Experimental Research On Effect Of Lanthanum Chloride On Osteoclastogensis And Related Gene Expression In RANkL-stimulated RAW264.7Cells

Objective：To observe the role of lanthanum chloride on RANKL（Receptor activator ofNF-κB ligand） which induce murine macrophage RAW264.7differentiate intoosteoclast. To observe expression of osteoclast gene（c-fos、trap、cathepsin K、MMP-9） and NF-kappaB pathway genes-mRNA（traf6、c-src、RANK、Fra1）.Toobserve influence of Tartrate-resistant phosphatase (TRAP) staining for the number ofosteoclast formation and MTT for activity of osteoclast.Cell apoptosis were measuredby flow cytometry. Preliminary analysis of the relationship between the lanthanumelements and osteoclast which was induced by RANKL.Methods：The experiment is mouse cell lines macrophage cell line RAW264.7whichpurchased from the cell bank of the Chinese Academy of Sciences (ATCC:TIB-71).Lanthanum chloride (LaCl3·7H20,99.9%) were purchased from Simga. RANKL(Receptor Activator of Nuclear Factor-kappa B ligand) were purchased from the R&D company. In accordance with the experimental design different of lanthanumchloride concentrations were divided into five groups:①The blank group:RAW264.7②The control group:RAW264.7+50ng/ml RANKL③The experimental group:lowconcentration of Lanthanum (RAW264.7+50ng/ml RANKL+2.5μmol/L LaCl3)④The experimental group:median concentration of Lanthanum (RAW264.7+50ng/mlRANKL+20μ mol/L LaCl3)⑤The experimental group:high concentration ofLanthanum(RAW264.7+50ng/ml RANKL+100μmol/L LaCl3).By MTT and flowcytometry,to observe lanthanum has activity effort of RAW264.7that was induced byRANKL.To investigate expression of osteoclasts by RT-PCR,to analysis of the role oflanthanum on the number of genesis osteoclast by TRAP staining.Results：1. It’s no obvious toxic effect that MTT test RAW264.7that was induced byRANKL in different concentration of lanthanum in24,48and72hours, it’s not statistical significance in difference OD value（P>0.05）.2. The TRAP staining test that RANKL induce RAW264.7to generate thenumber of osteoclast in different concentration of lanthanum. There is no matureosteoclasts in blank group.Compared with the control group（163.00±5.82）/cm2,inthe experimental group(137.60±4.54)/cm2、(102.70±3.23)/cm2、(72.20±5.28)/cm2,osteoclasts cells are reductive（P<0.05）.it’s no significant difference thatnumber of osteoclasts in different concentrations in the experimental group（P>0.05）.3. Osteoclasts apoptosis were measured by flow cytometry, there was nosignificant in statistically in different group（P>0.05）.4. osteoclast genes and pathways c-fos、 trap、 RANK、 traf6、 c-src、Fra1、cathepsin and MMP-9. Compared with Control group, the experimental groupincrease in the expression（P<0.05）, and it is obvious.mRNA has no obviousdifference in each experimental group（P>0.05）.Conclusions：1. RANKL can be separately used to induce murine macrophage-RAW264.7todifferentiate into mature osteoclast.2. It has no obvious toxic effect that RANKL induced RAW264.7todifferentiate, proliferate in different concentration of lanthanum chloride （2.5、20、100μmol/L）.3. Different concentration of lanthanum chloride （2.5、20、100μmol/L）reduce RAW264.7to differentiate into mature osteoclast which was induced byRANKL. But it has no obvious difference in different concentration.