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Technetium 99mTc Methylenediphosphonate Inhibits Differentiation of PBMCs Derived from Patients with Rheumatoid Arthritis and Its Molecular Mechanisms

Author: HuoXiaoCong
Tutor: ZhangHao
School: Central South University
Course:
Keywords: rheumatoid arthritis osteoclast NFATc1 Receptor activator of NF-κB ligand Technetium[99Tc]methylenediphosphonate
CLC: R593.22
Type: Master's thesis
Year: 2008
Downloads: 60
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Abstract


PartⅠ: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.PartⅡ: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.

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CLC: > Medicine, health > Internal Medicine > Systemic disease > Autoimmune diseases > Autoimmune diseases, connective tissue disease > Rheumatoid arthritis
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