关键词: 荧光素酶类, 脂肪组织, 间充质干细胞, 骨生成, 细胞分化

Abstract:

Objective：Human adipose-derived stem cells (hASCs) are a highly attractive source in bone tissue engineering. To generate a luciferase reporter system that could be used to quantitatively and rapidly examine osteogenic differentiation potential of human adipose-derived stem cells (hASCs) in vitro, and eventually make it possible to monitor the osteogenic differentiation of transplanted cells in vivo. Methods: The genomic DNA harboring promotor regions of osteocalcin and DNA sequences encoding luciferase genes were amplified by PCR and cloned into the pLVX-pTRE-puro vector to generate the OCpro-Luc-Puro construct. Then, the OCpro-Luc-Puro construct together with three assistant vectors: pMDLg/pRRE, pRSVREV, and pVSVG, were transiently transfected into HEK293T cells followed by viral supernatants collection, filtration and concentration. Next, the hASCs stably expressing luciferase repor-ter gene driven by osteocalcin promotor were created with the lentivirus carrying OCpro-Luc-Puro cassette under puromycin selection. The OCpro-Luc-hASCs were then cultured in the absence or presence of osteogenic differentiation medium. On the 7th and 14th days, after osteogenic induction, cellular extracts were collected and analyzed by luciferase reporter assay. Meanwhile, alizarin red staining and quantification as well as quantitative reverse transcription PCR (qRT-PCR) analysis of osteogenic associated genes osteocalcin (OC)， runt-related transcription factor 2 (Runx2) and  alkaline phosphatase (ALP) were used to assess the osteogenic differentiation ability of OCpro-Luc-hASCs. Results:OCpro-Luc-Puro plasmid and OCpro-Luc-hASCs were successfully generated. On the 7th and 14th days after osteogenic induction, the luciferase activity of the cellular extracts from OCpro-Luc-hASCs was dramatically increased. Consistently, the extracellular matrix mineralization, as shown by Alizarin red S (ARS) staining and quantification was also markedly intensified and a marked increase of the mRNA expression levels of OC, Runx2 and ALP, although to variable extent, was observed upon osteogenic differentiation. These results indicated that the observations from traditional experiments examining hASCs osteogenic differentiation were largely in agreement with that of our luciferase reporter assay in OCpro-Luc-hASCs. Conclusion: We established a luciferase reporter system that could be used to rapidly, quantitatively and specifically determine osteogenic differentiation ability of hASCs. Comparing with the traditional time-consuming methods, the system we generated here was highly effective. This system not only can be used to examine ostogenic differentiation of hASCs in a high throughput manner, but also provides a way to monitor ostogenic differentiation of cells in vivo.

Key words: Luciferases, Adipose tissue, Mesenchymal stromal cells, Osteogenesis, Cell differentiation