| Partâ… :The culture of osteoclast-like cell obtained from Peripheral blood mononuclear cells(PBMCs)of patients with rheumatoid arthritis in vitroObjecitive:Establish the system of osteoclast-like cell(OLC)cultural in vitro and appraise the cultured cells were osteoclasts.Methods:PBMCs were isolated by means of density gradient centrifugation from RA patients and were randomly assigned to 2 groups: group A(macrophage-colony stimulating factor,M-CSF,25μg/L),group B (Receptor activator of NF-κB ligand,RANKL,5,10,20,25,50μg/L,and 25μg/L M-CSF).After cultured for4,12,20 days,the cells onglass coverships were fixed and stained for tartrate resistant acid phosphatase,the bone slices were identified with toluidine blue stain.Result:1.The effect on the differentiation of OLC:RANKL induced a dose-dependent increase in TRAP positive OLC(P＜0.05).2.The effect on the activation of OLC: RANKL and M-CSF induced a dose-dependent increase in the number of bone resorptive lacuna on bone slices(P＜0.05),but we can not see bone resorptive lacuna on bone slices in group A.Conclusion:RANKL and M-CSF can induce the differentiation and activation of osteoclast derived from PBMCs of patient with rheumatoid arthritis,this cultural method is convenient,potent and repretitive for OLC formation in vitro.Partâ…¡:Effect of Technetium 99mTc methylenediphosphonate on the differentiation and activation of osteoclast-like cell and its molecular mechanismsObjecitive:To determine whether Technetium 99mTc methylenediphosphonate(99Tc-MDP)act directly on human osteoclast precursors obtained from patients with RA and influence monocyte-osteoclast differentiation induced by RANKL in vitro,the manner in which 99Tc-MDP affected the RANKL/RANK signaling pathway.Methods:PBMCs were isolated from RA patients by means of density gradient centrifugation and were randomly assigned to 4 groups with different concentration of 99Tc-MDP(final concentration is 0,5,10, 20mg/L)in the presence of RANKL(25μg/L)and M-CSF(25μg/L), Osteoclast formation was determined by assessing the number of TRAPstaining cells and measuring the extent of lacunar resorption after cultured for4,12,20 days.99Tc-MDP were added at the 4,8,12,16,20 day to the identify the stage at which differentiation was affected.The expression of osteoclast transcription factors,such as TNF receptor-associated factor 6 (TRAF6),nuclear factor of activated T cells c1(NFATc1),microphthalmia transcription factor(MITF)and PU.1 was assayed by reverse transcription-polymerase chain reaction.Result:1.The effect on the differentiation of OLC:99Tc-MDP induced a dose-dependent decrease in TRAP positive OLC(P＜0.05).2.The effect on the activation of OLC:99Tc-MDP induced a dose-dependent decrease in the number of bone resorptive lacuna on bone slices(P＜0.05).3.The effect on the RANKL/RANK signaling pathway:99Tc-MDP can suppress the mRNA expression of MITF,NFATc1(P＜0.05),while has on effect on the mRNA expression of TRAF-6,PU.1.Conclusion:99Tc-MDP acts directly on human osteoclast precursors in RA patients and exerts their immunosuppressive effects on human monocyte-osteoclast formation via targeting both the NFAT pathway and activation pathway for MITF,then has some protecting effect on patients with rheumatoid arthritis. |
