Multilineage differentiation of adipose-derived stromal cells from GFP transgenic mice
Multilineage differentiation of adipose-derived stromal cells from GFP transgenic mice
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DOI：
10.1007/s11010-005-9056-8
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发表时间：
2006-04-01
影响因子：
4.3
通讯作者：
Tian, Weidong
中科院分区：
文献类型：
--
作者：
Lin, Yunfeng;Chen, Xizhe;Tian, Weidong
肌肉骨骼组织的功能工程通常涉及祖细胞在体外的快速扩增，同时保留其进一步分化的潜力，然后在特定的培养条件下诱导。自体脂肪源性基质细胞（ASC）被认为含有多能间充质干细胞。利用绿色荧光蛋白（GFP）的表达进行细胞成像，有助于在体内外对ASCs分化为多种细胞系过程中的生理行为进行详细研究。在这项研究中，我们的目的是确认跨生殖可塑性的均匀标记的ASC从GFP转基因小鼠。同时，在ASC中的GFP表达的期限和强度也集中在变体诱导期间，当细胞与多种生长因子和佐剂一起孵育时。从转基因裸鼠的腹股沟脂肪垫中收获ASC，在单层培养物中传代3次，然后转移到成骨、成脂、神经和成肌培养基中。诱导细胞的形态特征进行了观察，使用相差显微镜和组织学染色，如茜素红的矿化结节和油红O的脂质积累。用RT-PCR和免疫细胞化学分析检测标记基因或蛋白的表达。成骨细胞I型胶原、骨桥蛋白（OPN）和骨钙素（OCN）阳性，成脂细胞过氧化物酶体增殖物激活受体（PPAR）-γ 2和脂蛋白脂酶（LPL）阳性，神经细胞胶质酸性蛋白（GFAP）和神经元特异性烯醇化酶（NSE）阳性，肌细胞α-平滑肌肌动蛋白（α-SMA）阳性。荧光显微成像结果显示，GFP在ASCs分化过程中的表达没有明显下降，直至分化的ASCs出现凋亡表型，GFP的表达水平一直保持稳定。因此，转基因ASCs的内源性GFP和多系分化潜能之间没有相互影响。由于GFP ASCs群体易于鉴定，因此，GFP ASCs有望成为进一步研究ASCs组织工程，特别是体内形成工程组织的候选种子细胞。
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)-gamma 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 alpha-smooth muscle actin (alpha-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 studies on ASCs tissue engineering, especially the study on engineered tissues formed in vivo.