750 [ ]1000-8861(2012)09-0750-05 5 * * [ ] 5 5 (GCSaDR 5 ScFv) H22 5 (adr 5 ScFv) GCS-aDR 5 ScFv GCS-aDR 5 Zeta H22 0.272 mg/ml GCS-aDR 5 ScFv 2 Western blot active-caspase8 active-caspase3 BAX adr 5 ScFv (M r 30 000)GCS-aDR 5 ScFv Zeta GCS-aDR 5 ScFv Western blot active-caspase8 active-caspase3 BAX GCS-aDR 5 ScFv H22 active-caspase8 active-caspase3 BAX [ ] GCS-aDR 5 ScFv; H22; [ ] R392.11 [ ] A Preparation and identification of adr 5 ScFv loaded GCS and assaying its antitumor effect in vivo HUANG Xiaoping ZHANG Jing ZHANG Minping LIU Bin YANG Jingjing WEN Qiang LUO Fanghong ZHUANG Guohong Anti-Cancer Research Center Medical College of Xiamen University Xiamen 361005 China [Abstract] In order to improve the effect of anti -human death receptor 5 single -chain antibodies in the treatment of liver cancer we assayed the therapeutic effect and mechanism of GCS-aDR 5 ScFv therapy in mouse hepatoma 22 (H22) model. GCS-aDR 5 ScFv was prepared and confirmed by electron microscopy and laser particle size analysis instrument then used to immunize H22 mouse every other day at dosage of 0.272 mg/ml for 2 weeks. We found that the body weight was improved but the tumor volume/weight decreased in immunized mice as compared with control mice. Furthermore Western blot showed that GCS-aDR 5 ScFv treatment could elevated the expression levels of active -caspase8 active -caspase3 and BAX in H22 mice. In conclusion GCS -adr 5 ScFv posses anti -tumor effects in H22 mice and its mechanism may related with upregulation of active -caspase8 active-caspase3 and BAX protein expression. [Key words] GCS-aDR 5 ScFv; H22; Anti-tumor effect [2] TRAIL(TNF [3] related apoptosis inducing ligand) 5 H22 [4] [1] DR4(death receptor 4) DR5(death receptor 5) [5] TRAIL : (81072472); (3502Z20124045) : 361005 ( );361021 ( ) * : E-mail: zhuangguohong@yahoo.com.cn; Email: luofanghong@163.com Δ [6] GCS-aDR 5 ScFv
751 GCS-aDR 5 ScFv 8 : ( PBS) GCS adr 5 ScFv H22 GCS-aDR 5 ScFv 200 μl 0.272 mg/ml GCS adr 5 ScFv GCS-aDR 5 ScFv 1 1.1 6~8 adr 5 ScFv 1.6.2 1 (glycol chitosangcssigma 82 100 89.2% ); ( ) : / (TPP) 6 ( )LEO1530 SEM ( 1.7 HE LEO ) SDS-PAGE BIO- RED RIPA ( 5 min; 10 min; 1% ) active- 1 min; 1 caspase8 active-caspase3 BAX Santa adr 5 ScFv 1.3 GCS GCS-aDR 5 ScFv GCS 500 μl RIPA 0.45 μm SDS-PAGE 2 mg/ml TPP PVDF PVDF 5% 10 mg/ml 2 h TBST 3 5 min Ag-Ab : 2 h TBST 3 5 min; HRP 4 ml GCS 2 h TBST 3 5 min ECL 25 ml 1.9 SPSS18.0 adr 5 ScFv ph 4.5 20~40 / t P < 0.05 min TPP 20 min 2 1.4 GCS GCS-aDR 5 ScFv Zeta 3 adr 5 ScFv 30 000 95% adr 5 ScFv Zeta 1.6 1.6.1 1% 30 s; 1 min; 20~ 1.2 adr 5 ScFv DR5 30 s; 1 min; 1 min;70 % 5 min; 80 % 5 min;95 % 5 min 2 ; 5 min [7] 2 ; 5 min 2 ; ; 1.8 Western blot Active-caspase8 activecaspase3 BAX 0.1 g 2.1 adr 5 ScFv adr 5 ScFv adr 5 ScFv 1 1.5 H22 2.2 GCS GCS-aDR 5 ScFv 2a H22 H22 GCS 0.01 mol/l PBS (ph7.2) 2b GCS-aDR 5 ScFv 1 10 7 /ml 1 ml 32 6~8 ( ) 1 10 6 ( 0.1 ml) 2.3 GCS GCS-aDR 5 ScFv Zeta 32 4 1 GCS 181.2 nm
752 2.4.2 : /6 [8] 14 H22 ( 3b) adr 5 ScFv GCS - adr 5 ScFv M) Marker; 1-3) respectively purified batches adr 5ScFv. 1 adr 5 ScFv Western blot Fig 1 Identification of adr 5 ScFv by Western blotting a) The shape of the GCS detected by SEM; b) The shape of the GCS-aDR 5ScFv tested by SEM. 2 GCS-aDR 5 ScFv GCS 203.7 nm 10~1 000 nm GCS-aDR 5 ScFv ( 4) Zeta GCS-aDR 5 ScFv Zeta GCS GCS-aDR 5 ScFv 2.4 2.4.1 1 1 4 HE ( 200) Fig 4 Hematoxylin-eosin staining of the tumor tissue( 200) ( 3a) 4 2.6 Western blotting active-caspase8 active- BAX Western blotting 5 12 GCS-aDR 5 ScFv caspase3 8.2 g PBS 2.5 g active-caspase8 active-caspase3 BAX (P < 0.05) GCS GCS-aDR 5 ScFv adr 5 ScFv activecaspase8 active-caspase3 BAX GCS GCS-aDR 5 ScFv Fig 2 The shape of the GCS and GCS-aDR 5 ScFv detected by SEM 1 Tab 1 Characteristics of the GCS nanoparticles Nanoparticles Average particle size(nm) Zeta potential (mv) Polydispersity index GCS 181.2 19.5 0.107 GCS-aDR 5 ScFv 203.7 24.2 0.203 GCS-aDR 5 ScFv adr 5 ScFv a) Comparison of body weight in H22 tumor bearing mice of each group; b) weight of mice tumor 14 days after treatment and volume of mice tumor treated per day. 3 Fig 3 Comparison of mouse body weight tumor height and tumor volume in H22 tumor bearing mice of each group 2.5 HE HE GCS-aDR 5 ScFv adr 5 ScFv GCS 5
753 3 DR5 BAX adr 5 ScFv DR5 DR5 active -caspase8 active - [9] DR5 caspase3 BAX DR5 GCS [13] GCS-aDR 5 ScFv GCS GCS-aDR 5 ScFv Zeta GCS-aDR 5 ScFv GCS GCS-aDR 5 ScFv GCS-aDR 5 ScFv Western blotting active-caspase8 activecaspase3 BAX GCS-aDR 5 ScFv adr 5 ScFv activecaspase8 active-caspase3 BAX adr 5 ScFv GCS-aDR 5 ScFv activecaspase3 BAX GCSaDR 5 ScFv active-caspase8 active-caspase3 BAX 5 Western blotting GCS-aDR 5 ScFv raav-s3c H22 active-caspase8 active-caspase3 [11] BAX DR5 Fig 5 Western blot analysis of active-caspase8 active-caspase3 TRAIL c-flip XIAP BCL-2 [12] and BAX expressions in GCS-aDR 5 ScFv-treated H22 mouse DISC caspase8 TRAIL active -caspase8 active -caspase3 H22 GCS GCS-aDR 5 ScFv [1] Lv F Qiu Y Zhang Y et al. Adeno-associated virus-mediated anti-dr5 chimeric antibody expression suppresses human 4 GCS -adr 5 ScFv tumor growth in nude mice[j]. Cancer Lett 2011 302(2): 119- adr 5 ScFv (P < 0.05)GCS GCS [2] 127. Lee SH Park DW Sung ES et al. Humanization of an [10] agonistic anti -death receptor 4 single chain variable GCS-aDR 5 ScFv adr 5 ScFv (P < 0.05) fragment antibody and avidity-mediated enhancement of its cell death-inducing activity[j]. Mol Immunol 2010 47(4): 816-824. GCS-aDR 5 ScFv [3]. 5 HE ZF1 H22 [J]. 2010 26(8):801-805. GCSaDR [4] Manning MC Patel K Borchardt RT. Stability of protein 5 ScFv pharmaceuticals[j]. Pharm Res 1989 6(11): 903-918. [5] Nam HY Kwon SM Chung H et al. Cellular uptake GCS-aDR 5 ScFv adr 5 ScFv mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles[j]. J Control Release 2009 135(3): 259-267. GCS-aDR 5 ScFv [6] Park JH Kwon SG Nam JO et al. Self -assembled nanoparticles based on glycol chitosan bearing 5β-cholanic acid for RGD peptide delivery[j]. J Control Release 2004 95(3): 579-588. [7]. DR5 [J]. 2009 25(6): 671-
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