1472 Journal of Experimental Hematology 2011 19 6 1472-1476 Article ID 1009-2137 2011 06-1472 - 05 1 510120 1 201102 BMMSC BMMSC GFP BMMSC BMMSC 4 BMMSC CD29 CD44 CD34 CD45 BMMSC BMMSC GFP BMMSC BMMSC GFP R392. 28 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 510120 Guangdong Province China 1 Department of Digestive System Children's Hospital of Fudan University Shanghai 201102 China Corresponding Author FANG Jian-Pei Professor. Tel 020 81332213. E-mail 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 2011 19 6 1472-1476 bone marrow mesenchymal 3 4 BMMSC stem cells BMMSC 5 BMMSC BMMSC 2010B060900027 1 2. 020 81332213. E-mail JPfang2005 BMMSC @ 163. com 1 /10 4-1 /10 5 2011-08 - 10 2011-08 - 25
BMMSC virus titre 1 10 8 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 -0. 02% EDTA PBS 2 1 80% 1 10 4 / 10 6 /ml Ep 24 1-8 3 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 4 6 1 10 4 /cm 2 2 10 5 / 2 ml / 37 5% CO 2 70% - 80% 2 ml / SD 4-6 100-150 g β- DMEM /F12 DMEM /F12 10% 0. 25% 3 3-0. 02% EDTA 100 U /ml / BMMSC Gibco CD29 BioLegend 4 6 1 CD44 Serotec CD45 BioLegend 10 4 /cm 2 2 10 5 / 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 -3- - 3 B 24 GFP lentivirus BMMSC 3 90% 2 10 5 / 250 g 10 PBS 6 40% - 1 2-3 ml 60% 1 ml 5 10 5 /cm 2 25 cm 2 1 ml 6 mg /ml 37 5% CO 2 polybrene 1 μl polybrene P03 3 15 25 35 μl 2-3 1 1 12-15 multiplicity of infection MOI 80% - 90% 16 1 2 25 cm 2 48 37 5% 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 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 2011 19 6 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. 12 5-BrdU DAPI CM -DiI CFSE 13 14 BMMSC GFP 27 kd 238 65-67 20-25 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 10 11 MSC 1 Sensebé L Krampera M Schrezenmeier H et al. Mesenchymal
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 2011 19 6 stem cells for clinical application. Vox Sang 2010 98 2 93-107 2 Uccelli A Moretta L Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008 8 9 726-736 3 Dazzi F Ramasamy R Glennie S et al. The role of mesenchymal stem cells in haemopoiesis. Blood Rev 2006 20 3 161-171 4 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 2005 52 8 2521-2529 5 Hong L Peptan I Clark P et al. Ex Vivo Adipose tissue engineering by human marrow stromal cell seeded gelatin sponge. Ann Biomed Eng 2005 33 4 511-517 6 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 2004 103 5 1662-1668 7 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 2007 179 3 1855-1863 8 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 2006 8 4 315-317 9 Horwitz EM Le Blanc K Dominici M et al. Clarification of the nomenclature for MSC The International Society for Cellular Therapy position statement. Cytotherapy 2005 7 5 393-395 10 Hoogduijn MJ Crop MJ Peeters AM et al. Human heart spleen and perirenal fat-derived mesenchymal stem cells have immunomodulatory capacities. Stem Cells Dev 2007 16 4 597-604 11 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 2006 24 2 376-385 12 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 2008 24 1 69-76 13.. CM-DiI. 2008 7 12 1-3 14. CFSE. 2009 29 1 148-150 15 Chalfie M Tu Y Euskirchen G et al. Green fluorescent protein as a marker for gene expression. Science 1994 263 5148 802-805 檹 檹 檹 檹 檹 檹 檹 檹 檹 檹 檹 檹 檹 檹 檹 Student t 2011 19 5 1230-1233 PTEN AGM Student t p < 0. 05 Student t p < 0. 05 2011 10 26