՠค‫ݤ‬ϩှ galtinaseǶԶ‫ځ‬ύ MMP13 Ξᆀࣁ collagenase-3Ƕ. 2.. GeltinaseǺ хࡴ MMP-2ǵ-9ǴΞᆀܴጤ䁙Ǵૈ୼ઇᚯ collagenaseǵ laminin ‫ۭ୷ک‬ጢǶ 11.

(20) 3.. StomelysinǺ хࡴ MMP-3ǵ-7ǵ-10 ฻ǴԜᜪ‫ ޑ‬MMPs ҁ‫ي‬όᛙ‫ۓ‬Ψό ܰӸӧಔᙃϣǴՠࢂࡐ৒ܰ೏΋٤ಒझᐟનǵဍዦӢη‫ک‬ғߏ Ӣη‫܌‬ᇨว (Matrisian, 1992)Ƕ. 4.. Membrane-type MMPǺ хࡴ MMP-14ǵ-15ǵ-16 ฻Ǵ೭ᜪ‫ ޑ‬MMPs ନΑёа཮ ϩှಒझѦ୷፦Ǵᗋ཮ᐟࢲ MMP-2 ‫ک‬-13Ƕ. 5.. ‫ځ‬дᜪ‫ ޑ‬MMPǺ хࡴ MMP-4ǵ-5ǵ-6ǵ-20 ฻฻ (Kuzuya and Iguchi, 2003)Ƕ. 12.

(21) 13.

(22) Fig. 3.. Schematic structure of MMPs.. (a) Matrix metalloproteinases (MMPs) are expressed as pro-proteins. A conserved Cys residue in the pro-domain coordinates the zinc ion, which would otherwise be used for catalysis. The pro-domain is removed by a combination of a cleavage in the domain and a cleavage between the pro-domain and the catalytic domain. (b) Most MMPs share a conserved domain structure of pro-domain, catalytic domain, hinge region and hemopexin domain (1) All MMPs are synthesized with a signal peptide, which is cleaved during transport through the secretory pathway. MMP-2 and MMP-9 have three fibronectin type II repeats in their catalytic domains (2) Membrane type MMPs (MT-MMPs) are linked to the plasma membrane. either. by. a. transmembrane. domain. or. by. a. glycosylphosphatidylinositol (GPI) linkage, attached to the hemopexin domain (3) Minimal MMPs lack the hinge and hemopexin domains (4) MMP-21 has a truncated hinge domain. Drosophila melanogaster DmMMP-2 has an insertion of 214 amino acids into its hinge domain. MMP-23 (not shown) has a nonconserved N-terminal domain that consists of an immunoglobulin IgC2 domain and a ShKT domain; it is unclear if MMP-23 contains a Cys residue switch (Page-McCaw et al., 2007).. 14.

(23) MMPs ନΑ཮ᆶ ECM ่ӝ଺ࣁಒझ౽୏‫ߟک‬ҍ‫ޑ‬ၡ৩ (Lochter et al., 1998) ѦǴനЬा‫ޑ‬բҔࢂӧϩှ ECM ᔅշ౽୏‫ޑ‬ಒझߟҍ‫ځډ‬ д‫ޑ‬ಔᙃϷᏔ‫۔‬ǶԖЎ᝘ࡰр MMPs ‫߄ޑ‬౜ໆନΑёа଺ࣁဍዦᙯ౽ ϷߟҍૈΚ‫ޑ‬኱૶ (McCawley and Matrisian, 2000) ѦǴᗋёа଺ࣁဍ ዦൾϯำࡋ‫ޑ‬ղᘐǴӵ MMP-1ǵ-2ǵ-3ǵ-9ǵ-13 ฻ (Stetler-Stevenson, 1996)ǴӢࣁόᆅӧΓᜪ‫ނ୏ࢂ܈‬ύ‫ޑ‬ဍዦಒझ MMPs ‫߄ޑ‬౜ໆ೿Ԗቚ у‫ޑ‬౜ຝ (Coussens et al., 2002)Ƕ ѤǵCCN3 CCN family (Cyr61-CTGF-Nov family)Ҟ߻ς‫ޕ‬ԖϤঁܴዴ‫ޑ‬ԋ ঩ Ǵ х ࡴ CCN1 (Cysteine-rich protein Ǵ Ξ ᆀ ࣁ Cyr61) ǵ CCN2 (Connective tissue growth factor Ǵ Ξ ᆀ ࣁ CTGF) ǵ CCN3 (Nephroblastoma overexpressedǴΞᆀࣁ Nov)ǵCCN4 (Wnt-induced secreted protein-1ǴΞᆀࣁ WISP-1)ǵCCN5 (Ψᆀࣁ WISP-2) аϷ CCN6 ( Ψ ᆀ ࣁ WISP-3) Ƕ CCN ೈ қ ่ ᄬ х ֖ ཱུ ଯ ‫ ޑ‬ӕ ྍ ‫܄‬ (Homology)ǴЬाࢂҗѤঁࡐ࣬՟‫ൂޑ‬ϡ (Domains) ‫܌‬ಔԋǴхࡴ insulin-like growth factor binding protein (IGFBP)ǵvon willebrand factor type. C. (VWC) ǵ thrombospondin. type. 1. repeat. (TSP1). ‫ک‬. carboxyl-teriminal domain (CT)Ǵ‫ځ‬ύ WISP-2 લϿ CT domain (Chen and Lau, 2009)ǶӢࣁ CCN family ԋ঩‫่ޑ‬ᄬ‫ ޑ‬N ᆄ (NH2-terminal). 15.

(24) ೿֖Ԗ secretory signal ‫ޑ‬਽୷ለූ୷Ǵ‫܌‬а CCN family ‫ޑ‬ԋ঩ឦ‫ܭ‬ ϩ‫ޑࠠݜ‬ೈқ፦ (Yang and Lau, 1991)Ǵёа߄౜ӧಒझ߄य़‫ࢂ܈‬ᆶ ಒझ୷፦ϐ໔ (Fig. 4)Ƕ. 16.

(25) 17.

(26) Fig. 4. Arrangement of CCN domains. (a) A diagram showing the signal peptide (SP), insulin-like growth factor binding domain (IGFBP) in red, von Willebrand factor C repeat (VWC) in blue, thrombospondin type-1 repeat (TSP-1) in yellow and cysteine knot (CT) in green. The protein is split into two halves separated by a variable ‘hinge’ region. Some of the known binding partners of each module are also listed: insulin-like growth factors (IGFs); bone morphogenic protein 4 (BMP4); transforming growth factor b (TGF-b); LDL receptor protein 1 (LRP-1); and heparin sulphated proteoglycans (HSPGs). (b) A sequence alignment of the CCN protein family. The sections of the sequence corresponding to each domain are shaded according to the colour scheme used in (a) The asterisks highlight the conserved residues and include the 38 cysteines that form part of the key motifs of each domain. The three regions of the sequence that have been implicated directly in integrin binding are also highlighted. These areas are highlighted in bold text. The V2 site binds integrin Įvȕ3; the T1 site binds Į6ȕ1; the H1 site also binds Į6ȕ1; and the H2 site binds HSPGs. The alignment was constructed by the T-Coffee server (Holbourn et al., 2008).. 18.

(27) CCN3 ന߃೏ว౜ࢂӢࣁӧച࿥ᜪύǴmyeloblastosis-associated viruses ᇨว‫ ޑ‬avian nephroblastomas (Zeng et al., 2004)ǴCCN3 ೏ᇡࣁ ୖᆶဍዦ‫׎‬ԋၸำǴԖЎ᝘ࡰрӧ΋٤ primary tumor cell lines хࡴǺ ឪៈဏᕎǵ໵ՅનዦϷମԺዦύ CCN3 ‫߄ޑ‬౜Ԗ೏ፓϲ‫ޑ‬౜ຝ (Maillard et al., 2001; Manara et al., 2002)Ǵӧ‫ځ‬ύ CCN3 ೏ࡰрёа྽ բੰΓႣࡕၨৡ‫ࡰޑ‬኱Ӣη (Perbal et al., 2008)ǴԜѦ CCN3 Ψࢂ΋ ঁख़ा‫ޑ‬ՈᆅཥғᇨᏤӢη (Novel angiogenic)ǴԖࣴ‫ࡰز‬рӧՈᆅ ϣ Ҝ ಒ झ ύ CCN3 ೸ ၸ integrin receptor ё а ߦ ຾ Ո ᆅ ‫ ׎‬ԋ (Proangiogenic)ǴԶӧᖏ‫׉‬΢Ψࡰр CCN3 ёаᇨᏤՈᆅ‫ޑ‬ཥғ (Neovascularization) (Lin et al., 2005)Ƕ ӧ೚ӭόӕ‫ޑ‬ಒझࠠᄊύǴCCN3 ёаբࣁ integrin ‫ ޑ‬ligandǴ ᙖ๱ᆶόӕ‫ ޑ‬integrin Į ᆶ ȕ subunit ಔӝǴӵ integrin Įvȕ3ǵĮvȕ5ǵĮ5ȕ1 ᆶ Į6ȕ1 ่ӝٰ୺Չፓ௓ಒझ‫ޑ‬фૈǴхࡴǺCCN3 ё่ӝԿಒझ߄ य़‫ ޑ‬Įvȕ3 Ϸ Į5ȕ1 integrin ٠ߦ຾ϣҜಒझ (Endothelial cell) ຠߕ‫܈‬ ౽୏ (Migration)ǵ೸ၸᆶ Į6ȕ1 Ϸ Įvȕ5 integrin ่ӝߦ٬ᠼᆢ҆ಒझ (Fibroblasts) ϷϣҜಒझຠߕ‫܈‬౽୏ǶନԜϐѦǴCCN3 ёа೸ၸ integrin Į6ȕ1 Ϸಒझ߄य़΢‫ ޑ‬HSPGs (Heparan sulfate proteoglycans) ٬ளಒझ‫ޑ‬ຠߕ‫׳‬уᛙ‫( ڰ‬Chen and Lau, 2009; Lin et al., 2005) (Table. 1)Ƕ. 19.

(28) ԜѦ CCN3 Ψёаፓ௓ࢌ٤୷Ӣ߄౜ǴхࡴᇨวՈᆅཥғϷว‫ݹ‬ ϸ ᔈ Ԗ ᜢ ‫ ޑ‬ಒ झ ᐟ ન ǵ MMPs а Ϸ cell adhesion receptors ቚ у (Benini et al., 2005)Ƕ. 20.

(29) Table 1. Specific CCN-integrin interactions and activities they mediate. (Chen and Lau, 2009). Integrins are cell adhesion receptors that also regulate other cellular functions. They serve as the principal receptors for CCN proteins.. 21.

(30) ϖǵġ ಒझᗹ๱‫ڙ‬ᡏ (Integrin) Integrin ࢂ΋ဂՏӧಒझ߄य़΢‫ޑ‬ௗ‫ڙ‬ᏔǴ཮௓‫ڋ‬ಒझମࢎ‫ނ‬፦ (Cytoskeletal elements) Ϸᆶಒझ୷፦ (Matrix) ϐ໔‫ޑ‬ҬϕբҔǴ‫܌‬ а integrin ӧಒझ౽୏Ϸᗹߕၸำύ‫ת‬ᄽ๱ख़ा‫ف‬ՅǶѬॺࢂҗ Į Ϸ ȕ ‫ঁٿ‬ԛൂϡ (Subunits) ‫܌‬ᄬԋ‫ ޑ‬heterodimer receptorǴҞ߻ς‫ޕ‬Ԗ 18 ᅿ Į ԛൂϡϷ 8 ᅿ ȕ ԛൂϡǴόӕ‫ ޑ‬Į ԛൂϡёа‫ ک‬ȕ ԛൂϡ่ ӝ‫׎‬ԋऊ 24 ᅿ‫ ޑ‬integrin receptor (Guo and Giancotti, 2004)Ƕ Integrin ᆶ ligand ่ӝਔЬाࢂᒤຎ ligand ύҗኧঁ਽୷ለ‫܌‬ಔ ԋ‫ޑ‬อ两 (Peptide) ‫ׇ‬ӈǴԶόӕ‫ ޑ‬Integrin ёаᒣձ࣬ӕ‫ׇ‬ӈ‫܈‬ӕ ΋ঁ ligand ύόӕ‫ޑ‬਽୷ለ‫ׇ‬ӈǴ‫ځ‬ύεठϩࣁ‫ٿ‬εᜪǺᒣຎ RGD (Arg-Gly-Asp) ‫ׇ‬ӈаϷߚ RGD ‫ׇ‬ӈǶ‫ځ‬ύD5ȕ1ǵDvȕ3ǵDvȕ5ǵDvȕ6ǵ Dɛbȕ3 ϷDvȕ1 ёа᛽ຎ ligand ϩηύ‫ ޑ‬RGD ‫ׇ‬ӈǹԶ‫ځ‬д߾ࢂᒣձ ligand ύ YGDLRǵKQAGDVǵDGEA ฻อ两‫ׇ‬ӈ (Ruoslahti, 1996) (Fig 5)Ƕ. 22.

(31) 23.

(32) Fig 5. The Integrin Receptor Family Integrins are Dȕ heterodimersǹeach subunit crosses the membrane once, with most of each polypeptide (>1600 amino acids in total) in the extracellular space and two short cytoplasmic domains (20-50 amino acids). The figure depicts the mammalian subunits and their Dȕ associationsǹ8ȕ subunits can assort with 18D subunits to form 24 distinct integrins. These can be considered in several subfamilies based on evolutionary relationships (coloring of D subunits), ligand specificity and , in the case of ȕ2 and ȕ7 integrins, restricted expression on white blood cells. D subunits with gray hatching or stippling have inserted I/A domains (see text). Such D subunits are restricted to chordates, as are D4 and D9 (green) and subunits ȕ2-ȕ8. In contrast, D subunits with specificity for laminins (purple) or RGD (blue) are found throughout the metazoan and are clearly ancient (see text). Asterisks denote alternatively spliced cytoplasmic domains. A few extracellular domains are also alternatively spliced (not shown). Furteher information on integrin subunit structures and details of lignad specificity are given in several extensive reviews (Hemler, 1999; Plow et al., 2000; van der Flier and Sonnenberg, 2001).. 24.

(33) ྽ integrin ᆶ፾྽‫ ޑ‬ligand ่ӝ‫ࢲډڙ‬ϯࡕǴ཮ஒಒझૻ৲җϣ ӛѦ (Inside-out) ϷҗѦӛϣ (Outside-in) ໺ሀǴନΑ཮‫ׯ‬ᡂಒझମ ࢎѐፓ࿯ಒझᆶಒझ໔ǵಒझѦ୷፦໔‫ߕޑ‬๱ૈΚ (Adhesion)ǵ୷Ӣ ᙯᒵ‫ޑ‬ፓ࿯฻Ǵ຾Զቹៜಒझ‫ޑ‬ғߏǵϩϯǵ౽୏ϷߟҍૈΚǵࣗԿ ཮‫ׯ‬ᡂಒझ‫ࠠޑ‬ᄊ (Hood and Cheresh, 2002; Hynes, 1992) Ϸୖᆶ໾ α‫ޑ‬অൺ (Wound healing)Ƕ ‫ځ‬ύ inside-out ‫ૻޑ‬৲໺ሀЬाࢂ‫ׯ‬ᡂಒझᗹ๱‫ڙ‬ᡏ‫ޑ‬ᒃ‫ک‬Κ (Affinity) (Hood and Cheresh, 2002; Zhang et al., 1996)ǹԶ outside-in ‫ޑ‬ ૻ৲໺ሀ߾ࢂࡰҗಒझᗹ๱‫ڙ‬ᡏ‫ࢲډڙ‬ϯࡕ‫܌‬Їଆ‫ૻޑ‬৲ᘣѲਏᔈ (Signaling cascade) (Hood and Cheresh, 2002)ǴԶ‫ځ‬ύ FAK (Focal adhesion kinase) ǵSrc kinase Ϸ paxillin ࢂΟঁख़ा‫ૻޑ‬৲໺ሀϩηǶ FAK ࢂ΋ঁߚ‫ڙ‬ᡏࠠႀữለᕗለᐟ䁙 (Non-receptor protein tyrosine kiase)Ǵϩηໆࣁ 125kDa (Kanner et al., 1990)Ǵ߄౜ӧεӭኧ ‫ޑ‬ಒझϷಔᙃύǶFAK ࢂ integrin receptor ౢғ outside-in signaling ύ Տ‫ܭ‬ന΢ෞΨࢂനख़ा‫ޑ‬ೈқǴԶ FAK ό‫ڀ‬ഢ SH2 ‫ ܈‬SH3 domains (SH2 domain for binding to phosphor-tyrosineǹSH3 domain for binding to proline-rich regions)Ƕ྽ૻ৲җ integrin ஒૻ৲۳Π໺ሀਔǴ཮ࢲϯ FAKǴ٬ள FAK ‫ ޑ‬C ᆄ (C-terminal) ཮ᆶ talin ‫ ک‬paxillin ่ӝࡕǴ ཮ӧ Y397 ೭ঁՏ࿼Ծ‫ך‬ᕗለϯࡕԶౢғࢲ‫܄‬Ǵௗ๱ஒૻ৲۳Π໺ଌ. 25.

(34) ຾ԶࢲϯΠෞ‫ૻޑ‬৲೼৩Ǵаፓ௓хࡴಒझᎂ౽ǵಒझӸࢲǵಒझቚ ғϷಒझϩϯ (Desgrosellier and Cheresh, 2010; Webb et al., 2004)ǶЎ ᝘ύࡰрӧ FAK. -/-. ‫ޑ‬ಒझύว౜‫ځ‬ಒझ‫ޑ‬౽୏ૈΚᆶғߏ೿཮ܴᡉ. ‫ޑ‬फ़ե (Cary et al., 1996; Westhoff et al., 2004)Ǵ೭ཀ‫ښ‬๱ FAK ӧᕎಒ झ‫ޑ‬ൾϯၸำԖ࣬ᜢ‫܄‬Ƕ. 26.

(35) ಃΟക ჴᡍ‫׷‬਑ϷБ‫ݤ‬. ಃ΋࿯ġ ჴᡍ‫׷‬਑. Ȑ΋ȑġ. ಒझ:. ҁჴᡍ‫܌‬٬Ҕ‫ࢂޑ‬Γᜪ೬ମዦಒझ (JJ012) җ Dr. Sean P Scully (University of Miami, School of Medicine, Miami, FL) ჴᡍ ࠻ග‫ٮ‬Ƕ. ȐΒȑġ. ၂Ꮚ:. A. ჴᡍᛰࠔ 1. CCN3 (Rocky Hill,NJ,USA,Peprotech) 2. WortmanninǵLY294992ǵAkt inhibitorǵPDTCǵTPCK ȐCalbiochem, San Diego, CA, USAȑ 3. NF-țB inhibitor peptide (Enzo) 4. RGDǵRAD (Louisville, KY). B. ಒझ୻Ꭶ၂Ꮚ 1. DMEM (Sigma-Aldrich, Madison, MA, USA) 2. Į-MEM (Sigma-Aldrich, Madison, MA, USA) 3. fetal bovine serum (FBS) (Invitrogen Corpoation, Califormia, 27.

(36) USA) 4. Gentamycin (Invitrogen, USA) 5. Hycobian (Invitrogen, USA) 6. Insulin (Invitrogen, USA). C. ႝ‫ݚ‬ϷՋБᏀᗺᙯᅄ၂Ꮚ 1. BASTM protein assay kit (Pierce, IL, USA) 2. AcrylamideǵTrisǵTris-HClǵSDSǵPMSFǵNa3VO4ǵ AprotininǵNaF (Ameresco Inc, Ohio, USA) 3. Tween20ǵGlycine (Ameresco Inc, Ohio, USA) 4. Tetramethylethylenediamine (TEMED)ǵAmmonium Persulfate (APS) ǵPVDF (Immobilon-p) (Millipore, MA, USA) 5. ECL (Kodak Inc, NY, USA) 6. Wester blot Develop & replenisherǵFix & replenisher ǵ BioMax light Film (Kodak Inc, NY, USA). D. ‫ל‬ᡏ 1. p-FAKǵp-PI3Kǵp-Aktǵp-IKKĮ/ȕǵp-IțBĮǵp-p65ǵȕ-actin (Cell signal, MA, USA) 2. FAK-siRNAǵAnti-mouseǵAnti-rabbitǵFAKǵPI3KǵAktǵ IKKĮ/ȕǵIkBĮǵp65 (Santa cruz Biotechnology Inc, CA, USA) 28.

(37) 3. Įvȕ3ǵĮvȕ5ǵĮ5ȕ1 integrin (Millipore). E. ջਔᆫӝ䁙ೱᙹϸᔈ (Real-time PCR) 1. TRLzol (Invitrogen, CA, USA) 2. DEPC (Invitrogen, CA, USA) 3. RNA ᙯ cDNA ԰಍ (Invitrogen, CA, USA) 4. IsopropanolǵChloroform ǵOligo dt (J.T Baker, NJ, USA). F. ಒझᙯࢉ‫( ݤ‬Cell transfection) 1. Lipofectamine 2000 (Invitrogen, CA, USA ) 2. Luciferase substrate (Promega, WI, USA). G. ႝ‫ݚ‬౽୏‫ׯ‬ᡂϩ‫( ݤ݋‬EMSA) 1. BCATM assay kit (Pierce, IL, USA) 2. EMSA kit (Thermo, MA, USA). H. ‫ځ‬Ѭᛰࠔ EDTAǵȕ-mecaptoethanolǵNaClǵDeoxycholateǵHClǵSodium bicarbonateǵbromophenol blue (Amresco Inc, Ohio, USA). ȐΟȑġ 1.. ჴᡍሺᏔᆶ઻‫׷‬ ༾ໆ֎ᆅ (Pippetman) 29.

(38) 2.. ё‫ـ‬Ӏ/๋ѦӀϩӀӀࡋी (UV/Visible spectrophotometer DU-800, Bechman Coulter, USA). 3.. ๋ѦӀᐩጃ (Gel analysis system, EverGene Biotechnology, UK). 4.. PCR у዗ᐒ (RoboCycler Temperature Cycler; Stratagene, La Jolla, CA). 5.. ջਔᆫӝ䁙ೱᙹϸᔈᏔ (Applied Biosystems 7300; Applied Biosystems prism 7900, Applied Biosystems Inc, CA, USA). 6.. ӭфૈϩӀӀࡋी (Muti-spectrophotometer). 7.. ಒझ୻Ꭶጃ (Incubator). ಃΒ࿯ġ ჴᡍБ‫ݤ‬. Ȑ΋ȑġ. ಒझ୻Ꭶ (Cell culture). ୻ᎦΓᜪ೬ମዦಒझ‫୻ޑ‬Ꭶనࣁ DMEMǺĮ-MEM =1Ǻ1Ǵଛ ࿼ ࡕ း Ε ྐ ๵ Ъ ੗ ଳ ၸ ‫ ࣒ ޑ‬ዟ ౟ Ƕ ଛ ࿼ ਔ ஒ ΋ х ‫ ޑ‬DMEM powder уΕ NaHCO3 3.7 լǴаϷ΋х‫ ޑ‬Į-MEM powder уΕ 2.2 լ‫ ޑ‬NaHCO3 ࡕϩձྋ‫ྐܭ‬๵‫ޑ‬ΒԛНύǴஒ pH ॶፓԿ pH 7.2Ǵ ံΒԛНԿᕴᡏᑈ 1 literǶӆа 0.22 ȝm ‫ޑ‬ᘠጢၸᘠϩးǴߥӸ‫ܭ‬ 4 ɗύǶ 30.

(39) A. ୻Ꭶనబу‫ނ‬Ǻ 1. Fetal bovine serum ஒհএ‫ޑ‬Ոమ߻΋ϺӃᓉ࿼ 4ɗӇጃǴ٬‫ځ‬ᄌᄌྋှࡕǴ႖ Ϻ‫ܫ‬ΕН੎ኲύа 55ɗύѐံᡏ 30 ϩដࡕǴӧค๵ᏹբᘚύϩ းԿ 50ml ค๵ᚆЈᆅύǴӆҔ parafilm хᙟ౟αǴߥӸ‫ܭ‬-20ɗǶ 2. Gentamycin ‫ ؂‬400 ml ‫୻ޑ‬ᎦనуΕ 450 ȝl ‫ ޑ‬gentamycinǶ(Final conc. 50 ȝg/ml) 3. Hycobian ‫ ؂‬400 ml ‫୻ޑ‬ᎦనуΕ 45 ȝl ‫ ޑ‬hycobianǶ 4. Insulin ‫ ؂‬400 ml ‫୻ޑ‬ᎦనуΕ 11 ȝl ‫ ޑ‬insulinǶ 5. PBS (Phosphate balanced solution) 1X PBS а 8 g NaClǵ1.2 g NaH2PO4ǷH2O уΒԛНଛᇙǴ ‫ ځ‬pH ፓԋ 7.4 ࡕံΒԛН‫ ډ‬1 ϲǴྐ๵ࡕߥӸ‫ ܭ‬4ɗǶ. B. ಒझ୻Ꭶ‫؁‬ᡯ 1. ಒझϩዬ (Subculture) ஒΓᜪ೬ମԺዦಒझ୻Ꭶ‫ ܭ‬culture medium (10% FBS)Ǵ‫ܫ‬࿼ ‫ܭ‬ಒझ୻Ꭶጃ (37ɗǴ5%. CO2)Ǵ฻ಒझԋߏऊΖԿΐϩᅈਔǴ. ຾Չϩዬ୻ᎦǶϩዬ߻཮ӧཥ‫୻ޑ‬Ꭶҝ΢኱ܴಒझਲ਼‫ޑ‬ӜᆀаϷ 31.

(40) ϩዬਔ໔Ǵϩዬਔஒᙑ‫୻ޑ‬Ꭶన֎‫و‬Ǵ‫؂‬ԛа 37ɗӣྕࡕ‫ྐޑ‬๵ 1X PBS 3~5 ml ؑࢱಒझǴӅమࢱΒԛǶӆуΕ 8~10 ml 37ɗӣྕ ࡕ‫୻ޑ‬ᎦనǴஒಒझӄ೽ؑΠٰǴ٠֎‫ܫ‬ኧԛ‫֡ډޔ‬Ϭؑණಒझ а 1Ǻ3 ‫ ܈‬1Ǻ4 ‫ޑ‬К‫ٯ‬ϩዬ୻ᎦǶ. 2. հএಒझǺ ऊΖϩᅈ‫ޑ‬Γᜪ೬ମԺዦಒझǴհএಒझ߻΋Ϻ‫׳‬ඤԋཥᗲ ‫୻ޑ‬ᎦనǶ२Ӄஒᙑ‫୻ޑ‬Ꭶన֎‫و‬Ǵ‫؂‬ԛа 3~5ml PBS ؑࢱಒझǴ ӅమࢱΟԛǶуΕ 1 ml tripsin ‫ܫ‬Ε incubator ϸᔈࡕуΕ 5ml ‫୻ޑ‬ ᎦనǴஒಒझӄ೽ؑΠٰǴӆஒಒझన֎‫ڗ‬࿼‫ ܭ‬15 ml ᚆЈᆅǴ ᚆЈ 1500 rpmǴ5 ϩដϐࡕஒಒझ؈ᐘΠٰǶѐନ΢మనǴஒ 1 ml ֖Ԗ 10%‫ ޑ‬DMSO ‫୻ޑ‬ᎦనؑΠಒझ٠း‫ܭܫ‬հএλᆅύǴ࿼ ‫ܭ‬-80ɗӇጃߥӸǶ. 3. ှএಒझǺ җ-80ɗӇጃύ‫ڗ‬рΓᜪ೬ମԺዦಒझǴِೲ‫ ډܫ‬37ɗ‫ޑ‬Н ੎ኲύ‫ז‬ೲӣྕࡕǴӧค๵ᏹբѠஒಒझྋ‫ܭ‬፾ໆ‫ޑ‬ಒझ୻Ꭶన ࡕǴ႖Ϻࡑಒझຠߕࡕӆඤ୻ᎦనǴջё຾ՉჴᡍǶ. ȐΒȑġ. Cell lysates ԏ໣. 32.

(41) A. ၂ᏊǺ Ьाྋన lysis buffer (RIPA buffer) Ǵёஒಒझ຋ှǶ٬Ҕ߻Ӄஒ Ѭଛ࿼ԋ modified RIPA bufferǴऩाଛᇙԋ 1 mlǴሡा 1ml RIPAǵ 50mM PMSF 20 ȝlǵ1000x protease inhibitor 1 ȝlǵ50mM Na3VO4 20 ȝl аϷ 1mM NaF 10 ȝlǴ೭٤ࢂೈқ፦Нှ䁙‫ڋ׭‬ᏊǴ‫ٯ‬ӵ sodium orthovannadate ࢂ phosphatase inhibitorǴ aprotinin аϷ PMSF ࢂ serine protease inhibitorǶ. B. ‫؁‬ᡯ : 1. ஒ medium ֎‫و‬ǴҔ 1X PBS మࢱಒझ‫ٿ‬ԛࡕǴӆஒ PBS ֎ଳǶ 2. уΕ 120 ȝl lysis buffer (6 well)ǴճҔ‫ڊ‬ήஒಒझ‫ڊ‬ΠǴ࿼‫ܭ‬Ӈ΢Ƕ 3. а 4ɗǵ13200 rpmǴᚆЈ 15 ϩដǴ‫ڗ‬΢మన‫ډ‬ཥ‫ ޑ‬eppendorfǴ ߥӸ‫ ܭ‬-20ɗǶ. ȐΟȑġ. ೈқ፦‫ۓ‬ໆෳ‫ۓ‬. A. ၂Ꮚ : 1. BSA (2 mg/ml) 2. Protein Reagent Assay Kit. 33.

(42) B. ‫؁‬ᡯ : ଛᇙ 1ǵ0.5ǵ 0.25ǵ0.125ǵ0.0625 Ϸ 0.03125 mg/mlǴ6 ᅿόӕ ᐚࡋ‫ ޑ‬BSA ࡕǴ଺рೈқ፦ᐚࡋ኱ྗԔጕǶ‫ ڗ‬10ȝl ‫ࡑޑ‬ෳኬࠔྋన ‫ ډ‬96 well ύࡕǴуΕ protein reagent assay kit ‫ߕ܌‬ϐ A ྋనᆶ B ྋన а 50Ǻ1 К‫ٯ‬షӝǴ٬നࡕᕴᡏᑈࣁ 200 ȝlǶ‫ܫ‬Ε 37ɗ incubator ύǴ ‫ܫ‬࿼ 30 ϩដǶ௦Ҕ Bradford protein assayǴෳӚᅿόӕໆϐ BSA ӧ ‫ ߏݢ‬595 nm ‫֎ޑ‬ӀॶǴฝр standard curveǶӆෳ sample ‫ ޑ‬O.D. ॶǴ‫؃‬р sample ‫ޑ‬ೈқ፦ᐚࡋǶ. ȐѤȑġ. ՋБᏀᗺ‫( ݤ‬Western blot analysis). A. ၂Ꮚ : 1. Sample buffer (5X) stacking buffer (0.5mM Tris-HClǴPH=6.8) SDS. 7.81 ml 2.5 g. glycerol. 12.36 ml. ȕ-meraptoethanol. 6.25 ml. bromophenol blue. 2 mg. ӆуΒԛНԿ 50 mlǶ. 2. Lysis buffer 34.

(43) RIPA buffer. 1 ml. 50 mM PMSF (8.71 mg in 1 mL DMSO). 20 ȝl. 50 mM Na3VO4 (9.195 mg in 1ml PBS). 20 ȝl. 1mM NaF (42 mg in 1ml PBS). 10 ȝl. 1ȝg/ml aprotinin. 10 ȝl. 1ȝg/ml leupeptin. 10 ȝl. 3. TBS-T (20X) Tris. 48.46 g. EDTA. 0.5 M. NaCl (PH=7.5). 58.44 g. уΒԛНԿ 1 ϲǶ. 4. Running buffer (10X) Tris-base. 30.2 g. Glycine. 140 g. SDS. 10 g. 5. Transfer buffer (10X) Tris. 30 g. Glycin. 144g. уΒԛНԿ 1 ϲǶ. 6. SDS-PAGE Stacking gel (5%) 35.

(44) d.d.H2O. 3 ml. 0.5 M Tris-HCl, pH 6.8. 1.75 ml. Acrylamide/bis (30%). 0.65 ml. 10% SDS. 0.1 ml. 10% APS. 0.1 ml. TEMED. 0.012 ml. Stacking gel (8%) ddH2O. 11.5 ml. 1.5 M Tris-HCl, pH 6.8. 6.3 ml. Acrylamide/bis (30%). 6.7 ml. 10% SDS. 0.025 ml. 10% APS. 0.025 ml. TEMED. 0.015 ml. Stacking gel (10%) d.d.H2O. 7.9 ml. 1.5 M Tris-HCl, pH 6.8. 5 ml. Acrylamide/bis (30%). 6.7 ml. 10% SDS. 0.2 ml. 10% APS. 0.2 ml. TEMED. 0.012 ml. Stacking gel (12%) d.d.H2O. 8.2 ml. 1.5 M Tris-HCl, pH 6.8. 6.3 ml 36.

(45) Acrylamide/bis ( 30%). 10.0 ml. 10% SDS. 0.25 ml. 10% APS. 0.25 ml. TEMED. 0.01 ml. 7. Blotting buffer ( 5%) TBS-T. 50 ml. 5% ಥિФѪ. 25g. B. ᇙጤǺ ٩΢Б‫ޑ‬ଛБᇙ Resolving gel ࡕ֡Ϭషӝ ( TEMED നࡕу)Ǵஒ ԜྋనॹΕς࿶ࢎ೛ӳ‫ޑ‬ጤኲύऊΖϩᅈࡕǴd.d.H2OǴ႖๊‫਻ޜ‬Ǵ ࡑᏉጤ (ऊ 25 ϩដ)Ƕ౽ѐ d.d H2O ࡕකΕකΕ combǴӆஒଛ࿼ӳ‫ޑ‬ stacking gel а pipette ‫ݙ‬ΕᗉխԖ਻‫ූޑݰ‬੮ǴࡑጤᏉջֹԋᇙጤ‫ޑ‬ ‫؁‬ᡯǶ. C. ႝ‫ݚ‬Ǻ ஒς࿶‫ۓ‬ໆӳ‫ޑ‬ೈқ፦уΕ 5x loading dyeǴ‫ܫ‬࿼ 95ɗу዗ 5 ϩដ٬ϐᡂ‫܄‬Ǵ‫ݙ‬Ε‫ډ‬ႝ‫ݚ‬ኲύǴа 100V າႝ‫ݚ‬Ǵֹԋࡕ‫ڗ‬р gelǶ D. ೈқ፦ᙯӑ‫ݤ‬Ǻ ஒ PVDF ጢа methanol ੆‫ݰ‬Ǵ‫ڗ‬рᙯӑ֨Ѻ໒ѳ‫ܫ‬Ǵ‫ܫ‬΢ੇᆟ. 37.

(46) პࡕᎎ΢΋஭ᘠરࡕǴ᠄΢ PVDF ጢࡕӆ‫ܫ‬΢ gel ӆᎎ΋஭ᘠરϷੇ ᆟǴးԖᙯӑ጗ፂన‫ޑ‬ᙯӑኲύǴа 400 mA ᙯӑΒλਔǴջё‫ڗ‬р ᙯӑֹԋ‫ ޑ‬PVDF ጢǴӧ࠻ྕύа 5%‫ޑ‬ಥિФѪ blocking ΋λਔࡕ ջёу΢΋‫לל‬ᡏ‫ྕ࠻ܭ‬Πϸᔈ΋λਔǴа TBS-T ྋనమࢱ 3 ԛ (15 ϩដ) ǴӆуΕΒ‫לל‬ᡏ࠻ྕΠϸᔈ΋λਔǴӆа TBS-T ྋనమࢱ 3 ԛ (15 ϩដ) Ǵ٩ membrane ελǴуΕ፾ໆ‫ ޑ‬ECL kit solution I ᆶ II ฻К‫֡ޑٯ‬Ϭష‫ک‬నǶϸᔈࡕа X-ray film གӀǶ. Ȑϖȑġ. ಒझӸࢲ౗ϩ‫( ݋‬MTT assay). A. ၂Ꮚ : MTT [3-(4,5-Dimethylthizol-2-yl)-2,5-diphenylterazolium bromide ] working solution (1mg/ml) A. চ౛Ǻ ࢲಒझύ‫ ޑ‬mitochondrial dehydrogenase ཮ஒ໳Յ‫ ޑ‬MTT (1 mg/ml)Ǵᙯඤԋόྋ‫๋ޑ܄‬Յ‫ ޑ‬formazanǴԶԝಒझ٠ό཮೷ԋӵԜ ‫ׯ‬ᡂǶࡺёҗԜа MTT assay ෳ‫ۓ‬ಒझӸࢲ౗‫܈‬ቚ෗բҔ‫ޑ‬Б‫ݤ‬Ƕ. B. ‫؁‬ᡯǺ ӧ 96 well ύᅿΠ 1000 ᗭΓᜪ೬ମԺዦಒझࡕࡑΐԋᅈࡕǴуΕ. 38.

(47) ‫ڋ׭‬Ꮚ‫ࢂ܈‬уΕόӕᐚࡋ‫ ޑ‬CCN3 ( 10ǵ30ǵ100 ng/ml ) ࡕǴ37ɗ incubator ୻Ꭶ 48hrs ࡕǴа PBS మࢱΒԛࡕуΕ 50ȝl ‫ ޑ‬MTT solution ‫ܫ‬࿼ 37ɗ incubator ϸᔈ 30 ϩដ (ᗉӀ) Ǵ֎ѐӭᎩ MTT ࡕуΕ DMSO 150 ȝl ᗉӀ 30 ϩដࡕǴа‫ ߏݢ‬570 nm ෳ‫֎ۓ‬ӀॶǶ. ȐϤȑġ. ಒझ౽Չϩ‫( ݋‬Migration assay). ճҔ Transwell (Costar, Corning Life Science, Acton, MA; pore size, 8ȝM) Ǵஒ‫ܫځ‬࿼‫ ܭ‬24 well ύ٬ҔǶӧ‫ ঁ؂‬transwell ύᅿΕаόӕ ‫ڋ׭ޑ‬ᏊхࡴǺLy294002ǵAkt inhibitorǵPDTCǵTPCKǵNF-țB inhibitor peptide Ϸ wortmannin ೀ౛ 30 ϩដ‫߻܈‬΋Ϻ٬Ҕಒझᙯࢉ‫ݤ‬ଌΕ MMP-13 siRNAǵFAK si-RNAǵFAK mutantǵp85 mutantǵAkt mutantǵ IKKD mutant Ϸ IKKȕ mutant ࡕǴஒ 15000 ᗭಒझᆶ 200 ȝl sermu-free ୻Ꭶన‫ ܭܫ‬transwell ‫ޑ‬΢ቫǴԶΠቫࣁ‫ܫ‬࿼ 300 ȝl ֖Ԗ CCN3 ‫ޑ‬ sermu-free ‫୻ޑ‬ᎦనǶϐࡕ‫ܫ‬Ε 37ɗ incubator ୻Ꭶ 16 Կ 18 ঁλਔ ࡕǴ٬Ҕ 1% formaldehyde ‫ ۓڰ‬5 ϩដ٠Ҕ 0.05% crystal violet ࢉՅ 30 ϩដࡕǴ٬Ҕ PBS మࢱӭᎩ‫ࢉޑ‬Ꮚࡕаා޸෇࡬ѐ PBS ٠౽ନ΢ ቫಒझࡕǴаᡉ༾᜔ᢀჸՉԿΠቫຠߕ‫ޑ‬ಒझ٠ीᆉ‫ځ‬ኧໆǴख़ፄჴ ᡍၲΟԛа΢Ƕ. 39.

(48) ȐΎȑġ. ಒझᙯࢉ‫ݤ‬. ٬Ҕ lipofectamine 2000 ᙯࢉ‫ݤ‬Ǵ‫ ע‬1 ȝl/well ‫ ޑ‬DNA ፦ᡏ‫ ܈‬siRNA (short interfering RNA) ‫ ܭܫ‬1.5 ml ‫ޑ‬ᚆЈᆅύǴуΕ 49 ȝl ‫ޑ‬ sermu-free mediumǴќѦྗഢ֖Ԗ 2 ȝl/well ‫ ޑ‬lipo-2000 уΕ 48 ȝl ‫ ޑ‬sermu-free medium ࡕǴϩձᓉ࿼ 5 ϩដǴ‫ ڗ‬50 ȝl lipo-2000 serum-free medium ‫ޑ‬షӝྋనуΕԿ֖Ԗ DNA ፦ᡏ‫ ޑ‬1.5 ml ‫ޑ‬ᚆЈ ᆅύǴ֡Ϭషӝࡕᓉ࿼ 25 ϩដǴஒ well ඤԋᡏᑈࣁ 900 ȝl ‫ ޑ‬sermu-free medium ࡕуΕ 100ȝl ‫ ޑ‬plasmid/lipo-2000 ፦ᡏషӝనǴ٬‫ځ‬ᕴᡏᑈ ࣁ 1 mlǴᇸਗ well ٬ྋన֡ϬϩթǴ‫ܫ‬࿼ 37ɗ incubator ୻Ꭶ 16~24 λਔࡕё٩όӕ‫ޑ‬ჴᡍ‫܌‬ሡԶӆ଺ೀ౛‫ޔ܈‬ௗஒಒझԏଆࡑෳǶ. ȐΖȑġ. ӄໆ RNA ‫ڗܜ‬. ‫ڗ‬рς࿶ֹԋᙯࢉ‫ࢂ܈‬уΕ‫ڋ׭‬ᏊϷ CCN3 ‫ ޑ‬6 wellǴ٬Ҕ PBS మ ࢱ ΋ ԛ ࡕ у Ε 0.5ml ‫ ޑ‬TRIzol reagent (Invitrogen Corpoation, California, USA)Ǵϸᔈ 5 ϩដࡕ‫ע‬ಒझԏ‫ ډ‬1.5 ml ‫ޑ‬ᚆЈᆅύǴуΕ 100 ȝl ‫ ޑ‬chloroform ٠ቃਗ਼འਗ 1 ϩដࡕᓉ࿼ 3 ϩដࡕǴᚆЈ 1100 rpmǴ15 ϩដǴஒ΢మన‫ډ‬ཥ‫ޑ‬ᚆЈᆅύуΕ 500 ȝl isopropyl alcohol ϸᔈ 10 ϩដǴᚆЈ 1100 rpmǴ10 ϩដǴѐନ΢మనࡕуΕ 1 ml 75% ଚᆒǴᚆЈ 7500 rpmǴ5 ϩដǴ॥ଳࡕ٬Ҕ DEPC НӣྋǶ. 40.

(49) Ȑΐȑġ. Complementary DNA, cDNA ϸᙯᒵբҔ. ς࿶‫ۓ‬ໆӳ‫ ޑ‬RNA ٬Ҕ M-MLV-RT kit (Invitrogen, Epicentre biotechnologies, Wisconsin, USA) ஒ total RNA ᙯԋ complementary DNAǶ ‫؁‬ᡯǺ 1. DEPC Н. 9 ȝl. oligo dT. 1 ȝl. dNTP. 1 ȝl. RNA. 1 ȝg. ᕴᡏᑈ 12 ȝLǶషӝࡕ‫ ܭܫ‬PCR у዗Ꮤ 65ɗǴ5 ϩដǵ4ɗǴ1 ϩដǴௗ๱࿼‫ܭ‬Ӈ΢Ƕ 2. 5X First-strand Buffer. 4 ȝl. 0.1M DTT. 2 ȝl. షӝࡕ࿼‫ ܭ‬PCR у዗Ꮤ 37ɗǴ2 ϩដǴௗ๱࿼‫ܭ‬Ӈ΢Ƕ 3. 1 ȝl. M-MLV. షӝࡕ‫ ܭܫ‬PCR у዗Ꮤ 37ɗǴ50 ϩដǵ70ɗǴ15 ϩដࡕջள‫ډ‬ cDNAǶёᓯӸ‫ܭ‬-20ɗаഢҔǶ. 41.

(50) ȐΜȑġ. RT-PCR. (Real-time Reverse Transcriptase-Polymerase Chain Reaction) A. চ౛ : ճҔ຾Չ PCR ӕ‫؁‬ୀෳ PCR ౢ‫ނ‬ᒿ๱ϸᔈቚу‫( ݩރޑ‬ջࣁ “Real-time”) ճҔ‫܌‬ୀෳ‫ޑډ‬ౢ‫ނ‬ӣ௢চኬࠔύ‫୷ޑ‬Ӣ߄౜ໆǶԶ Real-time PCR ё ϩ ࣁ ‫ ٿ‬ε ᜪ Ǻ TaqMan Ϸ SYBR¯GreenERTM (Invitrogen, Carlsbad, CA) Ƕ Զ ҁ ࣴ ‫ ࢂ ز‬٬ Ҕ ࣬ ჹ ‫ ۓ‬ໆ SYBR¯GreenERTM ‫ޑ‬চ౛ࢂӃ೛ी΋ঁ primerǴԶԜ primer ࢂ೛ी ӧ‫ॺך‬ा‫ޑ‬΋ࢤ DNA ‫ׇ‬ӈύǴёаҔٰᆶൂި DNA ᗹӝǴᙖҗ SYBR¯GreenERTM qPCR SuperMix ӧ PCR ၸำύǴᆶΠӈ DNA ‫ׇ‬ ӈᗹӝ‫ ޑ‬primer ( MDBio Inc)Ƕ B. primerǺ MMP-1Ǻ (sense) 5’-CGACTCTAGAAACACAAGAGCAAGA-3’ (antisense) 5’-AAGGTTAGCTTACTGTCACACGCTT-3’ MMP-2Ǻ (sense) 5’-GTGCTGAAGGACACACTAAAGAAGA-3’ (antisense) 5’-TTGCCATCCTTCTCAAAGTTGTAGC-3’ MMP3Ǻ (sense) 5’- GTTAGGAGAAAGGACAGTGGTCCTG-3’ (antisense) 5’- GGCATAGGCATGGGCCAAAACATT-3’ MMP-9Ǻ 42.

(51) (sense) 5’-CACTGTCCACCCCTCAGAGC-3’ (antisense) 5’-GCCACTTGTCGGCGATAAGC-3’; MMP-13Ǻ (sense)5’-TGCTCGCATTCTCCTTCAGGA-3’ (antisense) 5’-ATGCATCCAGGGGTCCTGGC-3’ Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)Ǻ (sense)5’-ACCACAGTCCATGCCATCAC-3’ (antisense) 5’-TCCACCACCCTGTTGCTGTA-3’. C. ‫؁‬ᡯǺ ଛᇙ mixture solutionǺ 0.7-1 ȝl. cDNA (10-100ng) SYBR ¯ GreenER T M SuperMix. 5 ȝl. primer R (10ȝM). 0.5 ȝl. primer F (10ȝM). 0.5 ȝl. షӝࡕஒኬҁ࿼‫ ܭ‬Applied Biosystems Inc prism 7900 (ABI prism 7900, CA, USA) ຾Չ࣬ჹ‫ۓ‬ໆϩ‫݋‬Ǵᒿ๱ PCR ౢ‫ޑނ‬ቚуǴߞဦΨ ཮ᒿ๱ቚமǴӢԜёа଺࣬ჹ‫ۓ‬ໆ‫ޑ‬ϩ‫݋‬Ƕ. ȐΜ΋ȑġ Luciferase ࢲ‫܄‬ෳ‫ۓ‬ ‫ڗ‬рς࿶ֹԋᙯࢉ‫ࢂ܈‬уΕ‫ڋ׭‬ᏊϷ CCN3 ‫ ޑ‬12 well ǴуΕ PBS మࢱΒԛࡕǴуΕ report lysis buffer ‫ڊ‬ΠಒझࡕӧӇ΢ᓉ࿼ 10 ϩដǴа 13200 rmp ᚆЈ 3 ϩដࡕǴ‫ڗ‬р΢మన 20 ȝl ࿼‫ ܭ‬96 well ‫ޑ‬. 43.

(52) қዬύ٠уΕ 80 ȝl ‫ ޑ‬luciferase substrate షӝࡕǴаհӀሺෳ‫ۓ‬ luciferase ࢲ‫܄‬Ǵ‫܌‬ள‫ޑ‬ኧॶаԭϩ౗ඤᆉࡕ߄ҢǶ. ȐΜΒȑġ Gelatin zymography A. ၂ᏊǺ 1. Non-reduced sample bufferǺ Stacking buffer (PH=6.8 , 0.5M Tris-HCl) SDS. 7.8 ml 2.5 g. Glycerol. 14.36 ml. уНԿ 50 mlǴу bromophenol bule (Ԗᡂᙔջё)Ƕ 2. Tris-HCl (500mM) Tris. 30 g. уНԿ 500 mlǴPH=7.5Ƕ 3. Coomassie blueǺ 0.125% Coomassie blue. 0.125 g. 40% MeOH. 40 g. 10% Acetic acid. 10 ml. уНԿ 100 mlǶ 4. Coomassie blue destain bufferǺ 40% d.d.H2O. 200 ml. 50% MeOH. 250 ml. 10% Acetic acid. 50 ml. 44.

(53) 5. Gelatin gelǺ d.d.H2O. 6.03 ml. 1.5M Tris-HCl (PH=8). 2.5 ml. 10% SDS. 0.1 ml. 30% Polyacrylamide. 2.83ml. Gelatin (20mg/ml). 0.5 ml. AP. 0.15 ml. TEMED. 0.009 ml. 6. Develop buffer compositionǺ Tris-HCl (50mMǴPH=7.5). 50 ml 5 ȝl. 10mM ZnCl2 1M CaCl2. 0.25 ml. Triton-100X. 0.5 ml. 10% NaN3. 0.1 ml. B. ‫؁‬ᡯǺ ஒଛᇙӳ‫ޑ‬ጤ‫ݙ‬Εࢎӳ‫ޑ‬ጤѠύǴsample у non-reduced sample buffer ࡕ‫ݙ‬Ε well ύа 100V າႝ‫ݚ‬Ǵֹԋࡕ‫ע‬ጤ‫ڗ‬рӃа d.d.H2O మࢱ‫ٿ‬ԛѐନӭᎩ‫ ޑ‬SDS ࡕǴӆ࿼‫[ ܭ‬Tris-HCl (50mMǴPH=7.5) + 2.5% Triton-100] ࠻ྕΠϸᔈ 30 ϩដǴӆ‫ܫ‬Ε Tris-HCl (50mMǴ PH=7.5)ύమࢱ‫ٿ‬ԛǴ΋ԛ 15 ϩដǴҞӦӧ‫ࡠܭ‬ൺ MMPs ‫܄ࢲޑ‬Ƕ ஒጤ‫ܫ‬Ε develop buffer composition buffer ύ‫ ܭ‬370CǴ50 rpm ‫ޑ‬ ୻ᎦጃϣǴऊ 15 λਔࡕǶጤҔ coomassie blue ࢉՅऊ΋λਔࡕӆ٬Ҕ 45.

(54) coomassie blue destain buffer ଏ‫ډ‬ёа࣮‫ ډޑ‬band ջё࠾ጤǶ. ȐΜΟȑġ ಍ीϩ‫݋‬ ೀ౛ಔᆶ௓‫ڋ‬ಔ‫ޑ‬ኧᏵ໔Ǵ߯௦Ҕ Student’s t-testǴp<0.05Ǵղ‫ۓ‬ ࢂցԖ಍ी΢ৡ౦ǴኧᏵ่݀а Mean±S.M. ߄ҢӚ໨ኧᏵǶ. 46.

(55) ಃѤക ่݀ ΋ǵġ. CCN3 ཮ߦ຾Γᜪ೬ମԺዦಒझ‫ޑ‬౽୏‫܄‬ ΋૓ᕎੱಒझ཮ӃԖ౽Չ‫ૈޑ‬ΚϐࡕǴω཮຾Չᙯ౽բҔ. (Metastasis)Ǵҗ‫ ܭ‬CCN3 ‫ڀ‬Ԗፓ࿯ಒझମࢎ‫ޑ‬ख़ಔᆶߦ຾‫ځ‬дᕎ ಒझ‫ޑ‬౽୏ૈΚǴӢԜ௢՗ CCN3 ёૈ཮‫ׯ‬ᡂಒझ‫ޑ‬౽Չ‫܄‬Ǵჴ ᡍ‫ޑ‬ҞӦࣁೀ౛όӕ‫ޑ‬ᐚࡋ‫ ޑ‬CCN3 (0Ǵ10Ǵ30Ǵ100 ng/ml) ࡕǴ ճҔ transwell migration assay ӧ‫ۓڰ‬ਔ໔ (16 λਔ) ᢀჸ CCN3 ჹ‫ܭ‬Γᜪ೬ମԺዦಒझԖค౽୏‫܄‬Ǵ่݀ว౜ӧೀ౛ 30 ng/ml ‫ޑ‬ ᐚࡋਔಒझ‫ޑ‬౽୏௃‫׎‬നࣁᡉ๱ (Fig. 6A)ǶԶԖЎ᝘ࡰр CCN3 ཮‫ڋ׭‬ᕎಒझғߏ (Poliferation)ǴӢԜӧҁჴᡍύ٬Ҕ MTT assay ѐୀෳ CCN3 ჹ‫ܭ‬Γᜪ೬ମԺዦಒझ‫ޑ‬ቚғำࡋаϷዴᇡ ಒझ‫ޑ‬౽୏‫܄‬ǴԶҗჴᡍ่݀ว౜όӕ‫ ޑ‬CCN3 ᐚࡋჹ‫ܭ‬Γᜪ೬ ମԺዦಒझ‫ޑ‬Ӹࢲ౗٠ؒԖ಍ी΢‫ޑ‬ཀကǴ߄ҢΑ CCN3 ཮ߦ຾ Γᜪ೬ମԺዦಒझ‫ޑ‬౽୏‫܄‬ǴՠόቹៜಒझӸࢲ౗(Fig. 6B)Ƕ. Βǵġ. CCN3 ཮೷ԋΓᜪ೬ମԺዦಒझϐ MMP-13 ‫ޑ‬εໆ߄౜ ಒझ‫ޑ‬ᙯ౽‫ک܄‬ಒझ‫܌‬ϩ‫ ޑݜ‬MMPs ӸԖࡐε‫ޑ‬ᜢᖄ‫܄‬Ǵ‫܌‬. а ӧ ჴ ᡍ ύ ٬ Ҕ Α RT-PCR ǵ Western blotting а Ϸ gelatin-zymography ϩ‫݋‬ಒझ‫܌‬ϩ‫ݜ‬ϐ MMP ‫߄ޑ‬౜ໆаϷࢲ‫܄‬Ǵ 47.

(56) ᢀჸӧೀ౛ CCN3 ࡕࢂց཮ቹៜಒझϩ‫ ݜ‬MMP ϷᅿᜪǶԖЎ᝘ ࡰрӧΓᜪ‫ޑ‬ᕎੱಒझύ‫ ځ‬MMP -1ǵ-2ǵ-3ǵ-9 ‫ک‬-13 ‫߄ޑ‬౜ᆶ ဍዦൾ‫܄‬ำࡋϷᙯ౽բҔԖᜢ (Egeblad and Werb, 2002; Tan et al., 2009)Ƕ‫܌‬аჴᡍӃа RT-PCR ѐᑔᒧ‫ ځ‬MMP -1ǵ-2ǵ-3ǵ-9 ‫ک‬-13 ‫߄ޑ‬౜Ǵว౜྽а CCN3 ೀ౛Γᜪ೬ମᕎ 24 λਔϐࡕǴа MMP-13 mRNA ‫߄ޑ‬౜ໆനࣁᡉ๱ (Fig. 7A)Ƕௗ๱ೀ౛ CCN3 όӕਔ໔ࡕ (0ǵ6ǵ12ǵ24 λਔ) ࡕǴ٬Ҕ Western blotting Ϸ gelatin-zymography ‫ޑ‬Б‫ݤ‬ᢀჸ MMP-13 ೈқ፦߄౜ᆶࢲ‫܄‬Ǵ่݀ࡰр MMP-13 ೿Ԗ time-dependent ‫ޑ‬౜ຝ (Fig. 7B)ǶନԜϐѦǴ‫׳ॺך‬຾΋‫ޑ؁‬٬ Ҕ MMP-13 si-RNA ٰຓܴ MMP-13 ࢂЬाቹៜΓᜪ೬ମԺዦಒ झ‫ޑ‬౽୏‫( ܄‬Fig. 7C)Ǵௗ๱٬ҔՋБᏀᗺ‫ݤ‬ຓܴǴᙯࢉ MMP-13 si-RNA Ԗԋф (Fig. 7D)Ƕҗ่݀ࡰр CCN3 ёа೷ԋΓᜪ೬ମ Ժዦಒझౢғ MMP-13 ຾Զ೷ԋಒझ‫ޑ‬౽୏‫܄‬Ƕ. Οǵġ. CCN3 ೸ၸ Įvȕ3 Ϸ Įvȕ5 integrin receptor ೷ԋΓᜪ೬ମԺዦ. ಒझ౽Չ Ў᝘ࡰр CCN3 ཮೸ၸ Į5ȕ1ǵĮvȕ3 Ϸ Įvȕ5 integrin receptor ፓ௓ಒझ‫ޑ‬౽୏‫܄‬ᆶߕ๱ΚǶ‫ॺךࢂܭ‬ଷ೛ Į5ȕ1ǵĮvȕ3 Ϸ Įvȕ5 integrin receptor ୖᆶӧ CCN3 ቹៜΓᜪ೬ମԺዦಒझ‫ޑ‬౽୏ၸ. 48.

(57) ำύǶ२Ӄ٬Ҕ Į5ȕ1ǵĮvȕ3 Ϸ Įvȕ5 ‫לޑ‬ᡏ 30 ϩដϐࡕǴӆу Ε CCN3Ǵ24 λਔࡕԏ໣ಒझճҔ Western blotting ᢀჸ MMP-13 ‫ޑ‬ೈқ፦߄౜Ǵว౜ಒझऩӃೀ౛ Įvȕ3 Ϸ Įvȕ5 ‫לޑ‬ᡏࡕ཮‫ڋ׭‬ CCN3 ౢғ MMP-13 (Fig 8A)Ƕќ΋Бय़٬Ҕ migration assay Ϸ RT-PCR ϩ‫݋‬ǴӕኬӃӧಒझύೀ౛ Į5ȕ1ǵĮvȕ3 Ϸ Įvȕ5 ‫לޑ‬ᡏ 30 ϩដϐࡕǴӆуΕ CCN3Ǵӧ migration assay ‫่݀ޑ‬ύว౜Ӄ ೀ౛ Įvȕ3 Ϸ Įvȕ5 ‫ל‬ᡏ‫ޑ‬ಒझ౽ՉૈΚ཮ܴᡉΠफ़ (Fig. 8B)Ǵ ќѦ RT-PCR ‫่݀ޑ‬ΨᡉҢ྽ಒझೀ౛ Įvȕ3 Ϸ Įvȕ5 ‫ל‬ᡏࡕǴ೷ ԋ MMP-13 ‫߄ޑ‬౜ໆܴᡉΠफ़ (Fig. 8C)ǴќѦҗ‫ ܭ‬integrin Ьा ᙖҗᒣ᛽ ligand ΢‫ ޑ‬RGD (Arg-Gly-Asp) ‫ׇ‬ӈԶ଺่ӝǴᚐѦ ٬Ҕ RGD peptide уаዴᇡǴҗ‫ܭ‬Ѭёаߔᘐ integrin ᆶ ligand ‫่ޑ‬ӝǶԶ RGD ёаߔᘐ CCN3 ‫܌‬ቚу MMP-13 ߄౜Ǵՠ RAD ؒԖԜբҔ (Fig. 8C)Ƕᆕӝа΢่݀ள‫ ޕ‬CCN3 ߦ຾Γᜪ೬ମ Ժዦಒझ౽Չᆶϩ‫ ݜ‬MMP-13 ࢂ೸ၸ Įvȕ3 Ϸ Įvȕ5 integrin receptorǶ Ѥǵġ. FAK ୖᆶӧΓᜪ೬ମԺዦಒझ౽Չύ Ў᝘ࡰр Focal adhesion kinase (FAK) ࢂ΋ᅿӸ‫ܭ‬ಒझ፦ύ. ‫ ޑ‬non-receptor protein tyrosine kiaseǴϩηໆεऊࣁ 125kDaǴ྽ ಒझ࿶җ integrin ߕ๱‫ܭ‬ಒझѦ୷፦ೈқ‫ޑ‬ਔࡕǴFAK ཮ࡐ‫ޑז‬. 49.

(58) ೏ᐟࢲǴ຾Զᆶ‫ځ‬дૻ৲ϩηբҔǴӢԜ FAK ೏ᇡࣁӧ integrin ‫܌‬ፓ࿯‫ૻޑ‬৲໺ሀၸำύǴ‫ת‬ᄽख़ा‫فޑ‬ՅǶ٬Ҕ Western blotting ᢀჸ๏ϒ CCN3 ‫ڈ‬ᐟ࿶ၸόӕ‫ޑ‬ਔ໔ (0ǵ10ǵ15ǵ30ǵ60ǵ120 ϩដ) ‫ ځ‬FAK (tyr397) ‫܄ࢲޑ‬Ǵว౜ FAK ‫ޑ‬ᕗለϯࢂอኩ‫ޑ܄‬Ǵ εऊӧ 10 ϩដ‫ ډ‬30 ϩដϐ໔ၲ‫ډ‬ଯঢ়ࡕǴӧ΋λਔߡ‫ז‬ೲΠफ़ (Fig. 9A)ǶԜѦᙯࢉΓᜪ೬ମԺዦಒझ FAK si-RNA ‫ ک‬FAK mutant 24 λਔࡕуΕ CCN3 ‫ڈ‬ᐟǴճҔ migration assay аϷ RT-PCR ‫ޑ‬ Б‫ݤ‬ϩ‫( ݋‬Fig. 9Bǵ9C)Ǵ೿ёᢀჸ‫ډ‬Γᜪ೬ମԺዦಒझ‫ޑ‬౽Չૈ Κᆶ MMP-13 ‫ޑ‬ౢғ೿Ԗܴᡉ‫ڋ׭ޑ‬բҔǶவ่݀ύள‫ ޕ‬FAK ‫ࢲޑ‬ϯୖᆶӧ CCN3 ‫ޑ‬բҔύǶ. ϖǵġ. PI3K/Akt ୖᆶӧΓᜪ೬ମԺዦಒझ౽Չύ җ integrin ‫܌‬ፓ࿯‫ૻޑ‬৲໺ሀၡ৩Ԗ PI3K/Akt ‫ୖ܌‬ᆶǴ‫ࢂ܈‬. ‫ځ‬дၡ৩ӵǺERKǵJNK Ϸ MEK/MAPKǶ‫܌‬а΋໒‫ॺךۈ‬Ӄ٬ Ҕ Western blotting ᢀჸӧ๏ϒಒझ CCN3 ࡕ‫ځ‬ၡ৩ೈқ‫ޑ‬ᕗለϯ ߄౜Ƕ྽๏ϒΓᜪ೬ମᕎಒझ CCN3Ǵ10 ϩដϐࡕǴPI3K ‫ޑ‬ᕗለ ϯԖᡉ๱‫ޑ‬ቚу (Fig. 10A)ǴӢԜӃ๱ख़ӧ PI3K/Akt ೭చၡ৩Ƕ ྽๏ϒ೬ମԺዦಒझ PI3K ‫ڋ׭‬Ꮚ (Ly294002 Ϸ wortmannin) 30 ϩដǴ‫ࢂ܈‬ᙯࢉ PI3K mutant (p85) 24 λਔࡕǴ຾Չ migration assay. 50.

(59) Ϸ RT-PCRǴёᢀჸ‫ډ‬Γᜪ೬ମԺዦಒझ‫ޑ‬౽ՉૈΚᆶ MMP-13 ‫ޑ‬ౢғ೿Ԗܴᡉ‫ڋ׭ޑ‬բҔ (Fig. 10Bǵ10Cǵ10Dǵ10E)ǶӢԜ PI3K ࢲϯୖᆶӧ CCN3 ϐբҔύǶௗΠٰ૸௖ Akt ࢂցΨୖᆶӧ ‫ځ‬ύǴ຾΋‫؁‬๏ϒಒझ Akt ‫ڋ׭ޑ‬Ꮚ (Akt inhibitor) 30 ϩដǴ‫܈‬ ࢂᙯࢉ Akt mutant 24 λਔࡕǴ٬Ҕ migration assay Ϸ RT-PCR ᢀ ჸ‫ډ‬Γᜪ೬ମԺዦಒझ‫ޑ‬౽ՉૈΚᆶ MMP-13 ‫ޑ‬ౢғ೿Ԗܴᡉ‫ޑ‬ ‫ڋ׭‬բҔ (Fig. 11Bǵ11Cǵ11Dǵ11E)ǶӢԜ Akt ࢲϯୖᆶӧ CCN3 ϐբҔύǶ. Ϥǵġ. NF-țB ୖᆶӧҗ CCN3 ፓ௓ቚуΓᜪ೬ମԺዦಒझ౽Չύ ϐ߻‫ࡰزࣴޑ‬р྽ CCN family ӧᇨวಒझᙯ౽ਔ཮೸ၸ. NF-țB ᙯᒵӢη (Lin et al., 2004)ǶӧԜࣁΑ௖૸Γᜪ೬ମԺዦ ಒझࢂց཮ჹ CCN3 ‫܌‬ᇨว‫ ޑ‬NF-țB ౢғፓ௓բҔǴ२Ӄ٬Ҕ Western blotting ᢀჸӧ๏ϒಒझ CCN3 ‫ڈ‬ᐟ࿶ၸόӕ‫ޑ‬ਔ໔ (0ǵ10ǵ15ǵ30ǵ60ǵ120 ϩដ) ࡕ‫ ځ‬IKKĮ/ȕǵIțB ೈқ፦‫ޑ‬ᕗ ለϯ߄౜Ǵჴᡍ่݀ࡰр‫ځ‬ᕗለϯ‫߄ޑ‬౜Ԗև౜ time-dependent ‫ޑ‬౜ຝ (Fig. 12C)Ƕௗ๱๏ϒಒझ NF-țB inhibitor (PDTCǵTPCK Ϸ NF-țB inhibitor peptide) 30 ϩដǴ‫ࢂ܈‬٬Ҕᙯࢉ IKKĮ Ϸ IKKȕ mutant 24 λਔࡕǴճҔ migration assay Ϸ RT-PCR ᢀჸǴว౜྽. 51.

(60) ೀ౛ NF-țB ‫ڋ׭‬Ꮚᆶ mutant ਔǴΓᜪ೬ମԺዦಒझ‫ޑ‬౽ՉૈΚ ᆶ MMP-13 ‫ޑ‬ౢғ೿Ԗܴᡉ‫ڋ׭ޑ‬բҔ (Fig. 12Aǵ12Bǵ12Dǵ 12E)Ƕ‫׳‬຾΋‫؁‬ஒΓᜪ೬ମԺዦಒझᙯࢉΕ țB-luciferase ྽բ NF-țB ࢲ‫ࡰޑ܄‬኱ӢηǴҗჴᡍ่݀ᡉҢǴ྽ಒझೀ౛ PI3K inhibitor (Ly294002 Ϸ wortmannin) ǵ Akt inhibitor ‫ ک‬NF-țB inhibitor (PDTCǵTPCK Ϸ NF- țB inhibitor peptide) ೿ૈफ़եҗ CCN3 ‫܌‬ᇨว‫ ޑ‬NF-țB ࢲ‫( ܄‬Fig. 13Aǵ13B)ǶќѦǴஒΓᜪ೬ ମዦಒझ΋ӕᙯࢉΕ p85ǵAktǵIKKDǵIKKȕ mutant Ψૈ‫ڋ׭‬ NF-țB ᙯᒵӢη‫܄ࢲޑ‬Ƕᆕӝа΢‫่݀ޑ‬ள‫ ޕ‬NF-țB ୖᆶӧ CCN3 ፓ௓Γᜪ೬ମԺዦಒझ‫ޑ‬ၡ৩ύǶ. 52.

(61) ಃϖക ૸ፕ. ဍዦಒझ཮ᙖҗϩ‫ݜ‬MMPsǴ٬ளဍዦಒझ཮ऀၸಒझѦ୷፦Ǵ ᒿ๱Ոన‫܈‬రЃ‫س‬಍ߟΕ‫ي‬ᡏ‫ځ‬д೽Տ‫ޑ‬ಔᙃ‫܈‬Ꮤ‫( ۔‬Bremnes et al., 2002; Page-McCaw et al., 2007) ࡕǴဍዦಒझ཮εໆቚғ೷ԋ҅தಒ झค‫ݤ‬ள‫ډ‬к‫ޑى‬ᎦϩԶᏤԿ҅தಔᙃᏔ‫ޑ۔‬фૈ෧১Ǵ೭Ψࢂ೷ԋ ᕎੱੰΓԝΫ‫ޑ‬ख़ाӢનϐ΋Ƕ೬ମԺዦ‫ڀ‬Ԗᙯ౽‫ޑ‬ወӧૈΚǴу΢ ೬ମԺዦჹ‫ܭ‬ϯᏢ‫ܫ܈‬ᕍ‫ݯޑ‬ᕍਏ݀٠όӳǴаϷલЮԖਏ‫ޑ‬ᇶշᕍ ‫ݤ‬Ǵ٬ளεӭኧ‫ޑ‬೬ମԺዦੰΓႣࡕૈΚৡǵ‫཮׳‬ԖൺวϷᙯ౽‫ޑ‬ё ૈ‫܄‬ǶӢԜऩૈவύᕕှ೬ମԺዦᙯ౽ၸำ‫ޑ‬ϩηᐒ‫ڋ‬Ǵ‫܈‬೚ёаග ‫ٮ‬҂ٰᜢ‫ݯܭ‬ᕍ೬ମԺዦᙯ౽ϐԖਏБ‫( ݤ‬Fong et al., 2007)ǶЎ᝘ࡰ рӧΓᜪ‫ޑ‬ᕎੱಒझύ‫ځ‬MMP-1ǵ-2ǵ-3ǵ-9‫ک‬-13‫߄ޑ‬౜ᆶဍዦൾ‫܄‬ ำࡋϷ࣬ᜢᙯ౽բҔԖᜢǶӢԜҁჴᡍ‫ޑ‬ҞӦӧ‫ܭ‬௖૸Γᜪ೬ମԺዦ ಒझᙯ౽‫ޑ‬ᐒ‫ڋ‬ၡ৩Ǵ‫׳‬຾΋‫૸ޑ؁‬ፕࢂٗ΋ᅿ‫ޑ‬MMPୖᆶӧ‫ځ‬ ύǴҗჴᡍ่݀ள‫ޕ‬ǴCCN3཮೷ԋΓᜪ೬ମԺዦಒझౢғεໆ‫ޑ‬ MMP-13ǶMMP-13ࢂᜢ࿯੯ੰύᏤԿ೬ମଏϯ‫ޑ‬ख़ाӢηϐ΋ǴԶ ྽೬ମр౜ୢᚒਔǴёૈ཮೷ԋӭᅿ‫ޑ‬ମᓝ੯ੰǴԶ‫ځ‬ύϣғ‫܄‬೬ମ ዦᗋԖёૈ཮ൾ‫܄‬ᙯϯࣁ೬ମԺዦ (Yu et al., 2003)Ƕ‫܌‬а‫ॺך‬ᇡࣁ MMP-13‫ޑ‬фૈନΑૈ୼ߦ຾ᕎಒझ‫ޑ‬౽୏ѦǴᗋёૈ཮೷ԋ‫ڬ‬ൎ҅. 53.

(62) த‫ޑ‬೬ମಒझᖿ‫ܭ‬ൾϯ‫׎‬ԋᕎಒझǶԶࣴ‫ز‬MMPନΑёаᔅշᕕှ ဍዦวғ‫ޑ‬ၸำѦǴ‫׆‬ఈёаவύѐࣴ‫ز‬໒วр‫ڋ׭‬MMPբҔ‫ޑ‬ᛰ ‫ݯٰނ‬ᕍᕎੱੰΓǴ‫܈‬೚೭཮ࢂफ़եੰΓᕎੱൺวϷᙯ౽‫ޑ‬ёૈ‫܄‬Ƕ ӧӃ߻‫زࣴޑ‬ύว౜CCN3཮߄౜ӧ҅தಔᙃǴхࡴ๝Ǵઓ࿶‫س‬ ಍Ǵ‫ޤ‬ǴԼԺǴ೬ମǴՠӧဍዦಒझύ‫߄ޑ‬౜ᗲϿࣁΓ‫( ޕ‬Manara et al., 2002)Ƕу΢ԖࣽᏢৎȐRudolf Virchowȑ ࡰрᕎಒझନΑԾ‫ڀي‬ ഢ‫ޑ‬೭٤ૈΚѦǴΨ཮‫ຼډڙ‬ᎁಒझϩ‫ޑݜ‬ಒझᐟનǵϯᏢᐟન‫܈‬ғ ߏӢηٰᇨ٬Ѭॺӛᕎಒझᆫ໣ (Coussens and Werb, 2002; DeNardo and Coussens, 2007)Ǵ٠٬Ѭॺ߄౜߄౜рԖճ‫ܭ‬ဍዦғߏǵᙯ౽‫ޑ‬ ‫׎‬ᄊǴ຾Զബ೷р΋ঁ፾ӝဍዦғߏ‫ޑ‬༾ᕉნ (Joyce and Pollard, 2009)Ƕҗ‫ܭ‬CCN fmailyёа྽଺integrin receptor‫ޑ‬ligandǴ่ӝϐࡕ ёаॄೢፓ௓ಒझ໔‫܈‬ಒझᆶಒझѦ୷፦‫ޑ‬բҔǴௗ๱ቹៜಒझମࢎ ‫׎‬ᄊǵፓ௓ಒझғߏǵϩϯǵ౽୏ǵߕ๱Ϸ໾α‫ޑ‬অൺǴӢԜ‫ॺך‬གྷ ‫ޕ‬ၰCCN3ࢂց཮೸ၸintegrin receptorٰ೷ԋΓᜪ೬ମԺዦಒझ‫ޑ‬౽ ୏‫܄‬аϷCCN3ᆶintegrin receptor‫ޑ‬ᜢ߯Ƕ२Ӄ‫ॺך‬Ӄࢂаtranswell ٰዴ‫ۓ‬ӧೀ౛CCN3ࡕΓᜪ೬ମԺዦಒझ‫ޑ‬౽୏‫܄‬Ǵ٠ճҔೀ౛ integrin receptor‫לޑ‬ᡏள‫ࣁ่݀ޑډ‬CCN3೸ၸĮvȕ3ϷĮvȕ5 integrin receptorፓ௓Γᜪ೬ମԺዦಒझ‫ޑ‬౽ՉૈΚǶ ӧ ၸ ѐ ‫ ز ࣴ ޑ‬ύ ࡰ р CCN3 ё ଺ ࣁ ମ ᕎ ੰ Γ Ǵ ‫ ٯ‬ӵ Ǻ Ewing. 54.

(63) sarcoma‫ک‬osteosarcomaύǴႣࡕၨৡ‫ࡰޑ‬኱Ӣη (Perbal et al., 2009; Perbal et al., 2008)ǴନԜϐѦӧChronic Myeloid Leukaemia (CML) Ϸ ໵Յનዦ (Melanoma) ύǴCCN3Ԗ೏down-regulated‫ޑ‬౜ຝǴ‫ځ‬фૈ ӧ‫ܭ‬फ़եဍዦಒझ‫ߟޑ‬ҍૈΚᆶϩ‫ݜ‬MMPsೈқ፦‫ࢂ܈‬ቚу঒Ϋբ ҔǴԖᗺᜪ՟ဍዦ‫ڋ׭‬Ӣη‫فޑ‬Յ(Fukunaga-Kalabis et al., 2008; McCallum et al., 2009)ǴฅԶӧମᕎಒझ‫ٯ‬ӵEwing's sarcomaύ཮ᢀჸ ‫ډ‬CCN3‫ڀ‬Ԗߦ຾ဍዦ‫ޑ‬౽ՉϷߟҍૈΚǴᆕӝа΢‫ޑ‬ፕᗺёа‫ޕ‬ ၰǴCCN3‫ܭ‬όӕ‫ޑ‬ಔᙃύ‫ת‬ᄽ๱όӕ‫فޑ‬Յ (Benini et al., 2005) Ƕ ߈ԃٰ‫ޑ‬Ў᝘ࡰрӧ΋٤ൾ‫ޑ܄‬ဍዦಒझύӵǺ‫ޤ‬ᕎǵ٢ᕎǵ໵ Յનዦǵ‫ط‬ᕎ…฻฻Ǵ೿ёаᢀჸ‫ډ‬NF-țB‫ޑ‬εໆ߄౜ǶќѦӧin vitro ‫ޑ‬ჴᡍύΨຓჴΑӧ೚ӭόӕ‫ޑ‬ಒझࠠᄊύǴಒझ߄य़‫ޑ‬integrin receptorsᆶligand่ӝࢲϯࡕǴ௴୏PI3K/Akt/NF-țB‫ૻޑ‬৲໺ሀǶ‫ٯ‬ ӵǴϣҜಒझӧՈᆅཥғၸำύёа࿶җ integrin Dvȕ3೸ၸNF-țBፓ ௓ ಒ झ ‫ ޑ‬ғ Ӹ ૈ Κ (Scatena and Giachelli, 2002) ‫ ࢂ ܈‬ᙖ җ ࢲ ϯ NF-țBߦ຾ಒझ‫ޑ‬౽୏ᆶߟҍૈΚ (Lin et al., 2004)Ƕௗ๱‫ॺך‬٬Ҕૻ ৲ ໺ ሀ ‫ ڋ ׭‬Ꮚ MMP-13 si-RNA ǵ Ly294002 ǵ wortmannin ǵ Akt inhibitorǵNF-țB inhibitor (PDTCǵTPCKϷNF-țB inhibitor peptide)ǵ p85 mutantǵAkt mutantǵIKKD mutant Ϸ IKKȕ mutantࡕǴ೿཮‫ڋ׭‬ CCN3‫ڈ‬ᐟΓᜪ೬ମԺዦಒझ‫ޑ‬౽ՉૈΚᆶMMP-13‫ޑ‬ౢғǶќ΋Б. 55.

(64) य़٬ҔRGD peptide ϷRAG peptideว౜RGD཮‫ڋ׭‬MMP-13‫ޑ‬ౢ ғǴՠ๏ϒRAG peptideਔؒԖᡉ๱‫ޑ‬ৡ౦Ǵ٠ଛӝ٬ҔհӀሇનࢲ ‫܄‬ෳ‫ۓ‬ᢀჸ‫ڋ׭‬Ꮚᆶmutantჹ‫ܭ‬ಒझϣNF-țB‫܄ࢲޑ‬Ǵว౜྽ೀ౛‫׭‬ ‫ڋ‬Ꮚᆶmutantਔ೿཮फ़եNF-țB‫܄ࢲޑ‬Ƕ೭٤่݀ࡰрCCN3཮࿶ၸ Įvȕ3ϷĮvȕ5 integrin receptorፓ௓Γᜪ೬ମԺዦಒझ‫ޑ‬౽Չᆶϩ‫ݜ‬ MMP-13Ǵࢂ೸ၸPI3K/Akt/NF-țB೭చၡ৩ (Fig. 14)Ƕ. 56.

(65) ಃϤക ่ፕ. ᆕӝа΢‫ޑ‬ჴᡍ่݀Ǵёаள‫ډ‬CCN3࿶җĮvȕ3ϷĮvȕ5 integrin receptor۳Π໺ሀૻ৲ǴࢲϯΠෞPI3K/Akt/NF-țB೭చၡ৩ፓ௓Γᜪ ೬ମԺዦಒझϩ‫ݜ‬MMP-13Ǵ຾Զፓ௓Γᜪ೬ମዦಒझ‫ޑ‬౽ՉૈΚǶ. 57.

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(71) კ߄. 63.

(72) Fig 6. CCN3 induced chondrosarcoma cells.. the. migration. activity. of. human. JJ012 cells were incubated with various concentrations of CCN3, and in vitro migration activities measured with the Transwell after 24 h showed that CCN3 (30 ng/ml) increased cell migration significantly (A). JJ012 cells sere stimulated by indicated concentraction intervals (0 ,10, 30 and 100 ng/ml) and proliferation was determined by MTT assay (B). Results are presented as Mean±S.E. (n=3). * p<0.05 was compared with control.. 64.

(73) 65.

(74) Fig 7. CCN3-directed migration activity of human chondrosarcoma cells involves upregulation of MMP-13. JJ012 cells were incubated with CCN3 (30 ng/ml) for 24h or for indicated time intervals, cell lysates were then collected and the mRNA level of MMP-1, -2, -3, -9 and -13 was determined using qPCR (A). Cells were incubated with CCN3 (30 ng/ml) for indicated time intervals .The cultured medium and cell lysates were then collected. Both the protein level of MMP-13 in cell lysates determined by Western blot analysis and the enzyme activity of MMP-13 in cell lysates and supernatant determined using zymography were increased in a time-dependent manner (B). Cells were transfected with MMP-13 or control siRNA for 24 h,and in vitro migration was measured with the Transwell after 24 h (C) Cells were transfected with MMP-13 or control siRNA for 24 h, and the mRNA and protein levels of MMP-13 were examined using Western blot analysis (D). Results are presented as Mean±S.E. (n=3). * p<0.05 was compared with control. # p<0.05 was compared with CCN3.. 66.

(75) 67.

(76) Fig 8. CCN3 increased human chondrosarcoma cells migration and MMP-13 expression through Įvȕ3 and Įvȕ5 integrin receptor. JJ012 cells were pretreated with Į5ȕ1ǵĮvȕ3 or Įvȕ5 antibody (5 ȝg/ml) after treating with CCN3 (30 ng/ml) for 24h, and MMP-13 expression was determined by Western blot analysis (A). JJ012 cells were pretreated with Įvȕ3 mAb (5 ȝg/ml), Įvȕ5 mAb (5 ȝg/ml) cyclic RGD (10 nM) and cyclic RAD (10 nM) for 30 min followed by stimulation with CCN3 (30 ng/ml). The in vitro migration activity measured after 24 h showed that Įvȕ3 mAbǵĮvȕ5 mAb could inhibit the cell migration (C). The qPCR result show that Įvȕ3 mAb, Įvȕ5 mAb and cyclic RGD but not Į5ȕ1 mAb and cyclic RAD could inhibit the MMP-13 expression (C). Results are presented as Mean±S.E. (n=3). * p<0.05 was compared with control. #. p<0.05 was compared with CCN3.. 68.

(77) 69.

(78) Fig 9. Involvement of FAK-signaling pathway in response to CCN3 in chondrosarcoma cells. (A) JJ012 cells were incubated with CCN3(30 ng/ml) for indicated time intervals, and p-FAK expression was determined by Western blot analysis. Note that CCN3 activated the FAK pathway in JJ012 cells. (B) Cells were transfected with mutant and siRNA of FAK for 24 h followed by stimulation with CCN3 (30 ng/ml), and in vitro migration was measured with the Transwell after 24 h. (C) JJ012 cells were transfected with mutant of FAK for 24 h followed by stimulation with CCN3 (30 ng/ml), and the mRNA level of MMP-13 were determined by using qPCR. Results are presented as mean±S.E. (n=3). * p<0.05 was compared with control. #p <0.05 was compared with CCN3.. 70.

(79) 71.