利用RNAi技术稳定抑制ERβ表达的人成骨细胞株hFOB 1.19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

中文关键词:  RNA干扰  雌激素受体β  人成骨细胞

英文关键词:RNA interference  Estrogen receptor β  Human osteoblast cell

中文摘要:

  【摘要】 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 receptor β，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ERβ mRNA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ERβ mRNA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，ERβ mRNA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。

英文摘要:

  【Abstract】 Objectives: To study the establishment of the cell model of human osteoblast cell line hFOB l.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 l.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 l.19 of the most efficient 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 MTT 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-1, ERβ-shRNA-2 and ERβ-shRNA-3 retroviral vectors, the inhibition rate of ERβ mRNA in hFOB l.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 l.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 MTT 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 l.19 was established in which ERβ expression was stably inhibited.