screening-identification-small-peptides-targeting-fibroblast-growth

Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library

The human Fibroblast Growth Factor Receptor (FGFR) family comprises four members, namely, FGFR1, FGFR2, FGFR3, and FGFR4, that are involved in various ...

Guangdong Provincial Key Laboratory of Bioengineering Medicine

&#x6D3B;&#x5316;&#x7684;&#x8840;&#x5C0F;&#x677F;&#x884D;&#x751F;&#x7684;&#x751F;&#x957F;&#x56E0;&#x5B50; &#xFF08;PDGF&#xFF09; &#x5DF2;&#x88AB;&#x94FE;&#x63A5;&#x5230;&#x6076;&#x6027;&#x764C;&#x75C7;&#x3002;&#x9AD8;&#x6C34;&#x5E73;&#x7684;&#x8840;&#x5C0F;&#x677F;&#x884D;&#x751F;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x5BFC;&#x81F4;&#x6FC0;&#x6D3B; (PDGFRs) &#x53CA;&#x5176;&#x53D7;&#x4F53;&#x4E0E;&#x7EC6;&#x80DE;&#x7684;&#x8FC7;&#x5EA6;&#x7E41;&#x884D;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x5E72;&#x6270;&#x6B64;&#x4FE1;&#x53F7;&#x8F6C;&#x5BFC;&#x901A;&#x8DEF;&#x5728;&#x80BF;&#x7624;&#x7EC6;&#x80DE;&#x4E2D;&#x53EF;&#x80FD;&#x5BF9;&#x6297;&#x764C;&#x836F;&#x7269;&#x7684;&#x53D1;&#x5C55;&#x5177;&#x6709;&#x91CD;&#x8981;&#x610F;&#x4E49;&#x3002;&#x5728;&#x4EE5;&#x5F80;&#x7684;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;&#x9759;&#x6001; CHO-k(1) &#x7EC6;&#x80DE;&#x8868;&#x8FBE;&#x7684; sPDGFR alpha-Fc &#x878D;&#x5408;&#x86CB;&#x767D;&#x8868;&#x660E;&#x5BF9;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7EC6;&#x80DE;&#x7684;&#x6297;&#x589E;&#x6B96;&#x4F5C;&#x7528;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x5F88;&#x96BE;&#x83B7;&#x5F97;&#x6B64;&#x878D;&#x5408;&#x86CB;&#x767D;&#x7684;&#x529F;&#x80FD;&#x7814;&#x7A76;&#x8FDB;&#x4E00;&#x6B65;&#x91CF;&#x5927;&#x3002;&#x5728;&#x672C;&#x7814;&#x7A76;&#x4E2D;&#xFF0C;sPDGFR &alpha;-Fc &#x878D;&#x5408;&#x86CB;&#x767D;&#x6682;&#x6001;&#x8868;&#x8FBE;&#x4E2D;&#x56FD;&#x4ED3;&#x9F20;&#x5375;&#x5DE2; (CHO) DG44 50 &#x6BEB;&#x5347;&#x8F68;&#x9053;&#x632F;&#x52A8;&#x751F;&#x7269;&#x53CD;&#x5E94;&#x5668;&#x4E2D;&#x7684;&#x5355;&#x5143;&#x683C;&#x3002;sPDGFR &alpha;-Fc &#x4F5C;&#x4E3A; 250 kDa &#x4E8C;&#x805A;&#x86CB;&#x767D;&#x4E0E;&#x6F5C;&#x5728;&#x7CD6;&#x57FA;&#x5316;&#x8868;&#x793A;&#x3002;SPDGFR &#x963F;&#x5C14;&#x6CD5;&#x754C;&#x529F;&#x80FD;&#x754C;&#x522B;&#x5728;&#x57F9;&#x517B;&#x4E0A;&#x6E05;&#x6DB2;&#x4E2D;&#x7684;&#x6700;&#x7EC8;&#x6536;&#x76CA;&#x7387;&#x9AD8;&#x8FBE; 16.68 mg/l&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x5728;&#x8F68;&#x9053;&#x632F;&#x52A8;&#x751F;&#x7269;&#x53CD;&#x5E94;&#x5668;&#x4E2D;&#x7684;&#x77AC;&#x65F6;&#x8868;&#x8FBE;&#x53EF;&#x80FD;&#x662F;&#x53EF;&#x884C;&#x7684;&#x7528;&#x4E8E;&#x4E34;&#x5E8A;&#x524D;&#x7814;&#x7A76;&#x7684;&#x91CD;&#x7EC4;&#x86CB;&#x767D;&#x5236;&#x5907;&#x3002;

&#x91CD;&#x7EC4; sPDGFR &alpha;-Fc CHO DG44 &#x7EC6;&#x80DE;&#x4F7F;&#x7528; 50 &#x6BEB;&#x5347;&#x8F68;&#x9053;&#x632F;&#x52A8;&#x4E00;&#x6B21;&#x6027;&#x751F;&#x7269;&#x53CD;&#x5E94;&#x5668;&#x4E2D;&#x7684;&#x77AC;&#x65F6;&#x8868;&#x8FBE;&#x3002;

Overactivity of platelet-derived growth factor (PDGF) has been linked to malignant cancers. High levels of PDGF result in the activation of its receptors (PDGFRs) and the over-proliferation of cells. Therefore, interfering with this signaling pathway in cancer cells could be significant for anti-cancer drug development. In a previous study, the sPDGFR alpha-Fc fusion protein expressed in static CHO-k(1) cells showed an anti-proliferative effect on vascular endothelial cells. However, it was difficult to obtain a large quantity of this fusion protein for further functional studies. In the present study, the sPDGFR alpha-Fc fusion protein was transiently expressed in Chinese Hamster Ovary (CHO) DG44 cells in 50-mL orbital shaking bioreactors. sPDGFR alpha-Fc was expressed as a 250-kDa dimeric protein with potential glycosylation. The final yield of sPDGFR alpha-Fc in the culture supernatant was as high as 16.68 mg/L. Our results suggest that transient expression in orbital shaking bioreactors may be feasible for preparation of recombinant proteins used for preclinical studies.

Transient expression of recombinant sPDGFR alpha-Fc in CHO DG44 cells using 50-mL orbitally shaking disposable bioreactors.

Suractivit&eacute; du facteur de croissance d&eacute;riv&eacute; des plaquettes (PDGF) a &eacute;t&eacute; li&eacute;e aux cancers malins. Des niveaux &eacute;lev&eacute;s de PDGF entra&icirc;ner l&#39;activation de ses r&eacute;cepteurs (PDGFRs) et l&#39;over-proliferation des cellules. Par cons&eacute;quent, interf&eacute;rer avec cette voie de signalisation dans les cellules canc&eacute;reuses pourrait &ecirc;tre importante pour le d&eacute;veloppement de m&eacute;dicaments anticanc&eacute;reux. Dans une &eacute;tude ant&eacute;rieure, la prot&eacute;ine de fusion alpha-Fc sPDGFR exprim&eacute;e dans les cellules CHO-k(1) statiques ont montr&eacute; un effet antiprolif&eacute;rative sur les cellules endoth&eacute;liales vasculaires. Cependant, il &eacute;tait difficile d&#39;obtenir une grande quantit&eacute; de cette prot&eacute;ine de fusion pour poursuivre ses &eacute;tudes fonctionnelles. Dans la pr&eacute;sente &eacute;tude, la prot&eacute;ine de fusion alpha-Fc sPDGFR a &eacute;t&eacute; transitoirement exprim&eacute;e dans des cellules ovariennes de Hamster chinois (CHO) DG44 50 mL orbitale secouant les bior&eacute;acteurs. sPDGFR alpha-Fc a &eacute;t&eacute; exprim&eacute;e en une prot&eacute;ine dim&eacute;rique 250 kDa avec glycosylation potentielle. Le rendement final de sPDGFR alpha-Fc dans le surnageant de culture s&#39;&eacute;l&egrave;ve &agrave; 16,68 mg/L. Nos r&eacute;sultats sugg&egrave;rent que l&#39;expression transitoire dans l&#39;orbitale secouant les bior&eacute;acteurs pourrait &ecirc;tre r&eacute;alisable pour la pr&eacute;paration de prot&eacute;ines recombinantes utilis&eacute;s pour les &eacute;tudes pr&eacute;cliniques.

Expression transitoire de recombinaison sPDGFR alpha-Fc dans les cellules CHO DG44 50 mL orbitalement secouant bior&eacute;acteurs jetables.

&Uuml;beraktivit&auml;t des Platelet-derived Growth Factor (PDGF) ist mit malignen Tumoren verbunden war. Hohe PDGF f&uuml;hren die Aktivierung der Rezeptoren (PDGFRs) und der J&auml;gerei der Zellen. Daher k&ouml;nnte dieses Signalweges in Krebszellen st&ouml;ren f&uuml;r Krebsmedikament Entwicklung bedeutsam sein. In einer fr&uuml;heren Studie zeigte die sPDGFR Alpha-Fc Fusionsprotein ausgedr&uuml;ckt in statischen CHO-k(1) Zellen eine antiproliferative Wirkung auf vaskul&auml;re Endothelzellen. Allerdings war es schwierig, eine gro&szlig;e Menge von dieser Schmelzverfahren Protein f&uuml;r weitere funktionelle Studien zu erhalten. In der vorliegenden Studie wurde die sPDGFR Alpha-Fc-Fusionsprotein vor&uuml;bergehend in Chinese Hamster Ovary (CHO) DG44 Zellen in 50 mL Orbital sch&uuml;tteln Bioreaktoren ausgedr&uuml;ckt. sPDGFR Alpha-Fc wurde als ein dimeren 250-kDa-Protein mit potenziellen Glycosylation ausgedr&uuml;ckt. Der endg&uuml;ltige Ertrag des sPDGFR Alpha-Fc in der Kultur&uuml;berstand war so hoch wie 16.68 mg/L. Unsere Ergebnisse legen nahe, dass Transienten Ausdruck in Orbital sch&uuml;tteln Bioreaktoren f&uuml;r Vorbereitung von rekombinanten Proteinen verwendet f&uuml;r Pr&auml;klinische Studien durchf&uuml;hrbar sein kann.

Transiente Ausdruck rekombinante sPDGFR Alpha-FC im DG44 CHO-Zellen, die mit 50 mL Orbital sch&uuml;tteln Einweg-Bioreaktoren.

Iperattivit&agrave; del fattore di crescita derivato dalle piastrine (PDGF) &egrave; stato collegato a tumori maligni. Alti livelli di PDGF provocare l&#39;attivazione dei suoi recettori (PDGFRs) e la proliferazione delle cellule. Di conseguenza, interferire con questa via di segnalazione in cellule tumorali potrebbe essere significativo per lo sviluppo di farmaci anti-cancro. In un precedente studio, la proteina di fusione alfa-Fc sPDGFR espressa nelle cellule CHO-k(1) statiche ha mostrato un effetto anti-proliferativo sulle cellule endoteliali vascolari. Tuttavia, era difficile ottenere una grande quantit&agrave; di questa proteina di fusione per studi funzionali. Nel presente studio, la proteina di fusione alfa-Fc sPDGFR era transitoriamente espresso nelle cellule di ovaio di criceto cinese (CHO) DG44 in 50 mL orbitale agitazione bioreattori. sPDGFR alfa-Fc &egrave; stata espressa come una proteina dimerica 250-kDa con potenziale glicosilazione. La resa finale di sPDGFR alfa-Fc nella cultura supernatante era alta come 16.68 mg/L. I nostri risultati suggeriscono che espressione transitoria in bioreattori di agitazione orbitale pu&ograve; essere fattibile per la preparazione di proteine ricombinanti utilizzati per studi preclinici.

Espressione transitoria di alfa-Fc sPDGFR ricombinante nelle cellule CHO DG44 utilizzando orbitally agitazione bioreattori monouso 50 mL.

&#x8840;&#x5C0F;&#x677F;&#x7531;&#x6765;&#x6210;&#x9577;&#x56E0;&#x5B50; (PDGF) &#x306E;&#x904E;&#x5270;&#x306A;&#x6D3B;&#x52D5;&#x306F;&#x3001;&#x60AA;&#x6027;&#x306E;&#x764C;&#x306B;&#x30EA;&#x30F3;&#x30AF;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;PDGF &#x306E;&#x9AD8;&#x30EC;&#x30D9;&#x30EB;&#x306F;&#x3001;&#x305D;&#x306E;&#x53D7;&#x5BB9;&#x4F53; (PDGFRs) &#x306E;&#x6D3B;&#x6027;&#x5316;&#x3068;&#x7D30;&#x80DE;&#x306E; over-proliferation &#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x304C;&#x3093;&#x7D30;&#x80DE;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x7D4C;&#x8DEF;&#x306B;&#x5E72;&#x6E09;&#x6297;&#x304C;&#x3093;&#x5264;&#x958B;&#x767A;&#x306E;&#x91CD;&#x8981;&#x306A;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x4EE5;&#x524D;&#x306E;&#x7814;&#x7A76;&#x3067;&#x306F;, &#x9759;&#x7684; CHO-k(1) &#x7D30;&#x80DE;&#x3067;&#x767A;&#x73FE;&#x3057;&#x305F; sPDGFR &#x30A2;&#x30EB;&#x30D5;&#x30A1; Fc &#x878D;&#x5408;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x8840;&#x7BA1;&#x5185;&#x76AE;&#x7D30;&#x80DE;&#x306F;&#x6297;&#x5897;&#x6B96;&#x52B9;&#x679C;&#x3092;&#x793A;&#x3057;&#x305F;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x3053;&#x306E;&#x878D;&#x5408;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x6A5F;&#x80FD;&#x7814;&#x7A76;&#x306E;&#x305F;&#x3081;&#x306E;&#x5927;&#x898F;&#x6A21;&#x306A;&#x91CF;&#x3092;&#x5F97;&#x308B;&#x3053;&#x3068;&#x304C;&#x56F0;&#x96E3;&#x3067;&#x3057;&#x305F;&#x3002;&#x672C;&#x7814;&#x7A76;&#x3067;&#x306F;&#x3001;sPDGFR &#x30A2;&#x30EB;&#x30D5;&#x30A1; Fc &#x878D;&#x5408;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306F;&#x4E00;&#x904E;&#x6027;&#x4E2D;&#x56FD;&#x30CF;&#x30E0;&#x30B9;&#x30BF;&#x30FC;&#x5375;&#x5DE3; (CHO) DG44 &#x7D30;&#x80DE;&#x30D0;&#x30A4;&#x30AA;&#x30EA;&#x30A2;&#x30AF;&#x30BF;&#x30FC;&#x3092;&#x63FA;&#x308C; 50 mL &#x8ECC;&#x9053;&#x3067;&#x8868;&#x73FE;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;sPDGFR &alpha;-Fc &#x306F;&#x3001;250 &#x6F5C;&#x5728;&#x7684;&#x306A;&#x7CD6;&#x9396;&#x306B;&#x3088;&#x308B;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306F;&#x4E8C;&#x91CF;&#x4F53;&#x3068;&#x3057;&#x3066;&#x8868;&#x73FE;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x6700;&#x5F8C;&#x306E; sPDGFR &#x30A2;&#x30EB;&#x30D5;&#x30A1;-Fc &#x306E;&#x57F9;&#x990A;&#x4E0A;&#x6E05; 16.68 mg/L &#x3068;&#x9AD8;&#x53CE;&#x7387;&#x30B8;&#x30E3;&#x30FC;&#x30D5;&#x30A1;&#x30FC;&#x30E1;&#x30F3;&#x30BF;&#x30FC;&#x578B;&#x57F9;&#x990A;&#x69FD;&#x632F;&#x52D5;&#x8ECC;&#x9053;&#x306B;&#x4E00;&#x6642;&#x7684;&#x306A;&#x5F0F;&#x3092;&#x524D;&#x81E8;&#x5E8A;&#x7814;&#x7A76;&#x306E;&#x305F;&#x3081;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x308B;&#x7D44;&#x63DB;&#x3048;&#x86CB;&#x767D;&#x8CEA;&#x306E;&#x6E96;&#x5099;&#x306E;&#x305F;&#x3081;&#x306B;&#x53EF;&#x80FD;&#x306A;&#x3053;&#x3068;&#x304C;&#x793A;&#x5506;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;

&#x4E00;&#x904E;&#x6027;&#x767A;&#x73FE;&#x7D44;&#x63DB;&#x3048; sPDGFR &#x30A2;&#x30EB;&#x30D5;&#x30A1; Fc 50 mL &#x8ECC;&#x9053;&#x632F;&#x52D5;&#x4F7F;&#x3044;&#x6368;&#x3066;&#x30D0;&#x30A4;&#x30AA;&#x30EA;&#x30A2;&#x30AF;&#x30BF;&#x30FC;&#x3092;&#x7528;&#x3044;&#x305F;&#x753A; DG44 &#x7D30;&#x80DE;&#x306E;&#x3002;

&#xD608;&#xC18C;&#xD310; &#xC720;&#xB798; &#xC131;&#xC7A5; &#xC778;&#xC790; (PDGF) overactivity &#xC545;&#xC131; &#xC554;&#xC5D0; &#xC5F0;&#xACB0; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. PDGF&#xC758; &#xC0C1;&#xBD80;&#xB294; &#xC218;&#xC6A9; &#xCCB4; (PDGFRs)&#xC758; &#xD65C;&#xC131;&#xD654; &#xBC0F; &#xC140; over-proliferation &#xACB0;&#xACFC;. &#xB530;&#xB77C;&#xC11C; &#xC554; &#xC138;&#xD3EC;&#xC5D0;&#xC11C;&#xC774; &#xC2E0;&#xD638; &#xD1B5;&#xB85C; &#xBC29;&#xD574; &#xD56D; &#xC554; &#xC2E0;&#xC57D; &#xAC1C;&#xBC1C;&#xC744; &#xC704;&#xD55C; &#xC911;&#xC694; &#xD55C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC774;&#xC804; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C;&#xB294; &#xC815;&#xC801; CHO-k(1) &#xC140; &#xB2E8;&#xC704;&#xB85C; sPDGFR &#xC54C;&#xD30C; Fc &#xC735;&#xD569; &#xB2E8;&#xBC31;&#xC9C8; &#xD608;&#xAD00; &#xB0B4; &#xD53C; &#xC138;&#xD3EC;&#xC5D0; &#xC99D;&#xC2DD; &#xBC29;&#xC9C0; &#xD6A8;&#xACFC; &#xBCF4;&#xC600;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xCD94;&#xAC00; &#xAE30;&#xB2A5; &#xC5F0;&#xAD6C;&#xC5D0; &#xB300; &#xD55C; &#xC735;&#xD569; &#xB2E8;&#xBC31;&#xC9C8;&#xC774; &#xB300;&#xB7C9;&#xC744; &#xC5BB;&#xAE30; &#xC5B4;&#xB824;&#xC6E0;&#xB2E4;. &#xD604;&#xC7AC;&#xC758; &#xC5F0;&#xAD6C;&#xC5D0;&#xC11C; sPDGFR &#xC54C;&#xD30C; Fc &#xC735;&#xD569; &#xB2E8;&#xBC31;&#xC9C8; transiently bioreactors &#xB5A8;&#xACE0; 50 mL &#xADA4;&#xB3C4;&#xC5D0; &#xC911;&#xAD6D; &#xD584;&#xC2A4;&#xD130; &#xB09C;&#xC18C; (&#xC870;) DG44 &#xC140;&#xC5D0; &#xD45C;&#xD604; &#xD588;&#xB2E4;. sPDGFR &#xC54C;&#xD30C;-Fc&#xC640; &#xC7A0;&#xC7AC;&#xC801;&#xC778; glycosylation dimeric 250 kDa &#xB2E8;&#xBC31;&#xC9C8;&#xC73C;&#xB85C; &#xD45C;&#xD604; &#xD588;&#xB2E4;. SPDGFR &#xC54C;&#xD30C;-Fc &#xC3DF;&#xACE0; &#xBB38;&#xD654;&#xC5D0;&#xC11C;&#xC758; &#xCD5C;&#xC885; &#xC218;&#xC775;&#xB960;&#xC740; &#xB192;&#xC740; 16.68 mg/l&#xC774; &#xC9C0; &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; &#xADA4;&#xB3C4; bioreactors &#xB5A8;&#xACE0; &#xACFC;&#xB3C4; &#xC2DD; &#xC804; &#xC784;&#xC0C1; &#xC5F0;&#xAD6C;&#xC5D0; &#xC0AC;&#xC6A9; &#xB418;&#xB294; &#xC7AC;&#xC870;&#xD569; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xC900;&#xBE44;&#xC5D0; &#xB300; &#xD55C; &#xAC00;&#xB2A5;&#xD55C; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xAC83;&#xC774; &#xC88B;&#xC2B5;&#xB2C8;&#xB2E4;.

&#xACFC;&#xB3C4; &#xC2DD; 50 mL orbitally &#xB5A8;&#xACE0; &#xC77C;&#xD68C;&#xC6A9; &#xC0DD;&#xBB3C; &#xBC18;&#xC751; &#xAE30;&#xB97C; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xC870; DG44 &#xC138;&#xD3EC;&#xC5D0;&#xC11C; &#xC7AC;&#xC870;&#xD569; sPDGFR &#xC54C;&#xD30C; Fc&#xC785;&#xB2C8;&#xB2E4;.

Hiperactividad de factor de crecimiento derivado de plaquetas (PDGF) se ha relacionado con c&aacute;ncer maligno. Altos niveles de PDGF producen la activaci&oacute;n de sus receptores (PDGFRs) y la excesiva proliferaci&oacute;n de c&eacute;lulas. Por lo tanto, interferir en esta v&iacute;a de se&ntilde;alizaci&oacute;n en c&eacute;lulas de c&aacute;ncer podr&iacute;a ser importante para el desarrollo de f&aacute;rmacos contra el c&aacute;ncer. En un estudio anterior, la prote&iacute;na de fusi&oacute;n alfa-Fc sPDGFR expresada en las c&eacute;lulas CHO-k(1) est&aacute;ticas demostraron un efecto anti proliferativo de las c&eacute;lulas endoteliales vasculares. Sin embargo, es dif&iacute;cil obtener una gran cantidad de esta prote&iacute;na de fusi&oacute;n para futuros estudios funcionales. En el presente estudio, la prote&iacute;na de fusi&oacute;n alfa-Fc sPDGFR transitoriamente se expres&oacute; en c&eacute;lulas de ovario de h&aacute;mster chino (CHO) DG44 en 50 mL orbital sacudiendo Biorreactores. sPDGFR alfa-Fc se expres&oacute; como una prote&iacute;na dim&eacute;rica de 250 kDa con potencial glicosilaci&oacute;n. El rendimiento final de sPDGFR alfa-Fc en el sobrenadante de cultivo fue tan alto como 16,68 mg/L. Nuestros resultados sugieren que la expresi&oacute;n transitoria en orbital sacudiendo Biorreactores puede ser factible para preparaci&oacute;n de prote&iacute;nas recombinantes utilizadas para los estudios precl&iacute;nicos.

Expresi&oacute;n transitoria de sPDGFR recombinante alfa-Fc en las c&eacute;lulas de CHO DG44 usando orbitally agitaci&oacute;n Biorreactores desechables 50 mL.

To study the functions of human Fibroblast growth factor receptor 2IIIc (FGFR2IIIc) gene in cancer cells, breast cancer cells MDA-MB-231 were infected by recombinant adenoviruses containing FGFR2IIIc and its S252W mutant, respectively. FGFR2IIIc gene was amplified from an existing plasmid and its S252W mutant was obtained by overlapping extension PCR. These two genes were separately cloned into the adenoviral shuttle plasmid pAdTrack-CMV, confivmed by DNA sequencing linearized, and co-transformed into Escherichia coli BJ-5183 with the adenoviral vector pAdEasy-1. The resulting recombinant expression vectors Ad-FGFR2IIIc and Ad-FGFR2IIIcS252W were linearized and transfected into HEK293A cells to get adenoviral particles. GFP was used to verify the gene expression. The recombinant adenoviral particles were harvested, titrated, and then infected MDA-MB-231 cells. The expression of FGFR2IIIc and its S252W mutant were examined by RT-PCR and Western blotting, and the effect of these recombinant adenoviruses on MDA-MB-231 cell proliferation was analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry. The results showed the recombinant adenoviral particles could infect MDA-MB-231 cells and express the target proteins. MTT showed that both FGFR2IIIc and its S252W mutant inhibited MDA-MB-231 cell proliferation, but the mutant was more effective. Flow cytometry showed that both FGFR2IIIc and its S252W mutant arrested MDA-MB-231 cell cycle at G0/G1 phase, resulting in low cell proliferation.

[Experssion of human FGFR2IIIc and its S252W mutant in MDA-MB-231 breast cancer cells with adenovirus vectors].

&#x4E00;&#x79CD;&#x65B0;&#x578B;&#x5149;&#x5B66;&#x4F20;&#x611F;&#x5668;&#x88AB;&#x56FA;&#x5B9A;&#x5728;&#x4E00;&#x6B21;&#x6027;&#x751F;&#x7269;&#x53CD;&#x5E94;&#x5668;&#xFF0C;Tubespin&#xFF0C;&#x4E00;&#x79CD;&#x65B0;&#x578B;&#x7684; &#xFF08;&#x5B9E;&#x65F6;&#xFF09; &#x7684;&#x5728;&#x7EBF;&#x76D1;&#x6D4B;&#x6EB6;&#x89E3;&#x7684;&#x6C27;&#x6D53;&#x5EA6;&#x5728;&#x7EC6;&#x80DE;&#x57F9;&#x517B;&#x8FC7;&#x7A0B;&#x3002;&#x7EC6;&#x80DE;&#x5BC6;&#x5EA6;&#x3001; &#x6D3B;&#x529B;&#x548C;&#x4F53;&#x79EF;&#x4F20;&#x8D28;&#x7CFB;&#x6570;&#x4E5F;&#x51B3;&#x5FC3;&#x8FDB;&#x4E00;&#x6B65;&#x63CF;&#x8FF0;&#x751F;&#x7269;&#x53CD;&#x5E94;&#x5668;&#x3002;K (L) &#x5728;&#x6807;&#x51C6;&#x6761;&#x4EF6; Tubespin &#x7684;&#x503C;&#x662F;&#x4E3A; 24 3 h(-1)&#xFF0C;&#x867D;&#x7136;&#x8FD9;&#x4E00;&#x5FAE;&#x8C03;&#x6846;&#x74F6;&#x53EA;&#x662F; 2.7 h(-1)&#x3002;Tubespin &#x751F;&#x7269;&#x53CD;&#x5E94;&#x5668;&#x4E2D;&#x7684;&#x6700;&#x5927;&#x7684;&#x7EC6;&#x80DE;&#x5BC6;&#x5EA6;&#x8FBE;&#x5230; 6 &times; 10(6) &#x7EC6;&#x80DE; mL(-1)&#xFF0C;&#x8FD9;&#x662F;&#x6BD4;&#x5FAE;&#x8C03;&#x6846;&#x74F6;&#x4E2D;&#x7684;&#x7EC6;&#x80DE;&#x5BC6;&#x5EA6;&#x9AD8;&#x51FA;&#x4E24;&#x500D;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x52A8;&#x6001;&#x7684;&#x6EB6;&#x89E3;&#x7684;&#x6C27;&#x6C34;&#x5E73;&#x7EF4;&#x6301;&#x5728;&#x4EE5;&#x4E0A; 90%&#x7A7A;&#x6C14;&#x9971;&#x548C;&#x5EA6;&#x7684; Tubespin&#xFF0C;&#x800C;&#x4EF7;&#x503C;&#x662F;&#x5FAE;&#x8C03;&#x6846;&#x74F6;&#x53EA;&#x6709; 1.9%&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x8868;&#x660E;&#x901A;&#x8FC7;&#x5FAE;&#x8C03;&#x6846;&#x70E7;&#x74F6;&#x5185;&#x4F7F;&#x7528; Tubespin &#x7CFB;&#x7EDF;&#x5DE5;&#x827A;&#x4F18;&#x5316;&#x53CA;&#x89C4;&#x6A21;&#x4E0B;&#x7814;&#x7A76;&#x7684;&#x6C27;&#x8F6C;&#x79FB;&#x53CA;&#x7EC6;&#x80DE;&#x751F;&#x957F;&#x7684;&#x7ADE;&#x4E89;&#x4F18;&#x52BF;&#x3002;

Tubespin&#x3001; &#x5C0F;&#x8BF4;&#x3001; &#x5C0F;&#x578B;&#x4E00;&#x6B21;&#x6027;&#x751F;&#x7269;&#x53CD;&#x5E94;&#x5668;&#x4E2D;&#x6C27;&#x7684;&#x5728;&#x7EBF;&#x76D1;&#x6D4B;&#x3002;

A novel, optical sensor was fixed in a new type of disposable bioreactor, Tubespin, for the on-line (real-time) monitoring of dissolved oxygen concentrations during cell culture. The cell density, viability and volumetric mass transfer coefficient were also determined to further characterize the bioreactors. The k(L)a value of the Tubespin at standard conditions was 24.3Â h(-1), while that of a spinner flask was only 2.7Â h(-1). The maximum cell density in the Tubespin bioreactor reached 6Â Ã—Â 10(6) cellsÂ mL(-1), which was two times higher than the cell density in a spinner flask. Furthermore, the dynamic dissolved oxygen level was maintained above 90% air-saturation in the Tubespin, while the value was only 1.9% in a spinner flask. These results demonstrate the competitive advantage of using the Tubespin system over spinner flasks for process optimization and scale-down studies of oxygen transfer and cell growth.

On-line monitoring of oxygen in Tubespin, a novel, small-scale disposable bioreactor.

Un roman, capteur optique a &eacute;t&eacute; corrig&eacute; dans un nouveau type de bior&eacute;acteur jetable, Tubespin, pour le contr&ocirc;le (temps r&eacute;el) en ligne de concentrations d&#39;oxyg&egrave;ne dissous au cours de la culture cellulaire. La densit&eacute; cellulaire, la viabilit&eacute; et le coefficient de transfert de masse volum&eacute;trique ont &eacute;t&eacute; &eacute;galement d&eacute;termin&eacute;es &agrave; mieux caract&eacute;riser les bior&eacute;acteurs. Le k (L) une valeur de la Tubespin dans les conditions normales &eacute;tait &eacute;vidence 24.3, tandis que celle d&#39;une fiole de fileur &eacute;tait seulement 2,7 &eacute;vidence. La densit&eacute; cellulaire maximale dans le bior&eacute;acteur de Tubespin atteint 6 &times; 10 6 cellules mL(-1), qui a &eacute;t&eacute; deux fois plus &eacute;lev&eacute;e que la densit&eacute; des cellules dans une fiole de c&ocirc;ne d&#39;h&eacute;lice. En outre, le niveau d&#39;oxyg&egrave;ne dissous dynamique a &eacute;t&eacute; maintenu au-dessus de 90 %-saturation de l&#39;air dans la Tubespin, tandis que la valeur n&#39;&eacute;tait que de 1,9 % dans une fiole de c&ocirc;ne d&#39;h&eacute;lice. Ces r&eacute;sultats d&eacute;montrent l&#39;avantage concurrentiel de l&#39;utilisation du syst&egrave;me Tubespin sur flacons spinner pour l&#39;optimisation des processus et &agrave; &eacute;chelle r&eacute;duite des &eacute;tudes de croissance cellulaire et de transfert d&#39;oxyg&egrave;ne.

Contr&ocirc;le en ligne de l&#39;oxyg&egrave;ne dans le Tubespin, un roman, le bior&eacute;acteur jetable &agrave; petite &eacute;chelle.

Roman, wurde eine neue Art von Einweg Bioreaktor, Tubespin, zur Online (Echtzeit) &Uuml;berwachung von gel&ouml;stem Sauerstoff-Konzentrationen w&auml;hrend der Zellkultur optischer Sensor behoben. Die Zelldichte, Lebensf&auml;higkeit und Volumetrische Massentransfer Koeffizient wurden auch bestimmt, um weiter die Bioreaktoren zu charakterisieren. Die k (L) der Wert der Tubespin unter Normbedingungen war 24.3 h(-1), w&auml;hrend der ein Spinner-Kolben nur 2,7 h(-1). Die maximale Zelldichte in Tubespin Bioreaktor erreicht 6 &times; 10 6-Zellen-mL(-1), die war 2 mal gr&ouml;&szlig;er als die Zellendichte in einen Spinner-Kolben. Dar&uuml;ber hinaus wurde das dynamische gel&ouml;sten Sauerstoff-Niveau &uuml;ber 90 % Luft-S&auml;ttigung in der Tubespin beibehalten, w&auml;hrend der Wert nur 1,9 % in einem Spinner-Kolben war. Diese Ergebnisse zeigen den Wettbewerbsvorteil der Verwendung des Tubespin-Systems &uuml;ber Spinner-Kolben f&uuml;r Prozessoptimierung und Scale-Down Studien des Sauerstoff-Transfer und Zelle Wachstum.

Online-Pr&uuml;fung von Sauerstoff in Tubespin, Roman, kleine Einweg-Bioreaktor.

Un romanzo, sensore ottico &egrave; stato fissato in un nuovo tipo di bioreattore monouso, Tubespin, per l&#39;on-line () monitoraggio in tempo reale delle concentrazioni di ossigeno disciolto durante la coltura delle cellule. La densit&agrave; delle cellule, la vitalit&agrave; e il coefficiente di trasferimento di massa volumetrica sono stati determinati anche per caratterizzare ulteriormente i bioreattori. K (L) un valore di Tubespin a condizioni standard era 24,3 h(-1), mentre quella di un pallone da spinner era solo 2,7 h(-1). La densit&agrave; massima delle cellule nel bioreattore Tubespin raggiunto 6 &times; 10 cellule mL(-1), che era due volte superiore rispetto la densit&agrave; delle cellule in un matraccio da spinner. Inoltre, il livello di ossigeno disciolto dinamico &egrave; stato mantenuto sopra il 90% dell&#39;aria-saturazione in Tubespin, mentre il valore era solo l&#39;1,9% in un pallone di spinner. Questi risultati dimostrano il vantaggio competitivo di utilizzando il sistema di Tubespin sopra boccette spinner per l&#39;ottimizzazione dei processi e studi a scala di crescita delle cellule e di trasferimento di ossigeno.

Monitoraggio on-line di ossigeno in Tubespin, un romanzo, su piccola scala bioreattore monouso.

&#x5C0F;&#x8AAC;&#x3001;&#x5149;&#x5B66;&#x30BB;&#x30F3;&#x30B5;&#x30FC; Tubespin&#x3001;&#x4F7F;&#x3044;&#x6368;&#x3066;&#x578B;&#x30D0;&#x30A4;&#x30AA;&#x30EA;&#x30A2;&#x30AF;&#x30BF;&#x30FC;&#x306E;&#x65B0;&#x3057;&#x3044;&#x30BF;&#x30A4;&#x30D7;&#x306E;&#x30AA;&#x30F3;&#x30E9;&#x30A4;&#x30F3; &#xFF08;&#x30EA;&#x30A2;&#x30EB;&#x30BF;&#x30A4;&#x30E0;&#xFF09; &#x6EB6;&#x5B58;&#x9178;&#x7D20;&#x6FC3;&#x5EA6;&#x306E;&#x57F9;&#x990A;&#x4E2D;&#x306B;&#x76E3;&#x8996;&#x3092;&#x4FEE;&#x6B63;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x7D30;&#x80DE;&#x5BC6;&#x5EA6;, &#x751F;&#x5B58;&#x7387;&#x53CA;&#x3073;&#x7269;&#x8CEA;&#x79FB;&#x52D5;&#x5BB9;&#x91CF;&#x4FC2;&#x6570;&#x3082;&#x3001;&#x30D0;&#x30A4;&#x30AA;&#x30EA;&#x30A2;&#x30AF;&#x30BF;&#x30FC;&#x3092;&#x3055;&#x3089;&#x306B;&#x7279;&#x6027;&#x3092;&#x6E2C;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30B9;&#x30D4;&#x30CA;&#x30FC; &#x30D5;&#x30E9;&#x30B9;&#x30B3;&#x306E;&#x3060;&#x3051; 2.7 h(-1) k (L) &#x6A19;&#x6E96;&#x7684;&#x306A;&#x6761;&#x4EF6;&#x3067; Tubespin &#x306E;&#x5024; 24.3 h(-1) &#x3042;&#x3063;&#x305F;&#x3002;Tubespin &#x30D0;&#x30A4;&#x30AA;&#x30EA;&#x30A2;&#x30AF;&#x30BF;&#x30FC;&#x306E;&#x30BB;&#x30EB;&#x306E;&#x6700;&#x5927;&#x5BC6;&#x5EA6;&#x306F; 6 &times; 10 (6) &#x7D30;&#x80DE;&#x306E; mL(-1)&#x3001;&#x30B9;&#x30D4;&#x30CA;&#x30FC; &#x30D5;&#x30E9;&#x30B9;&#x30B3;&#x306E;&#x7D30;&#x80DE;&#x5BC6;&#x5EA6;&#x306E; 2 &#x500D;&#x9054;&#x3057;&#x305F;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x5024;&#x3060;&#x3051; 1.9 &#xFF05; &#x30B9;&#x30D4;&#x30CA;&#x30FC; &#x30D5;&#x30E9;&#x30B9;&#x30B3;&#x4E2D;&#x52D5;&#x7684;&#x6EB6;&#x5B58;&#x9178;&#x7D20;&#x30EC;&#x30D9;&#x30EB; 90% &#x7A7A;&#x6C17;&#x98FD;&#x548C;&#x3001;Tubespin &#x306E;&#x4E0A;&#x306B;&#x7DAD;&#x6301;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x30D7;&#x30ED;&#x30BB;&#x30B9;&#x306E;&#x6700;&#x9069;&#x5316;&#x3001;&#x30B9;&#x30B1;&#x30FC;&#x30EB; &#x30C0;&#x30A6;&#x30F3;&#x7814;&#x7A76;&#x306E;&#x9178;&#x7D20;&#x4F1D;&#x9054;&#x304A;&#x3088;&#x3073;&#x7D30;&#x80DE;&#x5897;&#x6B96;&#x306E;&#x30B9;&#x30D4;&#x30CA;&#x30FC; &#x30D5;&#x30E9;&#x30B9;&#x30B3;&#x306B; Tubespin &#x30B7;&#x30B9;&#x30C6;&#x30E0;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x7AF6;&#x4E89;&#x4E0A;&#x306E;&#x512A;&#x4F4D;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30AA;&#x30F3;&#x30E9;&#x30A4;&#x30F3; Tubespin&#x3001;&#x5C0F;&#x8AAC;&#x3001;&#x5C0F;&#x898F;&#x6A21;&#x306A;&#x4F7F;&#x3044;&#x6368;&#x3066;&#x578B;&#x30D0;&#x30A4;&#x30AA;&#x30EA;&#x30A2;&#x30AF;&#x30BF;&#x30FC;&#x306B;&#x304A;&#x3051;&#x308B;&#x9178;&#x7D20;&#x306E;&#x76E3;&#x8996;&#x3002;

&#xC18C;&#xC124;, &#xC628;&#xB77C;&#xC778; (&#xC2E4;&#xC2DC;&#xAC04;) &#xBAA8;&#xB2C8;&#xD130;&#xB9C1; &#xC6A9; &#xC874;&#xB41C; &#xC0B0;&#xC18C; &#xB18D;&#xB3C4;&#xC758; &#xC138;&#xD3EC; &#xBC30;&#xC591; &#xC911;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xC720;&#xD615;&#xC758; &#xC77C;&#xD68C;&#xC6A9; bioreactor, Tubespin, &#xAD11;&#xD559; &#xC13C;&#xC11C; &#xC218;&#xC815; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC140; &#xBC00;&#xB3C4;, &#xC0DD;&#xC874; &#xBC0F; &#xCCB4;&#xC801; &#xC9C8;&#xB7C9; &#xC804;&#xB2EC; &#xACC4;&#xC218; &#xCD94;&#xAC00; &#xC0DD;&#xBB3C; &#xBC18;&#xC751; &#xAE30;&#xC758; &#xD2B9;&#xC131;&#xC744; &#xACB0;&#xC815; &#xD588;&#xB2E4;. &#xC2A4;&#xD53C;&#xB108; &#xD50C;&#xB77C;&#xC2A4; &#xD06C;&#xC758;&#xB294; &#xB2E8;&#xC9C0; 2.7 h(-1) k (L) &#xAC12;&#xC774; &#xD45C;&#xC900; &#xC870;&#xAC74;&#xC5D0;&#xC11C; Tubespin 24.3 h(-1) &#xC774;&#xC5C8;&#xB2E4;. Tubespin bioreactor&#xC5D0; &#xC788;&#xB294; &#xCD5C;&#xB300; &#xC140; &#xBC00;&#xB3C4; &#xB3C4;&#xB2EC; 6 &times; &#xC2DC;&#xBBFC;&#xAD8C;&#xC774; &#xC140; mL(-1), &#xC2A4;&#xD53C;&#xB108; &#xD50C;&#xB77C;&#xC2A4; &#xD06C;&#xC5D0; &#xC140; &#xBC00;&#xB3C4; &#xBCF4;&#xB2E4; &#xB450; &#xBC30; &#xB192;&#xC740; &#xD588;&#xB2E4;. &#xB610;&#xD55C;, &#xB3D9;&#xC801; &#xB179;&#xC740; &#xC0B0;&#xC18C; &#xC218;&#xC900; &#xAC12;&#xC740; &#xD68C;&#xC804;&#xC790; &#xD50C;&#xB77C;&#xC2A4; &#xD06C;&#xC5D0; 1.9% &#xD558;&#xB294; &#xB3D9;&#xC548; Tubespin&#xC5D0;&#xC11C; 90% &#xC5B4; &#xCC44;&#xB3C4; &#xC704;&#xC5D0; &#xC720;&#xC9C0; &#xD588;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xACB0;&#xACFC; &#xACF5;&#xC815; &#xCD5C;&#xC801;&#xD654; &#xBC0F; &#xC2A4;&#xCF00;&#xC77C; &#xB2E4;&#xC6B4; &#xC0B0;&#xC18C; &#xC804;&#xC1A1; &#xBC0F; &#xC138;&#xD3EC; &#xC131;&#xC7A5; &#xC5F0;&#xAD6C;&#xC5D0; &#xB300; &#xD55C; &#xD68C;&#xC804;&#xC790; &#xD50C;&#xB77C;&#xC2A4; &#xD06C;&#xB97C; &#xD1B5;&#xD574; Tubespin &#xC2DC;&#xC2A4;&#xD15C;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; &#xACBD;&#xC7C1; &#xC6B0;&#xC704; &#xC785;&#xC99D;.

Tubespin, &#xC18C;&#xC124;, &#xC18C;&#xADDC;&#xBAA8; &#xC77C;&#xD68C;&#xC6A9; &#xC0DD;&#xBB3C; &#xBC18;&#xC751; &#xAE30;&#xC5D0;&#xC11C; &#xC0B0;&#xC18C;&#xC758; &#xC628;&#xB77C;&#xC778; &#xBAA8;&#xB2C8;&#xD130;&#xB9C1;.

Una novela, sensor &oacute;ptico se fij&oacute; en un nuevo tipo de biorreactor desechable, Tubespin, para el seguimiento (tiempo real) on-line de concentraciones de ox&iacute;geno disuelto durante el cultivo celular. Tambi&eacute;n se determinan la densidad celular, la viabilidad y el coeficiente de transferencia de masa volum&eacute;trica para caracterizar los biorreactores. La k (L) un valor de la Tubespin en condiciones normales fue 24.3 h, mientras de un frasco de ruleta fue s&oacute;lo 2,7 h. La densidad m&aacute;xima de la c&eacute;lula en el biorreactor de Tubespin alcanzado 6 &times; 10 ml de c&eacute;lulas, que fue dos veces mayor que la densidad celular en un matraz de ruleta. Adem&aacute;s, el nivel de ox&iacute;geno disuelto din&aacute;mica se mantuvo por encima de 90% saturaci&oacute;n en el Tubespin, mientras que el valor fue de s&oacute;lo 1,9% en un matraz de ruleta. Estos resultados demuestran la ventaja de utilizar el sistema de Tubespin en frascos de spinner para optimizaci&oacute;n de procesos y estudios de escala desplegable de ox&iacute;geno transferencia y el crecimiento celular.

Seguimiento on-line de ox&iacute;geno en Tubespin, una novela, biorreactor desechable en peque&ntilde;a escala.

The soluble ectodomain of fibroblast growth factor receptor-IIIc (sFGFR2c) is able to bind to fibroblast growth factor (FGF) ligands and block the activation of the FGF-signaling pathway. In this study, sFGFR2c inhibited lung fibrosis dramatically in vitro and in vivo. The upregulation of α-smooth muscle actin (α-SMA) in fibroblasts by transforming growth factor-β1 (TGF-β1) is an important step in the process of lung fibrosis, in which FGF-2, released by TGF-β1, is involved. sFGFR2c inhibited α-SMA induction by TGF-β1 via both the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad3 pathways in primary mouse lung fibroblasts and the proliferation of mouse lung fibroblasts. In a mouse model of bleomycin (BLM)-induced lung fibrosis, mice were treated with sFGFR2c from d 3 or d 10 to 31 after BLM administration. Then we used hematoxylin and eosin staining, Masson staining and immunohistochemical staining to evaluate the inhibitory effects of sFGFR2c on lung fibrosis. The treatment with sFGFR2c resulted in significant attenuation of the lung fibrosis score and collagen deposition. The expression levels of α-SMA, p-FGFRs, p-ERK1/2 and p-Smad3 in the lungs of sFGFR2c-treated mice were markedly lower. sFGFR2c may have potential for the treatment of lung fibrosis as an FGF-2 antagonist.

Inhibition of α-SMA by the ectodomain of FGFR2c attenuates lung fibrosis.

Bone is one of the most common metastatic sites for breast cancer. In this study, we observed a promoting effect of osteoblast-conditioned medium (OCM) on the migration of MCF-7, a noninvasive cell line of breast cancer cells. Cytokine antibody array was used to compare the cytokines of OCM with the conditioned medium of non-differentiated osteoblast cells, which consequently revealed factors related to migration, such as IL8, IL6, CSF2 (G-CSF), CSF3 (GM-CSF), and TNFRSF11B (osteoprotegerin). The expression of genes related to migration was also estimated with a PCR array, which showed that 9 genes were upregulated and 26 genes downregulated. Moreover, activated p38, ERK, and AKT pathways were found in the OCM treatment group. This finding indicated the migration ability of breast cancer cells, which move toward the bone depending on the presence of specific cytokines in its surrounding microenvironment. 

Cytokines in osteoblast-conditioned medium promote the migration of breast cancer cells.

Insulin resistance (IR) is a physiological condition in which cells fail to respond to the insulin hormone. Despite advances in the diagnosis and treatment of IR, novel molecular targets are still needed to improve the accuracy of diagnosis and the outcomes of therapy. Here, we present a systems approach to identify molecular biomarkers for IR. We downloaded the gene expression profile of IR from the Gene Expression Omnibus (GEO), generated a regulatory network by mapping co-expressed genes to transcription factors (TFs) and calculated the regulatory impact factor of each transcription factor. Finally, we selected a list of potential molecular targets that could be used as therapeutic targets or diagnostic biomarkers, including ETS1, AR, ESR1 and Myc. Our studies identified multiple TFs that could play an important role in the pathogenesis of IR and provided a systems understanding of the potential relationships among these genes. Our study has the potential to aid in the understanding of IR and provides a basis for IR biomarker discovery. 

Constructing regulatory networks to identify biomarkers for insulin resistance.

Pituitary adenylate cyclase-activating peptide I receptor (PAC1R) is member of the B class of G protein-coupled seven-transmembrane receptors, with molecular functions associated with neural cell differentiation, regeneration and the inhibition of apoptosis. However, the integrity of the protein structure is difficult to be determined in vitro. In the present study, the physicochemical properties of PAC1R were analyzed, the extracellular, transmembrane and intracellular regions were constructed and a three-dimensional structure model of PAC1R was produced using extracellular loop region optimization and the energy minimization homology modeling method. Preliminary studies on the PAC1R protein and ligand interactions used a molecular docking method. The results indicated that the interaction sites of PAC1R were at Ile63, Ser100 and Gln105. These were the sites where the PAC1R combined with a hydrazide small molecule inhibitor. This study provides a theoretical basis for further studies on the model for the development of PAC1R target drugs.

Homology modeling and molecular docking of human pituitary adenylate cyclase‑activating polypeptide I receptor.

Pituitary adenylate cyclase‑activating polypeptide (PACAP) is a type of neuropeptide with multiple biological functions. However, it has a short half‑life period in the body, ~3 to 5 min, restricting its further development as a drug that can promote the recovery of nerve injury. In vitro and in vivo experiments have shown that PACAP can repair the epithelial cell on the surface of the injured cornea, as PACAP can act on the trigeminal nerve cell to secrete other active neurotransmitters, which can promote corneal epithelial cell proliferation and differentiation. In the present study, PACAP is connected to the N‑terminal agrin domain (NtA) with a genetic engineering method, which allows the function of repairing the injured nerve. Notably, the recombinant polypeptide can interact with laminin, improving the biological effect of PACAP in repairing the injured nerve. In the study, the recombinant protein was constructed by combining PACAP38 and NtA by genetic engineering, and it is expressed in the pronucleus escherichia coli. The recombinant protein, PACAP38‑NtA, is obtained with a two‑step purification method, including anion‑exchange chromatography and Ni‑affinity chromatography, with the purity reaching >90%. The in vitro experiment has shown that this recombinant protein not only has the neurotrophy and neural restoration function of PACAP, but also has the function of an anchoring protein as laminin interacts with NtA. According to the in vitro anti‑apoptosis, PC12 axon growth and ELISA experiments, this protein has the biological activity of a recombinant protein. PACAP38‑NtA also has an anchoring function as NtA and laminin interact with good biological activity.

Expression, identification and biological effects of the novel recombination protein, PACAP38-NtA, with high bioactivity.

We have previously published a report on the cloning and characterization of Harobin, a fibrinolytic serine protease. However, the broad application of this fibrinolytic enzyme is limited by its low expression level that was achieved in Pichia pastoris. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of Harobin. By screening 400 clones from random mutant libraries for enhanced fibrinolytic activity, two mutants were obtained: N111R, R230G. By performing site-directed mutagenesis, a Harobin double mutant, N111R/R230G, was constructed and can be functionally expressed at higher level than the wild type enzyme. In addition, it possessed much higher fibrinolytic and amidolytic activity than the wild type enzyme and other single mutants. The N111R/R230G expressed in basal salts medium was purified by a three step purification procedure. At pH of 6.0-9.0, and the temperature range of 40-90 °C, N111R/R230G was more active and more heat resistant. The fibrinolytic activities of Harobin mutants were completely inhibited by PMSF and SBTI, but not by EDTA, EGTA, DTT, indicating that Harobin is a serine protease. N111R/R230G showed much better anti-thrombosis effect than wild type Harobin and single mutants, and could significantly increase bleeding and clotting time. Intravenous injection of N111R/R230G in spontaneous hypertensive rats (SHR) led to a significant reduction in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) (p < 0.01), while heart rate (HR) was not affected. The in vitro and in vivo results of the present study revealed that Harobin double mutant N111R/R230G is an appropriate candidate for biotechnological applications due to its high expression level and high activity in area of thrombosis and hypertension.

Engineering of Harobin for enhanced fibrinolytic activity obtained by random and site-directed mutagenesis.

Recent studies indicate important roles for long noncoding RNAs (lncRNAs) as essential regulators of myogenesis and adult skeletal muscle regeneration. However, the specific roles of lncRNAs in myogenic differentiation of adult skeletal muscle stem cells and myogenesis are still largely unknown. Here we identify a lncRNA that is specifically enriched in skeletal muscle (myogenesis-associated lncRNA, in short, lnc-mg). In mice, conditional knockout of lnc-mg in skeletal muscle results in muscle atrophy and the loss of muscular endurance during exercise. Alternatively, skeletal muscle-specific overexpression of lnc-mg promotes muscle hypertrophy. In vitro analysis of primary skeletal muscle cells shows that lnc-mg increases gradually during myogenic differentiation and its overexpression improves cell differentiation. Mechanistically, lnc-mg promotes myogenesis, by functioning as a competing endogenous RNA (ceRNA) for microRNA-125b to control protein abundance of insulin-like growth factor 2. These findings identify lnc-mg as a novel noncoding regulator for muscle cell differentiation and skeletal muscle development.

Lnc-mg is a long non-coding RNA that promotes myogenesis.

Cancer-associated fibroblasts (CAFs) are believed to influence tumor behavior and clinical outcomes. We previously showed that conditioned medium (CM) from CAFs induces proliferation and motility of esophageal squamous cell carcinoma (ESCC) cells. Here, we investigated the molecular mechanisms by which the CAF-secreted proteins induce ESCC development and progression. Using antibody arrays, we identified urokinase plasminogen activator (uPA) as one of the main proteins whose release was increased in CAFs compared to normal fibroblasts (NFs). Immunohistochemical analysis of pathological sections showed that uPA-positive cells were localized at the boundaries of tumor and stroma tissues, in stroma between tumor nests, and within the tumors. Increased stromal uPA levels (132/146 cases) correlated with tumor invasion (p < 0.05) and overall survival of ESCC patients (p < 0.05). In vitro assays showed that uPA promotes ESCC cell proliferation, migration, and invasion via PI3K/AKT and ERK signaling pathways. In vivo, anti-uPA antibody suppressed tumor growth in ESCC xenografts. These results suggest that uPA released from stroma, and especially from CAFs, might be a predictive marker for ESCC diagnosis and prognosis, as well as an effective therapeutic target.

Urokinase plasminogen activator secreted by cancer-associated fibroblasts induces tumor progression via PI3K/AKT and ERK signaling in esophageal squamous cell carcinoma.

Bone morphogenetic protein 2 (BMP-2) has been reported to facilitate epithelial-to-mesenchymal transition (EMT) and bone metastasis in breast cancer xenograft models. To investigate the role of BMP-2 in the development of breast cancer stem cells (BCSCs), and to further elucidate the mechanisms underlying its influence on breast cancer metastasis, we conducted a comprehensive molecular study using breast cancer cell lines and clinical samples. Our results showed that downregulation of Rb by BMP-2 was associated with ubiquitin-mediated degradation activated by phosphorylation of Rb via the PI3K/AKT signal pathway. In addition, the Smad signaling pathways are implicated in upregulation of CD44 protein expression by BMP-2. It was suggested that cross-talk exists between Rb and CD44 signaling pathways, as recombinant human BMP-2 (rhBMP-2) was found to regulate CD44 expression partly through Rb signals. In clinical tissues, BMP-2 was positively and negatively correlated with CD44 and Rb expression, respectively. Based on the  and  results, we have established an integrated mechanism by which rhBMP-2 induces EMT and stemness of breast cancer cells via the Rb and CD44 signaling pathways, which then contribute to breast cancer metastasis. These findings may be helpful for developing new strategies for the treatment and prognosis of advanced breast cancer.

BMP-2 induces EMT and breast cancer stemness through Rb and CD44.

Resistance to platinum-based combination chemotherapy is the main cause of poor prognosis in patients with advanced esophageal squamous cell carcinoma (ESCC). Previously, we showed that CACNA2D3 (voltage-dependent subunit alpha 2 delta 3 of a calcium channel complex) was significantly downregulated and functioned as a tumor suppressor in ESCC, but its role in the chemosensitivity of ESCC to cisplatin remained unknown. Here, we found that the expression of CACNA2D3 was significantly associated with poor platinum response in ESCC patients from the Gene Expression Omnibus database. Overexpression of CACNA2D3 increased sensitivity to cisplatin in ESCC , whereas knockdown of CACNA2D3 increased cisplatin resistance. CACNA2D3 promoted cisplatin-induced apoptosis by modulating intracellular Ca stores.  experiments further showed that overexpression of CACNA2D3 enhanced cisplatin anti-tumor effects in a xenograft mouse model. CACNA2D3 overexpression also resulted in the attenuation of PI3K and Akt phosphorylation. Treatment with the PI3K/Akt inhibitor LY294002 restored the chemosensitivity of CACAN2D3-knockdown cells to cisplatin. In conclusion, the results of the current study indicate that CACAN2D3 enhances the chemosensitivity of ESCC to cisplatin via inducing Ca-mediated apoptosis and suppressing PI3K/Akt pathways. Therefore, regulating the expression of CACNA2D3 is a potential new strategy to increase the efficacy of cisplatin in ESCC patients.

CACNA2D3 Enhances the Chemosensitivity of Esophageal Squamous Cell Carcinoma to Cisplatin via Inducing Ca-Mediated Apoptosis and Suppressing PI3K/Akt Pathways.

Esophageal cancer is the eighth most common malignant tumor worldwide, of which esophageal squamous cell carcinoma (ESCC) is the dominant histological subtype. A drug shortage for ESCC therapy triggered us to explore the roles of fibroblast growth factor receptor 2 (FGFR2) and its upstream regulator miR-671-5p in ESCC progression. We compared the levels of FGFR2 and miR-671-5p between human ESCC tissues and their matched normal esophageal tissues and found an association between higher levels of FGFR2 and lower levels of miR-671-5p in ESCC tissues. High levels of FGFR2 resulted in the activation of the ERK and AKT pathways and a promotion of ESCC progression. High levels of miR-671-5p specifically reduced the expression of FGFR2 and suppressed ESCC progression in both in vitro nd in vivo models. Therefore, suppressing FGFR2 and enhancing miR-671-5p expression may be the right approaches for ESCC therapy.

Article	Year
Transient expression of recombinant sPDGFR alpha-Fc in CHO DG44 cells using 50-mL orbitally shaking disposable bioreactors. Protein and peptide letters\| PubMed ID: 20205651	2010
[Experssion of human FGFR2IIIc and its S252W mutant in MDA-MB-231 breast cancer cells with adenovirus vectors]. Sheng wu gong cheng xue bao = Chinese journal of biotechnology\| PubMed ID: 20518350	2010
On-line monitoring of oxygen in Tubespin, a novel, small-scale disposable bioreactor. Cytotechnology\| PubMed ID: 21574065	2011
Inhibition of α-SMA by the ectodomain of FGFR2c attenuates lung fibrosis. Molecular medicine (Cambridge, Mass.)\| PubMed ID: 22451267	2012
Cytokines in osteoblast-conditioned medium promote the migration of breast cancer cells. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine\| PubMed ID: 24026883	2014
Constructing regulatory networks to identify biomarkers for insulin resistance. Gene\| PubMed ID: 24512691	2014
Homology modeling and molecular docking of human pituitary adenylate cyclase‑activating polypeptide I receptor. Molecular medicine reports\| PubMed ID: 25069645	2014
Expression, identification and biological effects of the novel recombination protein, PACAP38-NtA, with high bioactivity. International journal of molecular medicine\| PubMed ID: 25483608	2015
Engineering of Harobin for enhanced fibrinolytic activity obtained by random and site-directed mutagenesis. Protein expression and purification\| PubMed ID: 26363113	2017
Lnc-mg is a long non-coding RNA that promotes myogenesis. Nature communications\| PubMed ID: 28281528	2017
Urokinase plasminogen activator secreted by cancer-associated fibroblasts induces tumor progression via PI3K/AKT and ERK signaling in esophageal squamous cell carcinoma. Oncotarget\| PubMed ID: 28404945	2017
BMP-2 induces EMT and breast cancer stemness through Rb and CD44. Cell death discovery\| PubMed ID: 28725489	2017
CACNA2D3 Enhances the Chemosensitivity of Esophageal Squamous Cell Carcinoma to Cisplatin via Inducing Ca-Mediated Apoptosis and Suppressing PI3K/Akt Pathways. Frontiers in oncology\| PubMed ID: 31001468	2019
miR-671-5p Blocks The Progression Of Human Esophageal Squamous Cell Carcinoma By Suppressing FGFR2. International journal of biological sciences\| PubMed ID: 31523191	2019

An Hong

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