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.

切取4只雄性SD大鼠鼠蹊部皮下脂肪，通过机械分割和组织消化法获得脂肪基质细胞，用人内皮细胞培养基进行定向诱导，倒置显微镜下观察细胞生长状况，并以透射电镜及Ð因子免疫细胞化学方法对诱导后的细胞进行鉴定。SD大鼠脂肪基质细胞经过定向诱导后呈典型的铺路石样单层融合贴壁生长；细胞浆中Ð因子免疫细胞化学染色阳性；透射电镜显示细胞内具有内皮细胞特征性的Weibel-Palade氏小体，细胞边缘可见较多指状突起，胞浆内可见溶酶体等结构。结果证实从SD大鼠皮下脂肪获取得基质细胞经过定向诱导后，可以分化成为内皮细胞，该细胞具有与其它来源的血管内皮细胞相同的特征。皮下脂肪可以成为内源性血管内皮细胞的又一细胞来源。

Functional engineering of musculoskeletal tissues generally involves rapid expansion of progenitor cells in vitro while retaining their potential for further differentiation and then induction in specific culture conditions. The autologous adipose-derived stromal cells (ASCS) are considered to contain pluripotent mesenchymal stem cells. Imaging with expression of green fluorescent protein (GFP) facilitates the detailed research on ASCs physiological behavior during differentiation into a variety of cell lineages both in vitro and in vivo. In this study, we aimed to confirm the trans-germ plasticity of homogeneously marked ASCs from GFP transgenic mice. Simultaneously, the term and intensity of GFP expression in ASCs were also focused on during variant inductions, when cells were incubated with multiple growth factors and adjuvant. ASCs were harvested from inguinal fat pads of transgenic nude mice, passaged 3 times in monolayer cultures, and then transferred to osteogenic, adipogenic, neurogenic, and myogenic medium. The morphological characterization of inductive cells was observed using phase-contrast microscopy and histological staining such as alizarin red for mineralization nodules and oil red O for lipid accumulation. The expression of marker genes or proteins was measured using RT-PCR and immunocytochemical analysis. Collagen type I, osteopontin (OPN), and osteocalcin (OCN) were positive in osteogenic lineages, peroxisome proliferator-activated receptor(PPAR)-γ 2 and lipoprotein lipase (LPL) were positive in adipogenic ones, glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) were positive in neurogenic ones, and α-smooth muscle actin (α-SMA) was positive in myogenic ones. Moreover, the results of fluorescence microscopic imaging suggested that there was no significant decline of GFP expression during ASCs differentiation and the level of GFP maintained stable till differentiated ASCs showed apoptotic phenotype. So the endogenous GFP and multilineage potential of transgenic ASCs had no influences on each other. Since the population of GFP ASCs can be easily identified, it is proposed that they may be promising candidate seed cells for further studieson ASCs tissue engineering, especially the study on engineered tissues formed in vivo. (Mol Cell Biochem xxx: 1–10, 2005)

tomesenchymal cells in the first branchial arch were enzymatically isolated from the mandibular processes of BALB/c mice and were maintained in an intact state in a medium containing leukaemia inhibitory factor. Here, we first evaluated the proliferative activity of the cells after the third passage, using bromodeoxyuridine labelling and in situ hybridization of telomerase mRNA. Positive staining for expression of HNK-1, S-100 and vimentin confirmed that the population of stem cells originated from the ectomesenchyme, which did not express cytokeratin. Then we investigated the molecular and cellular characteristics of the ectomesenchymal cells during their differentiation towards neurogenic, endothelial, myogenic and odontogenic lineages. Expression of multiple lineagespecific genes and proteins was detected by utilizing a range of molecular and biochemical approaches when the cells were transferred to inductive medium. Histological and immunohistochemical analysis of the induced cells at various intervals indicated obvious phenotypic alteration and presence of specific proteins for the differentiated lineages, for example nestin, factor VIII, α-SMA and dentin sialophosphoprotein (DSPP), respectively. Correlatively, results of reverse transcription–PCR corroborated at Mrna level the expression of the characteristic molecules during differentiation. Therefore, it is suggested that the ectomesenchymal cells derived from the first branchial arch may represent a novel source of multipotential stem cells capable of undergoing expansion and variant differentiation in vitro.

目的研究绿色荧光蛋白（GFP）基因体外转染人脂肪基质细胞的方法，并检测基因转染后细胞的生物学特性及分化潜能。方法体外分离培养人脂肪基质细胞，用重组腺病毒载体Ad-GFP及脂质体介导质粒pEGFP-C1两种方法转染GFP基因，通过流式细胞术观察比较GFP转染和表达的结果;倒置显微镜下观察细胞生长情况；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GFP基因的腺病毒Ad-GFP转染的脂肪基质细胞的感染率达（42.5±1.5）%，16h即有GFP表达，7d时表达趋于稳定，感染6周时仍可见有GFP表达，明显优于脂质体转染组（转染效率最高为11.4%）。感染Ad-GFP后的脂肪基质细胞与未感染的对照组脂肪基质细胞成骨诱导分化后碱性磷酸酶（ALP）表达均逐渐增高，两组间差异无统计学意义（P>0105）；诱导21d后两组均见到茜素红钙盐染色阳性。结论重组GFP基因的腺病毒载体Ad-GFP可高效率感染脂肪基质细胞，基因转染后细胞的增殖分化能力未受到影响，可以作为一种高效可靠的人脂肪基质细胞标记方法。

Pluripotent stem cells within the adipose stromal compartment, termed adipose-derived stromal cells (ASCs), have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. Imaging with expression of exogenous or endogenous green fluorescent protein (GFP) reporters facilitates the detailed research on ASCs’ physiological behavior during differentiation in vivo. This study was aimed to confirm whether ASCs expressing GFP still could be induced to chondrogenesis, and to compare the expression of exogenous or endogenous GFP in ASCs during chondrogenic differentiation. ASCs were harvested from inguinal fat pads of normal nude mice or GFP transgenic mice. Monolayer cultures of ASCs from normal mice were passaged three times and then infected with replication-incompetent adenoviral vectors carrying GFP genes. Allowed to recover for 5 days, Ad/GFP infected ASCs were transferred to chondrogenic medium as well as the ASCs from transgenic mice cultured in vitro over the same passages. The level of GFP in transgenic ASCs maintained stable till 3 months after chondrogenic induction. Whereas, high level of GFP expression in Ad/GFP infected ASCs could last for only 8 weeks and then declined stepwise. Important cartilaginous molecules such as SOX9, collagen type I, collagen type II, aggrecan, collagen type X were assessed using immunocytochemistry, RT-PCR, andWestern Blot. The results indicated that no matter the GFP was exogenous or endogenous, it did not influence the chondrogenic potential of ASCs in comparison with the normal controls. Moreover, chondrogenic lineages from ASCs also underwent phenotypic modulation called dedifferentiation as a result of long-term culture in monolayers similar to normal chondrocytes. (Mol Cell Biochem 277: 181–190, 2005)