Nurr1基因在大鼠体外培养骨髓基质细胞的表达

目的 :为获得高效表达孤儿核受体 (orphannuclearreceptor,Nurr1)基因的骨髓基质细胞。 方法 :采用分子克隆技术 ,构建携带Nurr1基因的重组腺相关病毒 (adeno associatedvirus,AAV)载体 ;与包装质粒 pAAV RC和辅助质粒phelper一起用磷酸钙法转染包装细胞HEK2 93制备具有感染活性的AAV Nurr1病毒粒子 ;用病毒上清液感染原代培养的大鼠骨髓基质细胞 (Marrowstromalcells,MSCs) ,并用免疫细胞化学方法检测阳性细胞。 结果 :经酶切鉴定和DNA测序 ,得到了序列正确的重组pAAV Nurr1;感染HT10 80细胞进行病毒滴度测定 ,每mL病毒贮存液可达 10 12 个阳性细胞 ;获得了Nurr1阳性的骨髓基质细胞 ,阳性率在 6 0 %以上。结论 :重组AAV携带的Nurr1基因能够在MSCs中表达 ,本文为进一步探讨将该细胞用于帕金森病基因治疗的可能性研究奠定了基础。

The expression of Nurr1 gene from bone marrow stromal cells of the rats in vitro

Objective: To obtain the genetically modified marrow stromal cells expressing the gene of Nurr1. Methods: A recombinant adeno-associated virus vector encoding Nurr1( pAAV-Nurr1) was constructed with the technique of molecular cloning. High-titer viral particles of AAV-Nurr1 were obtained after co-transfection of HEK293 with the other two helper vectors by calcium-phosphate-mediated transfection. Primary cultured marrow stromal cells from the rat were infected with viral stock and identified by the method of immunocytochemistry. Results: pAAV-Nurr1 was verified by restriction endonuclease digestion and DNA sequencing. The titer of recombinant AAV viral stock could be reached up to 10 12 positive cells per milliliter viral stock. Primary cultured marrow stromal cells from the rat infected with viral stock were proved to be the genetically modified MSCs expressing Nurr1 by the method of immunocytochemistry. Conclusions: These genetically modified MSCs might be used for further research on gene therapy of Parkinson disease.