骨髓间充质干细胞经慢病毒转染绿色荧光蛋白后的体内观察.doc

骨髓间充质干细胞经慢病毒转染绿色荧光蛋白后的体内观察 　　作者：樊守刚，吴小涛，王运涛，李果 作者单位：(东南大学附属中大医院 骨科，江苏 南京 210009) 　　【摘要】 目的:观察经慢病毒转染绿色荧光蛋白(green fluorescence protein，GFP)的骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMSCs)移植注入兔椎间盘后的荧光时效性。方法:体外培养并纯化BMSCs，并用含有GFP标记的重组慢病毒载体(LentivirusGFP)转染后移植入兔椎间盘，在移植后2、4、6、8、10、12周用激光共聚焦显微镜观察BMSCs荧光强度及细胞增殖情况，考察慢病毒转染GFP体内荧光维持时间。结果:经慢病毒转染GFP后的BMSCs，荧光显微镜下48 h开始显现绿色荧光，72 h荧光亮度达到最高。植入体内后8周内均能用激光共聚焦显微镜观察到绿色荧光,8周后荧光强度开始逐渐减弱，第2、4、6、8、10、12周6个时间点GFPBMSCs的阳性率分别是(21.75±1.32)%、(33.07±3.53)%、(33.20±3.60)%、(29.90±4.23)%、(19.64±2.93)%和(10.91±3.43)%。结论:BMSCs在椎间盘微环境中能够稳定增殖，用慢病毒转染GFP的方法能在至少8周内可靠示踪其在椎间盘内的存活状态，8周后荧光强度开始逐渐减弱。 　　【关键词】 椎间盘退变; 骨髓间充质干细胞; 慢病毒; 绿色荧光蛋白 　　Tracing of bone marrow mesenchymal stem cells with green fluore 　　scence protein labelled in rabbit intervertebral disc 　　FAN Shougang, WU Xiaotao, WANG Yuntao, LI Guo(Department of Orthopaedics, Zhongda Hospital, Southeast University, Nanjing 210009, China ) 　　[Abstract] Objective: To observe the fluorescence timeliness of bone marrow mesenchymal stem cells(BMSCs) transfected green fluorescent protein(GFP) in the rabbit intervertebral disc after transplanted. Methods: BMSCs were cultured and purified, and transfected with GFPcontained recombinant lentiviral vector(LentivirusGFP) in vitro. The fluorescence intensity of BMSCs and cell proliferation were studied after transplanted into the rabbit intervertebral disc in vivo by confocal laser significantly microscope. Results: Green fluorescence of GFP in BMSCs was collected 48 hours after transfected with GFP, and got the highest fluorescent light at 72hour. In vivo green fluorescence could be obtained by confocal laser microscope within 8 weeks after implantation, and the fluorescence intensity began to weaken after 8 weeks. The positive rates of GFPBMSCs at 2,4,6,8,10,12 weeks were(21.75±1.32)%,(33.07±3.53)%,(33.20±3.60)%,(29.90±4.23)%,(19.64±2.93)% and(10.91±3.43)%. Conclusion: The stabile proliferation of BMSCs can be obtained in the inter