利用RNAi技术稳定抑制ERβ表达的人成骨细胞株hFOB1.19细胞模型的建立

Establishment of the cell model of human osteoblast cell line hFOB 1.19 in which ERβ expression stably inhibited by RNAi

目的:探讨利用RNA干扰(RNA interference,RNAi)技术稳定抑制雌激素受体β(Estrogen receptorβ,ERβ)表达建立人成骨细胞株hFOB 1.19细胞模型的可行性。方法:设计3种特异性ERβ-shRNA,体外合成后将其克隆人pRNAT-H1.4/Retro逆转录病毒质粒中,并包装成逆转录病毒;ERβ-shRNA逆转录病毒瞬时感染hFOB l.19细胞后,通过流式细胞仪检测感染效率,并使用半定量RT-PCR和Western blot检测对ERβmRNA和蛋白表达的抑制效率;然后取ERβ抑制效率最高的hFOB l.19细胞,通过抗性筛选,将得到稳定感染的细胞扩大培养,再通过半定量RT-PCR和Western blot检测ERβ稳定抑制的效率;并应用MTT法检测ERβ稳定抑制后对细胞增殖的影响。结果:成功构建了3种ERβ-shRNA逆转录病毒载体;流式细胞仪检测结果显示,瞬时感染效率均高达70%以上;ERβ-shRNA-1、ERβ-shRNA-2、ERβ-shRNA-3逆转录病毒载体对hFOB l.19细胞中ERβmRNA的抑制率分别为(54.56±0.95)%、(69.60±1.12)%、(76.49±1.15)%,蛋白的抑制率分别为(59.21±4.44)%、(78.35±2.00)%、(85.60±2.66)%(均P<0.05);成功筛选出稳定感染ERβ-shRNA-3逆转录病毒载体的hFOB l.19细胞,ERβmRNA和蛋白的抑制率分别为(83.23±2.45)%和(93.11±0.57)%(均P<0.05),MTT法检测显示ERβ稳定抑制后对细胞的增殖没有明显影响(P>0.05)。结论:利用RNAi技术可成功建立ERβ稳定抑制的hFOB l.19细胞模型。

Objectives： To study the establishment of the cell model of human osteoblast cell line hFOB 1.19 in which estrogen receptor β （ERβ） expression was stably inhibited by RNA interference （RNAi）. Methods： Three designed ERβ-shRNA sequences were synthesized and cloned into the expression vectors of pRNAT-H1.4/Retro, then packaged into retrovirus. After transfection of hFOB 1.19 by ERβ-shRNA retroviral vectors, the transfection rates were measured by flow cytometry, and the inhibition rates of ERβ mRNA and protein were measured by semi-quantitative RT-PCR and Western-blot respectively. The hFOB 1.19 of the most effi-cient inhibition of ERβ was selected by hygromycin and cultured for proliferation. The stable inhibition rates of ERβ were measured by semi-quantitative RT-PCR and Western-blot respectively. The MTF method was used to measure the influence on the cell proliferation when ERβ expression was stably inhibited. Results： Three ERβ-shRNA retroviral vectors were constructed successfully and packaged into high efficient retrovirus. The results of flow cytometry showed that the transfection rates were all higher than 70%. By ERβ-shRNA-L ERβ-shRNA-2 and ERβ-shRNA-3 retroviral vectors, the inhibition rate of ERβ mRNA in hFOB 1.19 was （54.56±0.95）%, （69.60±1.12）% and （76.49±1.15）% respectively, while the inhibition rate of ERβ protein was （59.21±4.44）%, （78.35±2.00）% and （85.60±2.66）% respectively（all P〈0.05）. The hFOB 1.19 stably transfected by ERβ-shRNA-3 retroviral vector was selected successfully, the stable inhibition rate of ERβ mRNA and protein was （83.23±2.45）% and （93.11±0.57）% respectively（all P〈0.05）, and the result of MTF method showed that there was no significant influence on the cell proliferation when ERβ expression was stably inhibited （P〉 0.05）. Conclusions： The cell model of hFOB 1.19 was established in which ERβ expression was stably inhibited.