BMMSC virus titre TU /ml Cyagen BMMSC A DMEM / PBS F12 2 O 3 5 ml DMEM /F12 10% 100 U /ml 1473 BMMSC 90% 4 BMMSC 0. 25% 4 BMMSC BMMSC % E

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1 1472 Journal of Experimental Hematology Article ID BMMSC BMMSC GFP BMMSC BMMSC 4 BMMSC CD29 CD44 CD34 CD45 BMMSC BMMSC GFP BMMSC BMMSC GFP R A Culture In Vitro and Lentivirus Transfection of Rat Mesenchymal Stem Cells ZHANG Hong-Shan FANG Jian-Pei SU Hao-Bin YANG Min 1 Department of Pediatric Sun Yat-Sen Memorial Hospital Sun Yat-Sen University Guangzhou Guangdong Province China 1 Department of Digestive System Children's Hospital of Fudan University Shanghai China Corresponding Author FANG Jian-Pei Professor. Tel JPfang2005@ 163. com Abstract This study was purposed to establish the methods for isolation culture identification and labeling of bone marrow mesenchymal stem cells BMMSC so as to provide quantified seed cells for cell transplantation. Bone marrow was collected from SD rat by flushing femur and tibias under sterile condition and BMMSC were purified by adherent culture and amplified in vitro. The immunophenotypes of BMMSC were identified by flow cytometry the ability of differentiation to osteogenic and adipogenic lineages was detected by alizarin red and oil red O respectively. The BMMSC were transfected by using lentivirus with green flurescence protein GFP gene so as to determine GFP expression in BMMSC. The results demonstrated that the method of adherent culture could effectively isolate and purify rat BMMSC which displayed homogenous fibro-like morphology. The flow cytometry showed that BMMSC expressed CD29 CD44 not expressed CD34 CD45. The BMMSC could differentiated into osteoblasts and adipocytes two mesenchymal lineages when grown in specific medium for each lineage. After being transfected by lentivirus BMMSC could express GFP. It is concluded that the adherent culture is simple effective feasible method to separate MSC from the bone marrow of adult rats the separated and cultured cells exhibit the biological characteristics of BMMSC and differentiating potential. BMMSC can express GFP efficiently and stably in vitro after being transfected by lentivirus which can be used to label cells for tracing in vivo. Key words bone marrow mesenchymal stem cell lentivirus transfection adherent culture J Exp Hematol bone marrow mesenchymal 3 4 BMMSC stem cells BMMSC 5 BMMSC BMMSC 2010B JPfang com 1 / /

2 BMMSC virus titre TU /ml Cyagen BMMSC A DMEM / PBS F12 2 O 3 5 ml DMEM /F12 10% 100 U /ml 1473 BMMSC 90% 4 BMMSC 0. 25% 4 BMMSC BMMSC % EDTA PBS % / 10 6 /ml Ep PE anti-rat /mouse CD29 PE anti- rat CD44 PE anti-rat CD45 PE anti-rat CD34 PE anti-rat CD11b 4 30 PBS 6 BMMSC BMMSC BMMSC /cm / 2 ml / 37 5% CO 2 70% - 80% 2 ml / SD g β- DMEM /F12 DMEM /F12 10% 0. 25% % EDTA 100 U /ml / BMMSC Gibco CD29 BioLegend CD44 Serotec CD45 BioLegend 10 4 /cm / 2 ml / CD34 Santa Cruz SD 37 5% CO BMMSC 2 80% - 90% 2 ml / Cyagen Polybrene Sigma GFP lentivirus A 1- lentiviral vector plv /EX2D-Neo-EF1A-eGFP B 24 GFP lentivirus BMMSC 3 90% / 250 g 10 PBS 6 40% ml 60% 1 ml /cm 2 25 cm 2 1 ml 6 mg /ml 37 5% CO 2 polybrene 1 μl polybrene P μl multiplicity of infection MOI 80% - 90% cm % CO 2 BMMSC BMMSC

1474 结果细胞培养及诱导分化骨髓细胞接种于培养瓶后在培养体系中以悬浮折 1 2 天后可见有极少量圆光性强的圆形细胞为主形细胞贴壁 72 小时换液可见少量贴壁细胞单个或多个散在出现形态不规则有长梭形多角形等原代培养约 15 天左右细胞可达 90% 以上融合主要成纺锤形细胞整体排列更趋于规律性呈漩涡状排列此时开始传代扩增传代后的细胞贴壁及生长更加迅速

3 1474 结果细胞培养及诱导分化骨髓细胞接种于培养瓶后在培养体系中以悬浮折 1 2 天后可见有极少量圆光性强的圆形细胞为主形细胞贴壁 72 小时换液可见少量贴壁细胞单个或多个散在出现形态不规则有长梭形多角形等原代培养约 15 天左右细胞可达 90% 以上融合主要成纺锤形细胞整体排列更趋于规律性呈漩涡状排列此时开始传代扩增传代后的细胞贴壁及生长更加迅速 24 内细胞完全贴壁伸展随着传代次数的增多细胞形态逐渐单一呈成纤维细胞样形态图 1A 大鼠 BM M SC 经成骨诱导后细胞体积增大呈多角形诱导 2 周后胞质内充满颗粒细胞间可见钙质沉积诱导 3 周后钙结节形成明显经茜素红染色呈红色结节图 1B 成脂诱导后细胞变为圆形或多边形细胞内有小脂滴出现诱导 2 周后脂滴数量增加并相互融合油红 O 染色显示有大量脂质沉淀图 1C 中国实验血液学杂志 J Exp Hematol CD44 表达阳性 CD45 表达阴性图 2 而 CD34 Figure 2 Expre ssion of rat BMMSC marke rs 大鼠 BMMSC 的慢病毒转染从 15 μl 的病毒添加量开始转染率能达到 90% 以上最佳 MOI 为 12第 3 代 BMMSC 用慢病毒转染后 48 小时细胞表达 GFP 荧光倒置显微镜下可观察到细胞表达绿色荧光转染后的细胞形态与转染前相细胞生长情况良好随着体外培养时间的延长传同代次数的增加荧光强度未见明显变化图 3 Figure 1 Morphology and diffe re ntiation of rat BMMSC unde r light microscope A fourth passage of rat BM M SC 40 B osteogenic differentiation alizarin red 100 C adipogenic differentiation oil red O 200 Figure 3 Morphology of BMMSC transfe cte d by le ntivirus unde r inve rte d fluore sce nce microscope 100 A morphology of BM M SC transfected by lentivirus for 48 hours B morphology of BM M SC transfected by lentivirus for 4 w eeks 大鼠 BMMSC 的表型流式细胞术检测结果显示第 4 代 BMMSC CD29 及 BMMSC 生长曲线生长曲线显示慢病毒转染的 BM M SC 与正常的

1475 BMMSC 1 2 3 4 6 7 CD34 CD45 BMMSC CD29 CD44 BMMSC BMMSC CD29 CD44 4 CD34 CD45 BMMSC BMMSC 5-5-BrdU Figure 4. Growth curve of rat BMMSC.

4 1475 BMMSC CD34 CD45 BMMSC CD29 CD44 BMMSC BMMSC CD29 CD44 4 CD34 CD45 BMMSC BMMSC 5-5-BrdU Figure 4. Growth curve of rat BMMSC BrdU DAPI CM -DiI CFSE BMMSC GFP 27 kd BMMSC Ser-Tyr-Gly 3 7 enhenced green BMMSC fluorescent protein EGFP BMMSC BMMSC BMMSC 15 MSC BMMSC EGFP BMMSC BMMSC 3 EGFP 6-7 BMMSC 15 90% BMMSC EGFP BMMSC BMMSC BMMSC BMMSC 8 9 MSC MSC BMMSC SH2 SH3 Sca1 CD29 CD44 CD71 CD90 CD120a CD124 CD34 CD45 CD14 CD11b CD80 CD86 HLA-Ⅱ MHC-Ⅱ MSC 1 Sensebé L Krampera M Schrezenmeier H et al. Mesenchymal

5 檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹 1476 J Exp Hematol stem cells for clinical application. Vox Sang Uccelli A Moretta L Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol Dazzi F Ramasamy R Glennie S et al. The role of mesenchymal stem cells in haemopoiesis. Blood Rev Sakaguchi Y Sekiya I Yagishita K et al. Comparison of human stem cells derived from various mesenchymal tissues superiority of synovium as a cell source. Arthritis Rheum Hong L Peptan I Clark P et al. Ex Vivo Adipose tissue engineering by human marrow stromal cell seeded gelatin sponge. Ann Biomed Eng Peister A Mwllad JA Larson BL et al. Adult stem cells from bone marrow MSCs isolated from different strains of inbred mice vary in surface epitopes rates of proliferation and differentiation potential. Blood Gupta N Su X Popov B. Intrapulmonary delivery of bone marrow -derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice. Immunol Dominici M Le Blanc K Mueller I et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy Horwitz EM Le Blanc K Dominici M et al. Clarification of the nomenclature for MSC The International Society for Cellular Therapy position statement. Cytotherapy Hoogduijn MJ Crop MJ Peeters AM et al. Human heart spleen and perirenal fat-derived mesenchymal stem cells have immunomodulatory capacities. Stem Cells Dev Mitchell JB McIntosh K Zvonic S et al. Immunophenotype of human adipose-derived cells temporal changes in stromalassociated and stem cell-associated markers. Stem Cells Kobayashi T Ochi M Yanada S et al. A novel cell delivery system using magnetically labeled mesenchymal stem cells and an external magnetic device for clinical cartilage repair. Arthroscopy CM-DiI CFSE Chalfie M Tu Y Euskirchen G et al. Green fluorescent protein as a marker for gene expression. Science 檹檹檹檹檹檹檹檹檹檹檹檹檹檹檹 Student t PTEN AGM Student t p < Student t p <

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無投影片標題幹細胞和臍帶血的介紹中國醫藥大學附設醫院兒童醫院小兒血液腫瘤科主任巫康熙醫師幹細胞具有兩大特色一. 有自行再生的能力幹細胞自行再生的能力, 使它可以不斷產生與自己一模一樣的後代二. 有分化成各種細胞的潛能幹細胞