[In vitro study of combination rhOPG-Fc and alendronate on inhibition osteoclast].
OBJECTIVE
To study the effect of combination rhOPG-Fc and alendronate on mature osteoclasts.


METHODS
Recombinant human osteoprotegerin secretory expression in P. pastoris was performed. Osteoblasts were got from new born mouse skeletal bone and proved by ALP staining and incubated together with osteoclasts precursor cell line Raw 264.7 in 96 well plate. After 9 d, 10 micromol/L ALN, 10(-5) g/L rhOPG-Fc, 10 micromol/L ALN + 10(-5) g/L rhOPG-Fc, 5 micromol/L ALN + 5 x 10(-6) g/L rhOPG-Fc were added to these coculture systems. Osteoblasts cultured without the drugs mentioned above served as controls. TRAP stain positive cells counting and cortical bone pit formation counting were preformed in the following the 3rd and 7th d.


RESULTS
SDS-PAGE and Western blot showed that molecular weight of the expressed protein was about 55 KD, and it could reach specifically with anti-IgG antibody. Many multi-nuclear TRAP stain positive cells were found in the coculture control group after 9 d incubation, and proved to be mature osteoclasts by TRAP stain. In the 3rd and 7th d after the addition of rhOPG-Fc, ALN or both, TRAP stain positive cells counting and cortical bone pit formation counting decreased significantly in the rhOPG-Fc, ALN or both groups than in the control group, and the combine group (10(-5) g/L rhOPG-Fc + 10 micromol/L ALN) decreased most significantly when compared with rhopG-FC or ALN single.


CONCLUSIONS
rhOPG-Fc can decrease the number of osteoclasts and inhibit their function. The combination of both rhOPG-Fc and ALN shows the significant inhibition effect on mature osteoclasts.
Huang
Peng
P
Department of Orthopaedic Surgery, People's Liberation Army General Hospital, Beijing 100853, China.
Wang
Yan
Y
Chi
Zhi-yong
ZY
Yang
Zi-yi
ZY
Ni
Jian
J
Yang
Wu-jian
WJ
Wang
Ran-dong
RD
Bai
Jin-zhu
JZ
D019386:Alendronate / Q000494:pharmacology*; D000818:Animals; D004357:Drug Synergism; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008815:Mice, Inbred Strains; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects*; D053244:Osteoprotegerin / Q000096:biosynthesis / Q000494:pharmacology*; D010843:Pichia / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology
D053244:Osteoprotegerin; D011994:Recombinant Proteins; C506030:TNFRSF11B protein, human; D019386:Alendronate