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

English Keywords:RNA interference  Estrogen receptor β  Human osteoblast cell

English Abstract:

  【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.