Wang Qian, Steigelman Megan B, Walker John A, Chen Shuo, Hornsby Peter J, Bohnenblust Mary E, Wang Howard T
Plastic and Reconstructive Surgery, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA.
J Craniofac Surg. 2009 Nov;20(6):2193-9. doi: 10.1097/SCS.0b013e3181bf04af.
Adipose-derived stem cells (ASCs) have the potential to differentiate into osteogenic cells that can be seeded into scaffolds for tissue engineering for use in craniofacial bone defects. Green fluorescent protein (GFP) has been widely used as a lineage marker for mammalian cells. The use of fluorescent proteins enables cells to be tracked during manipulation such as osteogenic differentiation within three-dimensional scaffolds. The purpose of this study was to examine whether ASCs introduced with GFP-encoding lentivirus vector exhibit adequate GFP fluorescence and whether the expression of GFP interfered with osteogenic differentiation of ASCs in both monolayer and three-dimensional scaffolds in vitro.
Primary ASCs were harvested from the inguinal fat pad of Sprague Dawley rats. Isolated ASCs were cultured and infected with a lentiviral vector encoding GFP and plated into both monolayers and three-dimensional scaffolds in vitro. The cells were then placed in osteogenic medium. Osteogenic differentiation of the GFP-ASCs was assessed using alizarin red S, alkaline phosphate staining, and immunohistochemistry staining of osteocalcin with quantification of alizarin red S and osteocalcin staining.
The efficacy of infection of ASCs with a lentiviral vector encoding GFP was high. Cell-cultured GFP-ASCs remained fluorescent over the 8 weeks of the study period. The GFP-ASCs were successfully induced into osteogenic cells both in monolayers and three-dimensional scaffolds. Whereas the quanitification of alizarin red S revealed no difference between osteoinduced ASCs with or without GFP, the quantification of osteocalcin revealed increased staining in the GFP group.
Transduction of isolated ASCs using a lentiviral vector encoding GFP is an effective method for tracing osteoinduced ASCs in vitro. Quantification data showed no decrease in staining of the osteoinduced ASCs.
脂肪来源干细胞（ASC）有分化为成骨细胞的潜力，可接种到组织工程支架中用于颅面骨缺损修复。绿色荧光蛋白（GFP）已被广泛用作哺乳动物细胞的谱系标记。荧光蛋白的使用能够在诸如三维支架内成骨分化等操作过程中追踪细胞。本研究的目的是检测导入编码GFP的慢病毒载体的ASC是否表现出足够的GFP荧光，以及GFP的表达是否会干扰体外单层和三维支架中ASC的成骨分化。
从Sprague Dawley大鼠腹股沟脂肪垫中获取原代ASC。分离得到的ASC进行培养，用编码GFP的慢病毒载体感染，然后接种到体外的单层和三维支架中。接着将细胞置于成骨培养基中。使用茜素红S、碱性磷酸酶染色以及骨钙素免疫组化染色并对茜素红S和骨钙素染色进行定量分析，以评估GFP-ASC的成骨分化情况。
用编码GFP的慢病毒载体感染ASC的效率很高。在为期8周的研究期间，细胞培养的GFP-ASC一直保持荧光。GFP-ASC在单层和三维支架中均成功诱导分化为成骨细胞。虽然茜素红S定量分析显示有或没有GFP的成骨诱导ASC之间没有差异，但骨钙素定量分析显示GFP组染色增加。
使用编码GFP的慢病毒载体转导分离的ASC是体外追踪成骨诱导ASC的有效方法。定量数据显示成骨诱导ASC的染色没有减少。
