目的体外研究绿色荧光蛋白（GFP）基因转染骨髓间充质干细胞（MSCS）的方法，并检测基因转染后细胞的生物学特性及分化潜能。方法体外分离培养大鼠MSCS，用重组GFP的腺病毒载体Ad-GFP及脂质体介导质粒PEGFP-C1两种方法转染GFP，流式细胞术观察比较基因转染和表达结果；将腺病毒介导的基因转染细胞经成骨定向诱导后，检测碱性磷酸酶表达和钙结节形成情况。结果重组Ad-GFP对细胞的感染率达（4113±114）%，感染6周后仍有表达；脂质体组转染效率最高为1215%。病毒感染组阳性细胞与未感染组细胞经成骨诱导分化后碱性磷酸酶表达均逐渐增高；诱导3周后茜素红钙盐染色均为阳性。结论重组GFP的腺病毒载体Ad-GFP可高效感染MSCS，基因转染后细胞的增殖分化能力与未转染细胞差异无统计学意义（P>0105），可以作为一种高效的大鼠MSCS的标记方法。

目的研究绿色荧光蛋白（GFP）转基因小鼠骨髓间充质干细胞（MSCS）体外定向诱导下的多向分化潜能。方法取6周龄GFP转基因小鼠1只，用密度梯度离心法结合贴壁法体外培养获得GFP转基因小鼠骨髓MSCS有限细胞系（GFP-MSCS）。取第3代GFP-MSCS进行体外多向分化诱导：成骨诱导后进行碱性磷酸酶增殖活性检测及茜素红钙盐染色;成脂肪诱导20d后进行油红O染色鉴定;成神经诱导6h后用免疫组织化学方法检测神经元烯醇化酶（NSE）的表达。结果GFP-MSCS经成骨诱导后ALP表达较对照组明显升高，20d后茜素红染色可见有橘红色钙盐沉积;经成脂诱导后油红O染色阳性;经成神经诱导后，细胞形态由梭形转变为星状细胞，NSE染色呈强阳性表达。结论GFP转基因小鼠骨髓MSCS在体外诱导下具有多向分化能力，对GFP稳定表达无影响，可以作为研究MSCS多向分化潜能机制的一个有效示踪工具。

目的:分离人体脂肪基质细胞并诱导培养其成骨表型。方法:将脂肪抽吸术获取的人体脂肪进行机械分割，通过Ⅰ型胶原酶消化后得到脂肪基质细胞，在BGJb培养基中原代培养10d，消化传代后用含体积分数10%FBS及10-8mol/L地塞米松，50mg/L左旋抗坏血酸，10nmol/L维生素D3，10mmol/Lβ2磷酸甘油钠的DMEM/F12培养基中诱导培养14d，观察细胞形态，绘制细胞生长曲线并对其成骨表型进行鉴定。结果：脂肪基质细胞呈成纤维细胞样贴壁生长，其中的成体干细胞经诱导培养后体积明显增大，胞核大面圆，胞浆丰富，群体倍增时间为66h。Gomori萘酚磷酸酯法染色显示其胞浆内富含碱性磷酸酶颗粒，vonKossa染色表明聚集的细胞团能形成矿化结节。结论：脂肪基质细胞中的成体干细胞经过矿化诱导培养后可向成骨细胞分化，并具有明显的成骨表型。

目的研究分离的人体脂肪基质细胞经过诱导培养后向骨骼肌细胞分化的潜能。方法通过脂肪抽吸术获取健康成人脂肪，机械分割后用Ⅰ型胶原酶消化，得到脂肪基质细胞。将脂肪基质细胞在BGJb培养基中原代培养10d，在DMEM/F12培养基中进行成肌诱导培养20d，获得细胞爬片。细胞爬片经4%多聚甲醛固定后，用甲苯胺蓝、Mallory磷钨酸苏木素染色观察细胞形态；Myosin单克隆抗体免疫细胞化学染色观察肌球蛋白表达情况。结果经过成肌诱导培养的细胞与常规培养的脂肪基质细胞在形态上有明显的差异，表现为细胞体积增大，单个核的细胞融合形成多核的肌管样结构，胞浆内细胞器发达，肌浆网扩张并形成肌原纤维骨骼肌特异性的Myosin表达阳性。未经诱导的脂肪基质细胞在相同时间点无此现象。结论人类脂肪基质细胞中存在着成体干细胞，在成肌诱导培养条件下具有向骨骼肌细胞分化的潜能。

Musculoskeletal tissues regeneration requires rapid expansion of seeding cells both in vitro and in vivo while maintaining their multilineage differentiation ability. Human adipose-derived stem cells (ASCs) are considered to contain multipotent mesenchymal stem cells. Monolayer cultures of human ASCs were isolated from human lipoaspirates and passaged 3 times and then infected with replication-incompetent adenoviral vectors carrying green fluorescent protein (Ad/GFP) genes. Then, Ad/GFP infected human ASCs were transferred to osteogenic, chondrogenic, adipogenic, and myogenic medium. The morphological characterization of induced cells was observed using phase-contrast microscopy and fluorescence microscopy. The expression of marker proteins or genes was measured by immunocytochemical and RT-PCR analysis. Osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, aggrecan and SOX9 were positive in chondrogenic ones, peroxisome proliferatoractivated receptor (PPAR-γ 2) and lipoprotein lipase (LPL) were positive in adipogenic ones, and myogenin and myod1 was positive in myogenic ones. At the same time, the results of fluorescence microscopic imaging proved that the high level of GFP expression during ASCs differentiation maintained stable nearly 2 months. So the exogenous GFP and multilineage potential of human ASCs had no severe influences on each other. Since the human ASCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of GFP facilitates the research on ASCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.