[Increasing alcohol-induced osteogenesis of human bone marrow-derived mesenchymal cells using siRNA transient suppression of peroxisome proliferator activated receptor gamma: an in vitro experiment study].
OBJECTIVE
To investigate the potential of small interfering RNA (siRNA) against human peroxisome proliferator activated receptor gamma (PPARgamma) to suppress the adipogenic effect of alcohol on human bone marrow-derived mesenchymal cells (hBMSCs) and increase osteogenesis.


METHODS
hBMSCs collected from joint replacement surgery were cultured, passaged, and transfected with PPARgamma-siRNA by using liposomal-based strategy. Then the cells were maintained in culture and treated with alcohol 50 mmol/L and dexamethasone, an osteogenic inducer, for 24 days. Oil red O staining was used to observe the fat drops in the cells so as to count the number of adipocytes. Histochemistry was performed to detect the protein expression of alkaline phosphatase, osteocalcium, and type 1 collagen on days 24 and 28 after treatment. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of osteoblast-specific transcription factor-2 (Osf2)/core-binding factor alpha subunit 1 (Cbfa1) on day 10.


RESULTS
The adipocyte number of the PPARgamma-siRNA group was significantly lower than those of the alcohol-induced group and the controls (adipogenic group, liposomal group, and negative siRNA group) (all P < 0.05). The Calcium nodule formation rate of the PPARgamma-siRNA group was significantly higher than alcohol-induced group and the controls (adipogenic group, liposomal group, and negative siRNA group) (all P < 0.05). PCR and Western blotting showed significantly higher mRNA and protein expression of the human adipocyte-specific markers:Cbfa1, ALP, type 1 collagen, and osteocalcin, than the other groups.


CONCLUSIONS
The adipogenic effect of alcohol on hBMSCs can be inhibited with a concomitant increase in osteogenesis by using siRNA technique to specific targeted human PPARgamma gene in vitro cell culture systems. PPARgamma-siRNA strategy may be a useful tool for studying the mechanisms of alcohol-induced osteoporosis and provides theoretical basis for its genetic therapy.
Zhang
Hui
H
Department of Orthopedics, West China Hospital, Sichuan University, Chengdu 610041, China.
Cheng
Jing-Qiu
JQ
Huang
Qiang
Q
Yang
Jing
J
Shen
Bin
B
Zhou
Zong-Ke
ZK
Kang
Peng-de
PD
Lian
Yong-Yun
YY
Pei
Fu-Xing
FX
D001854:Bone Marrow Cells / Q000166:cytology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D000431:Ethanol / Q000494:pharmacology; D006801:Humans; D010006:Osteoblasts / Q000166:cytology*; D010012:Osteogenesis; D047495:PPAR gamma / Q000235:genetics*; D034741:RNA, Small Interfering; D017154:Stromal Cells / Q000166:cytology
D047495:PPAR gamma; D034741:RNA, Small Interfering; D000431:Ethanol