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目的 :实现绿色荧光蛋白 (greenfluorescentprotein ,GFP)报告基因在骨髓基质细胞 (BMSC)中的高效、稳定、持久表达 ,直接示踪BMSC在关节软骨缺损内的分布及分化。方法 :构建重组逆转录病毒表达载体RV GFP ,通过PT6 7包装细胞克隆、G4 18筛选及扩增 ,获得大量含GFP基因的假病毒液 ,并直接转染BMSC。将含有GFP基因转染标记的BMSC与生物可降解材料复合后 ,植入猪自体关节软骨缺损处 ,7个月后共聚焦显微镜检测修复组织中GFP标记的BMSC的分布及分化。结果 :经双酶切鉴定证实重组逆转录病毒表达载体RV GFP构建成功 ,转染PT6 7细胞后可在荧光激发波长下 ,发出明亮的绿色荧光 ,转染率达 2 0 %～ 5 0 % ,经G4 18筛选后可达 10 0 %。筛选、扩增后 ,PT6 7细胞的上清培养液可成功地转染BMSC ,筛选后能稳定、持久、高效表达GFP。将标记的BMSC植入关节软骨缺损 7个月后 ,修复组织仍能高效表达GFP ,激光共聚焦显微镜下显示 ,多数新生软骨陷窝内有GFP标记的细胞。结论 :构建的重组逆转录病毒GFP载体 ,能持久标记骨髓基质细胞 ,用于细胞动态变化的研究及细胞转归的示踪 ,该方法简便、灵敏、可靠、直观。标记的BMSC可在关节软骨缺损内分化为成熟的软骨细胞 ,并在软骨缺损的修复中发挥重要作用
OBJECTIVE: To achieve efficient, stable and sustained expression of green fluorescent protein (GFP) reporter gene in bone marrow stromal cells (BMSCs), to directly observe the distribution and differentiation of BMSCs in articular cartilage defects. Methods: Recombinant retroviral vector RV GFP was constructed and cloned by PT6 7. G418 was selected and amplified. A large number of pseudovirions containing GFP gene were obtained and directly transfected into BMSCs. The BMSC labeled with GFP gene was composted with biodegradable material and implanted into the autologous articular cartilage defects in pigs. Seven months later, the distribution and differentiation of GFP-labeled BMSCs in the repaired tissues were detected by confocal microscopy. Results: The recombinant retroviral vector RV GFP was successfully constructed by double enzyme digestion. After transfected into PT6 7 cells, it could emit bright green fluorescence under the fluorescence excitation wavelength with the transfection rate of 20% ~ 50% After G4 18 screening up to 10%. After screening and amplification, the supernatant medium of PT6 7 cells can be successfully transfected into BMSC, which can stably, stably and efficiently express GFP after screening. Seven months after the labeled BMSCs were implanted into articular cartilage defects, the repaired tissues could still express GFP efficiently. Confocal laser scanning microscopy showed GFP-labeled cells in most of the nascent cartilage lacunae. CONCLUSION: The recombinant retrovirus GFP vector can permanently label bone marrow stromal cells for the study of cell dynamic changes and tracing of cell turnover. The method is simple, sensitive, reliable and intuitive. Labeled BMSC can differentiate into mature chondrocytes within the articular cartilage defect and play an important role in the repair of cartilage defects