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。

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。

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.

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.

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)