Construction of human osteoblast-like cell model with the down-regulated estrogen receptor α subunit gene by RNA interference / 中华老年医学杂志

ABSTRACT

Objective To construct the human osteoblast-like cell model with estrogen receptor α(ERα)subunit gene knocked down. Methods According to the computer-aided design(CAD),ERα-specific small interference RNA(siRNA)gene was synthesized and cloned into the expression vector pSilencer 4.1-CMV. The recombinant ERα siRNA plasmid was transfected into human osteoblast-like cell line MG63 by lipofectin,the cloned MG63 ceils were selected by hygromycin,and the cloned MG63 cell was cultured more than 20 passages after transfection.The expression of ERα mRNA in MG63 cells was detected by reverse transcription-polymerase chain reaction(RT-PCR).The expression and location of ERα protein were identified by immunocytochemistry.Compared with control groups,proliferation rate,growth cycle and expression did not show significant difference.Results The recombinant eukaryote plasmid vector was constructed.Furthermore,the recombinant plasmid knocked down ERα protein in human osteoblast-like cells. Conclusions The human osteoblast-like cell model with RNAi-knocked down ERα gene is constructed successfully.This model appears to be very useful for the future research on ERα biological characters and on molecular mechanism of bone metabolism.