摘要： 目的：利用慢病毒载体介导小鼠颏舌肌成肌细胞ERα基因沉默，筛选鉴定后建立ERα基因稳定下调的细胞群。方法：设计合成4组针对小鼠ERα基因的shRNA序列（ERα-321、ERα-693、ERα-978、ERα-1297）及1组阴性对照序列(shNC)，连接到经双酶切处理过的pGLV3/H1/GFP载体上，转染293T细胞包装产生慢病毒。用慢病毒转染体外培养的小鼠颏舌肌成肌细胞，流式细胞仪筛选后提取细胞RNA，RT-PCR法鉴定各组细胞中ERα的RNA干扰效果。结果：细胞转染5种慢病毒后荧光率分别为21.4%、25.1%、30.7%、23.6%和56.4%，经流式细胞仪无菌分选后荧光率接近100%。与阴性对照组相比，4组细胞的ERαmRNA表达均显著降低（P＜0.05），其中ERα-978组的干扰效率最高，为82.7%。结论：本实验成功筛选并建立了ERα基因稳定下调的成肌细胞群，为研究ERα下游信号通路奠定了重要基础。

关键词:  雌激素受体α；  , 慢病毒载体；  , RNA干扰；  , 成肌细胞；  , 鼠

Abstract: Objective： To establish a stable ERα gene silenced myoblasts of mouse genioglossus muscle by using shRNA lentiviral vector. Methods: Four pairs of complementary short hairpin RNA(shRNA) oligonucleotides targeting the mouse ERα gene and a pair of oligonucleotides as negative control were designed and synthesized, and then were inserted into pGLV3/H1/GFP plasmid vectors. The recombinant plasmids were transfected with packaging vectors into 293T cells. The cell culture supernatant were obtained to infect the myoblasts of mouse genioglossus muscle which were cultured before hand in vitro. The ERα mRNA level were detected by RT-PCR after all groups of cells were screened by the flow cytometer. Results: The myoblasts cultured in vitro were testified by immunocytochemical staining. The efficiency of transfection was highly improved by the flow cytometer when assessed under fluorescent microscope. The expression of ERα was significantly decreased at mRNA level in 4 experimental groups（P＜0.05）, as compared with negative control group. The interferential efficiency of ERα-978 shRNA is the highest(82.5%). Conclusion: A stable and effectively reduced ERα gene silenced cell group of myoblast was successfully established in this study, which will provide an effective cell model for future study on the ERα downstream signaling pathways.

Key words: estrogen receptor α(ERα), lentiviral vector, RNAi, myoblast, mouse

中图分类号: 

• Q78