构建一种可评价脂肪间充质干细胞成骨向分化的荧光素酶报告系统

Establishing a luciferase reporter system to evaluate osteogenic differentiation potential of human adipose-derived stem cells

目的:构建一种可用于评价人脂肪间充质干细胞(human adipose-derived stem cells,h ASCs)成骨向分化的荧光素酶报告系统,以期能够快捷、特异且定量的检测出细胞在体外的成骨向分化情况,并且使将来活体监测小型实验动物外源移植细胞的成骨分化成为可能。方法:将骨钙素(osteocalsin,OC)基因的启动子克隆到荧光素酶luciferase报告基因的上游,构建p LVX-OC_(pro)-Luc-Puro载体,进行慢病毒包装后感染人脂肪间充质干细胞,嘌呤霉素(puromycin)筛选阳性克隆,获得稳定整合OC_(pro)-Luc-Puro的细胞株,将该细胞在成骨诱导培养基中培养的第7和14天,观察其在成骨诱导环境下分化的情况。在成骨诱导培养的第7和14天检测荧光素酶的活性,同时进行茜素红(Alizarin red S)染色定量及成骨相关基因表达水平的检测,通过比较验证该荧光素酶报告系统用于成骨评价的可行性。结果:成功构建p LVX-OC_(pro)-Luc-Puro载体获得稳定整合报告基因的h ASCs细胞株。在OC_(pro)-Luc-Puroh ASCs成骨诱导的第7和14天,茜素红钙化结节染色结果均为阳性,且第14天的染色及定量结果明显强于第7天。OC、Runx2及碱性磷酸酶(alkaline phosphatase,ALP)基因的mRNA水平在成骨分化刺激下,其表达水平逐渐上升,同时,荧光素酶被激活,且荧光素酶的活性在第14天明显强于第7天,即荧光素酶表达的趋势与茜素红染色定量及成骨相关基因表达水平一致。结论:成功构建了一种可用于评价成骨向分化的荧光素酶报告系统,并通过与传统的检测成骨向分化方法(茜素红染色定量、成骨相关基因表达)对比,验证了该系统的有效性。

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 stern cells （hASCs） in vitro, and eventually make it possible to monitor the osteogenic differentiation of transplanted cells in vi-vo. 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： pM- DLg/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 osteo- genic 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 osteo- calcin （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 OC ro-Luc-hASCs was dramatically increased. Consistently, the extracellular matrix mineralization, as shown my 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 osteo- genic 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 differentia- tion of hASCs in a high throughput manner, but also provides a way to monitor ostogenic differentiation of cells in vivo.