Inhibition of retinoblastoma binding protein 2 promotes osteogenic differentiation of human adipose-derived stromal cells
10.3760/cma.j.issn.1002-0098.2011.03.006
- VernacularTitle:抑制视网膜母细胞瘤结合蛋白2基因表达促进人脂肪基质细胞成骨向分化的研究
- Author:
Wen-Shu GE
1
;
Yong-Sheng ZHOU
;
Hai-Lan FENG
;
Gui-E MA
;
Yun-Song LIU
;
Yong-Wei XU
Author Information
1. 北京大学口腔医学院口腔医院
- Keywords:
RNA,small interfering;
Retinoblastoma protein;
Lentivirus;
Human adipose-derived stromal cells
- From:
Chinese Journal of Stomatology
2011;46(3):148-152
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the effect of retinoblastoma binding protein 2 (RBP-2), a histone H3K4 demethylase, on osteogenic differentiation of human adipose-derived stromal cell(hASC).Methods According to the GenBank sequence information of RBP-2, four different small interfering RNAs (siRNA) targeting RBP-2 gene were designed and the corresponding short hairpin RNAs (shRNA) were cloned into pLL 3.7 lentivirus RNA interference vector. The lentivirus with RBP-2-siRNA was packaged in 293T cells. The effective sequence was examined and selected by Western blotting and real-time PCR. The lentiviruses with efficient knockdown effects were used to infect hASC. On the 14th day after osteogenic differentiation, alkaline phosphatase (ALP) activities of hASC were quantitatively tested and at the same time, ALP staining and alizarin red staining were performed to assess the difference of osteogenic differentiation between the knockdown group and the control group. Results The recombinant lentivirus siRNA targeting RBP-2 was successfully constructed and the expression of RBP-2 mRNA and protein were dramatically suppressed by infection with RBP-2-siRNA lentivirus. On the 14th day after osteogenic induction, ALP activity of hASC in the knockdown group[(299.2 ±22.7), (224.3± 17.7) U/g]was much stronger than that in the control group[( 129.9 ± 12.9) U/g, P ＜ 0. 05]and the same result was achieved for the ALP staining and alizarin red staining. Conclusions The constructed RBP-2-siRNA lentivirus could markedly decrease the expression of RBP-2 and promote osteogenic differentiation of hASC.It indicated that RBP-2 can repress the osteogenic differentiation of hASC.
