摘要
目的:构建转录辅助因子LBH(limb-bud and heart)过表达的人间充质干细胞(MSC)株,研究Lbh基因对人MSC功能的调控。方法:通过分子克隆的方法构建人Lbh基因的慢病毒表达质粒p CDHP-Lbh,经限制性内切酶酶切和核酸测序鉴定,利用293T细胞包装表达Lbh基因的慢病毒及对照;将包装的病毒感染人MSC,利用表达载体携带的GFP报告基因,采用流式细胞术分选GFP阳性目的细胞;通过油红O染色鉴定阳性细胞的成脂分化能力,通过茜素红和硝酸银染色鉴定阳性细胞的成骨分化能力。结果:双酶切和测序结果表明正确克隆了人Lbh基因的全长CDS序列,定量PCR和Western印迹结果表明构建的p CDHP-Lbh重组质粒能够表达Lbh基因;流式分选获得LBH稳定表达的人MSC细胞株,该细胞株经成骨和成脂诱导分化培养后,油红O染色、茜素红染色和硝酸银染色结果均呈阳性。结论:构建了人Lbh基因的慢病毒表达质粒p CDHP-Lbh,并获得稳定表达Lbh基因的人MSC细胞株,该细胞株保留了向脂肪细胞和成骨细胞定向分化的能力。本研究为进一步探讨Lbh基因对人MSC功能的调控提供了重要的实验基础。
Objective： To establish human mesenchymal stem celI（MSC） line overexpressing transcription cofac- tor limb-bud and heart （LBH） and to study Lbh gene function in human MSC cells. Methods： The lentiviral vec- tor pCDHP-Lbh expressing human Lbh gene was constructed through molecular cloning. The recombinant clones were validated by restricted enzyme digestion and DNA sequencing. Lentivirus expressing Lbh and the control vi- rus were packaged in 293T cells and were used to transfect human MSC. The stably transfected cells were sorted through flow cytometry based on the expression of reporter gene GFP on the vector. The MSC overexpressing hu- man LBH was induced for adipogeneic or osteogeneic differentiation respectively. Adipocytes were detected by oil red O staining and osteoblasts were detected by alizarin red S and von Kossa staining. Results： Full length hu- man Lbh CDS sequence was successfully cloned into lentiviral vector pCDHP. Over expression of Lbh was con- firmed by real-time PCR and Western blot. The human MSC line that stably overexpressing LBH was established and has been validated with its adipogeneic and osteogeneic differentiation capacities. Conclusion： Human MSC line toverexpressing human Lbh gene has been constructed and induced differentiation cuhure confirmed the cell line retained the multi-lineage differentiation potential. The human MSC line established by present study has pro- vided important basis for the future investigation of the role of Lbh gene on and human MSC functions.
出处
《生物技术通讯》
CAS
2016年第1期40-43,共4页
Letters in Biotechnology
基金
国家重点基础研究发展计划(2012CB966504)
国家自然科学基金(81170459)
人事部留学人员择优资助项目(2012年)
