稳定表达PAsRed2-N1-LC3基因的肺泡上皮细胞株的建立

Establishment of alveolar epithelial cell model for stably expressing PAsRed2 - N1 - LC3 gene

目的建立能稳定表达红色荧光真核表达载体（PAsRed2-N1-LC3）基因的大鼠肺泡上皮细胞株（CCL149）。方法构建PAsRed2-N1-LC3，应用转染技术将该质粒导入CCL149细胞，筛选出可稳定表达的细胞株。真核细胞中PAsRed2-N1-LC3的表达分别用荧光显微镜、聚合酶链反应（PCR）测序及Western blot法检测，观察稳定表达细胞株缺氧性自噬。结果成功获得4株可稳定表达PAsRed2一N1一LC3的细胞株，在倒置荧光显微镜下观察CCL149PAsRed2-N1-LC3转染细胞株的红色荧光率在90％[（91．13±1．85）％]以上。Western blot结果证实在缺氧条件下，CCL149 PAsRed2-N1-LC3转染细胞株的LC3相对表达量[LC3／β-肌动蛋白（β—actin）]为0．61670±0．03394，正常细胞LC3相对表达量（LC3／β-actin）为0．16000±0．00635，差异有统计学意义（P〈0．05），且实验组LC3表达率平均是对照组的3．854倍；运用PCR技术检测CCL149 PAsRed2-N1-LC3转染细胞株的LC3基因相对扩增量是正常CCL149细胞LC3基因相对扩增量的4．289863倍。结论用含有PAsRed2-N1-LC3真核表达载体转染CCL149细胞，可成功建立PAsRed2-N1-LC3稳定表达细胞株。

Objective To establish a alveolar epithelial cell model for stably expressing PAsRed2 - N1 - LC3 gene. Methods The PAsRed2 - N1 - LC3 plasmid was constructed and transfected into CCL149 cells with transfection reagent. PAsRed2 - N1 - LC3 expression was analyzed by fluorescence microscope, Western blotting and PCR techiques. In addition, autophagy could be observed and confirmed under the condition of hypoxia. Results Four transfected cell lines showed high level of PAsRed2 - N1 - LC3 expression. More than 90% [ （91.13 ±1. 85）% ] ceils showed positive fluorescent signals under inverted fluorescence microscope. The result of Western blotting confirmed that the amount of protein expressed by LC3 genes in CCL149 PAsRed2 - N1 - LC3 （0. 616 70 ±0. 033 94） was higher than that of CCL149 （0. 160 00 ±0. 006 35） （P 〈0. 05）, which was 3. 854 times higher than that of normal CCL149 cells on average. The transcription of LC3 in CCL149 PAsRed2 - N1 - LC3 was 4. 289863 times higher than that of CCL149 detected by PCR technique. Conclusion A CCL149 ceil line stably expressing RFP -LC3 was constructed successfully after transfection by PAsRed2 - N1 - LC3 plasmid.