Ficoll. MTT Sigma 1. 3

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Βαρνάβας Καραμήτσος
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1 776 PLCL / PLCL / poly L-lactide-co-ε-caprolactone PLCL PLCL / SEM PLCL / PLCL / bone marrow mesenchymal stem cells BMSCs MTT 1 PLCL / 305 ± 87 nm ± PLCL / 2 kg 1. 2 PLCL R R A Ficoll DMEM /F12 Hyclone CD29-FITC / CD44- FITC CD45-PE HLA-DR-PE 100 ebioscience 0. 25% 1-2 MTT Sigma DMSO Amresco 3 Olympus Thermo Multiskan FC Kato-Tech M mail huhejie@ hotmail. com E- Sigma-Aldrich HFIP Stereoscan 360 SEM Cambridge Dataphysics BMSCs action PCR. The mir-194 gene was cloned into PIRESneo3 vector directionally after enzyme cut thus constructing the eukaryotic expression plasmid of PIRESneo3 / mir-194. Then the recombinant PIRESneo3 / mir-194 plasmid was transfected into the MCF-7 cell line by Lipofectamine and mir-194 highly expressed cells was selected by G μg /ml. The expression level of mir-194 was detected by real-time quantitative PCR. Results The recombinant PIRESneo3 /mir-194 plasmid was constructed and the MCF-7 cell line with highly mir-194 gene was screened. Conclusion mir-194 highly expressed MCF-7 cell line is established sucessfully which will be a perfect target of mir-194 gene therapy and for observing its function in breast cancer cells. Key words mirna transfection breast cancer plasmid

2 777 Ficoll 40 ImageJ NIH /ml 5 25 cm μl / ml 20% FBS 100 U /ml 100 U /ml DMEM /F % CO 2 48 h 75% 30 min PBS 2 ~ 3 d min 24 h 80% 96 1 ~ μl 80% 3 BMSCs CD29 CD44 CD45 HLA-DR /ml 100 μl BMSCs 4 12 h 20 μl MTT 5 mg /ml h PBS μl DM /ml μl SO 10 min 24 h SO 10 min 490 nm d 9 d d OD /ml HFIP 1. 4 SPSS % w /v PLCL HFIP DMEM /F12 x 珋 ± s Student t mg /ml 2 PLCL BMSCs h ml h 20 kv PLCL / 2 ml /h cm 20 cm 1A 15 cm 500 r /min 1B 5 ~ 6 1C 10 mm /min 7 ~ 8 90% 1D 48 h 1E BMSCs CD29 CD44 CD45 μl MTT 5 mg /ml 37 5% CO 2 OD h PBS μl DM PLCL / HLA-DR CD29 CD44 Ste- CD45 HLA-DR reoscan 360 SEM BMSCs 490 nm h OD 490 2

3 778 1 BMSCs BMSCs CD29 CD44 CD45 HLA-DR 3 BMSCs 2. 2 BMSCs 1 3 BMSCs 305 ± 87 nm ± BMSCs 4 12 h ku 6 BMSCs 7 BMSCs BMSCs S ~ 3 3 ~ 7 3 S 2. 3 PLCL 2 / 4 PLCL 8% w 3 ~ 5 /v 100 mg /ml 2 1 6

4 779 4 PLCL / A ~ D E 5 3 BMSCs 3 BMSCs 14 PLCL / BMSCs CD29 CD44 CD90 CD105 BMSCs 8 - CD14 CD34 CD45 CD11b 9 BMSCs 1 ~ 4 CD29 CD44 CD45 HLA-DR CD29 CD44 CD45 HLA-DR BMSCs ± 87 nm 50 ~ 500 nm ± PLCL 14 PLCL 15 1 Chung S Ingle N P Montero G A et al. Bioresorbable elasto-

5 780 meric vascular tissue engineering scaffolds via melt spinning and electrospinning J. Acta Biomater Uttayarat P Perets A Li M et al. Micropatterning of three-dimensional electrospun polyurethane vascular grafts J. Acta Biomater Aquirre A Planell J A Engel E. Dynamics of bone marrow-derived endothelial progenitor cell / mesenchymal stem cell interaction in co-culture and its implications in angiogenesis J. Biochem Biophys Res Commun Hang C Chen R Ke Q et al. Electrospun collagen-chitosan- TPU nanofibrous scaffolds for tissue engineered tubular grafts J. Colloids Surf B Biointerfaces Soletti L Hong Y Guan J et al. A bilayered elastomeric scaffold for tissue engineering of small diameter vascular grafts J. Acta Biomater Sell S A McClure M J Garg K et al. Electrospinning of collagen / biopolymers for regenerative medicine and cardiovascular tissue engineering J. Adv Drug Deliv Rev Kitano Y Radu A Shaaban A et al. Selection enrichment and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cycling adherent bone marrow cells J. Exp Hematol Wulf G G Jackson K A Goodell M A. Somatic stem cell plasticity current evidence and emerging concepts J. Exp Hematol Lee J W Gupta N Serikov V et al. Potential application of mesenchymal stem cells in acute lung injury J. Expert Opin Biol T- her Ju Y M Choi J S Atala A et al. Bilayered scaffold for engineering cellularized blood vessels J. Biomaterials Chung S Moghe A K Montero G A et al. Nanofibrous scaffolds electrospun from elastomeric biodegradable poly L-lactide-co-ε-caprolactone copolymer J. Biomed Mater Jeong S L Kim S Y Cho S K et al. Tissue-engineered vascular grafts composed of marine collagen and PLGA fibers using pulsatile perfusion bioreactors J. Biomaterials Zhao H Ma L Gong Y et al. A polylactide /fibrin gel composite scaffold for cartilage tissue engineering fabrication and an in vitro evaluation J. J Mater Sci Mater Med Fang Z D Fu W G Dong Z H et al. Preparation and biocompatibility of electrospun poly L-lactide-co-ε-caprolactone /fibrinogen blended nanofibrous scaffolds J. Appl Surf Sci Ku S H Park C B. Human endothelial cell growth on mussel-inspired nanofiber scaffold for vascular tissue engineering J. Biomaterials In vitro biocompatibility between electrospun nanofibrous films of PLCL / fibrinogen and rabbit bone marrow mesenchymal stem cells Jin Liang Hu Hejie Fang Zhengdong Dept of General Surgery The Affiliated Provincial Hospital of Anhui Medical University Hefei Abstract Objective To investigate the biocompatibility between electrospun nanofibrous films of PLCL / fibrinogen nanofibrous films and rabbit bone marrow mesenchymal stem cells BMSCs in vitro. Methods Electrospun nanofibrous films were fabricated from poly L-lactide-co-ε-caprolactone PLCL and characterized by scanning electron microscopy SEM and water contact angle analyzer. Take the cells seeded on PLCL /fibrinogen nanofibrous films as experimental group and seeded on polystyrene culture plate as control group. The adhesion and proliferation results of rabbit BMSCs in two groups were detected by MTT assay. Results The cells used in this assay had the characteristics of mesenchymal stem cells MSCs. The average diameter and water contact angle of PLCL / fibrinogen nanofibrous was 305 ± 87 nm and ± respectively. Compared with control group rabbit BMSCs in experimental group were exhibited similar adhesion and proliferation. Conclusion Electrospun PLCL / fibrinogen nanofibrous films have good performance in biocompatibility aspect and great potential applications in vascular tissue engineering field. Key words electrospinning nanofibrous tissue engineering artificial vascular mesenchymal stem cells

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