The function and meaning of receptor activator of NF-κB ligand in arterial calcification / 华中科技大学学报（医学）（英德文版）

Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead.

Comparison of MMP-9 Expression in Osteoclasts Obtained by Different Methods / 昆明医科大学学报

Objective To investigate the differences of protein and mRNA expression of matrix metalloproteinase 9 (MMP-9) in osteoclasts (OC) and osteoclast-like cells (OLC) obtained by mechanical and inducement methods. Methods Mechanical separation method was used to separate mature osteoclasts from long bones of SD rats aged one-day;and inducement culture method was applied to induce OLC by using RANKL (100 ng/mL) and M-CSF (100 ng/mL) . The protein expression of MMP-9 was measured by immunocytochemistry and mRNA expression of MMP-9 was assayed by in situ hybridization. Results The integral optical density (IOD) and average optical density (AOD) of positive cells in the visual field were higher in the 12-d group of inducement method as compared with the 3 d-group of mechanical method. Conclusions It is suggested that the protein and mRNA expression of MMP-9 in OLC obtained by 12 d inducement method is much high than in OC obtained by 3 d mechanical method. OLC obtained by inducement method can be applied in the study of osteoporosis.

Effects of pioglitazone on differentiation and function of cultured osteoclast-like cells of rats / 中华内分泌代谢杂志

Objective To study the effect of pioglitazone on the differentiation and function of rat osteoclast-like cells (OLC), and to probe the relationship between activated PPARγ2 and osteoclasts. Methods On day 1 of OLC formation from nonadherent bone marrow ceils (BMC) obtained from rats induced by M-CSF and receptor activator of NF-кB ligand (RANKL), 1, 5 and 10μmol/L pioglitazone hydrochloride was added. RT- PCR was performed to determine the mRNA expressions of PPARγ2 and receptor activator of NF-кB (RANK) on day 3, 5 and 7 during incubation, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells,the number of bone resorption pits and the ratio of its area on dentin slice were counted, the activity of TRAP and the mean fluorescence intensity of integrin β3 (CD61) of OLC were also measured. Results (1) The effect on the differentiation of OLC: The addition of pioglitazone at the start of the culture period induced a dose-dependent decrease in TRAP-positive OLC and the activity of TRAP (P < 0.01 or P < 0.05) ; the mRNA expression of PPARγ2 was up-regulated by 5 and 10 μmol/L pioglitazone in the early stage of incubation and attenuated with thematuration of OLC on the contrary, however, the expression of RANK was down-regulated by 5 and 10 μmol/L piolitazone in every stage of incubation (P < 0.05 or P < 0.01), combined with decrease in TRAP-positive OLC from day 3 by 10 μmol/L pioglitazone. (2) The effect on the function of OLC: the number of bone resorption pits and the ratio of its area on dentin slice were decreased in groups of 5 and 10 μmol/L pioglitazone (P < 0.01 orP < 0.05), no obvious change was noted in the group with 1 μmol/L pioglitazone compared with the control group; the mean fluorescence intensity of CD61 were down-regulated in groups of 5 and 10 μmol/L pioglitazone (P < 0.05 or P <0.01). Conclusion Activation of PPARγ2 pathway by pioglitazone could partially inhibit differentiation and function of OLC derived from rat BMC.

Effect of Two Different Cultural Methods on Osteoclast Bone Absorption Activity and Expression of ATPase a3 Gene / 浙江中医药大学学报

[Objective] To investigate the difference of bone Absorption function of the rat osteoclast(OC) in different cultural methods, and the difference in the levels of mRNA expression of bone Absorption key gene, ATPase a3 gene, and provide the basis for further investigation on the OC in vitro. [Methods] with mechanical separation method, mature osteoclast was separated from inner wall of 24 hours newborn rat long bones medullary cavity; and induction culture methods, bone marrow cell was induced to osteoclast like cell(OLC) by using 1,25(OH)2D3.The morphological and functional change of osteoclast was observed and the difference in the level of ATPase a3 mRNA expression was determined.[ Result]OC and OLC are multinucleated giant cells, which can be stained positive by potartrate resistant acid phosphatase(TRAP), and can form bone Absorption lacuna. The number of OLC in induction method is more than in mechanical separation method, but bone Absorption lacuna is smaller and shallower than the early stage of induction. The late stage of OCL is extremely similar to OC. There is no significant difference of ATPase a3 mRNA expression from the cells between mechanical separation 8 hour and induction culture 6 days, but either of them is significantly less than inducing 8 days. [Conclusion]Induction method can produce a large number of OLC which is superior to mechanical separation method, but its bone resorption function is weaker in the earlier stage, because bone resorption function is associated with the number of nucleus. The late stage of OCL is extremely similar to OC of mechanical separation method. And it can be used in any experiments.