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目的构建稳定表达由6OSE2启动子驱动的萤火虫荧光素酶的小鼠C2C12成肌细胞,并筛选出能定量反映Runx2活性的细胞株。方法双酶切pUC57-6OSE2,获得启动子片段;插入到消化后的pGL4.14,构建真核表达载体;随后将其转染到C2C12细胞中;用潮霉素B筛选获得稳定转染的细胞株C2C12-6OSE2-Luc。将Runx2瞬时转染到该细胞株中,鉴定其对Runx2的响应性。利用不同浓度的IGF-I和BMP2处理,通过Luc报告基因的活性增高,筛选出能够响应BMP2的细胞株。结果构建了p6OSE2-Luc表达载体,利用p6OSE2瞬时转染C2C12-Runx2细胞的相对荧光素酶活性是同样处置后的C2C12细胞的4倍。通过筛选获得C2C12-6OSE2-Luc细胞株。转染Runx2质粒后细胞的相对荧光素酶活性是转染空载体细胞的7倍。利用荧光素酶活性筛选出一株报告基因活性随BMP2处理增强的细胞株。结论构建了能定量反映成骨细胞Runx2活性的细胞模型,可以用于进一步Runx2的转录活性及信号传导途径的研究中。
Objective To construct a mouse C2C12 myoblast stably expressing firefly luciferase driven by the 6OSE2 promoter and screen out cell lines that can quantitatively reflect Runx2 activity. Methods Double enzyme digestion of pUC57-6OSE2 to obtain a promoter fragment; inserted into the digested pGL4.14 to construct a eukaryotic expression vector; then transfected into C2C12 cells; and stably transfected cells were screened with hygromycin B Strain C2C12-6OSE2-Luc. Runx2 was transiently transfected into this cell line and its responsiveness to Runx2 was identified. By using different concentrations of IGF-I and BMP2, the activity of Luc reporter gene was increased, and the cell lines that responded to BMP2 were screened out. Results The p6OSE2-Luc expression vector was constructed and the relative luciferase activity of transient transfection of C2C12-Runx2 cells with p6OSE2 was 4-fold higher than that of C2C12 cells treated with the same treatment. The C2C12-6OSE2-Luc cell line was obtained by screening. The relative luciferase activity of cells transfected with Runx2 plasmid was 7 times that of transfected empty vector cells. A luciferase activity-screened a reporter gene activity with BMP2 enhanced cell lines. Conclusion A cell model that can quantitatively reflect the Runx2 activity of osteoblasts was constructed and could be used for the further study of the transcriptional activity and signal transduction pathway of Runx2.