The Effect Of Rhein On Formation And Differentiation Of Osteoclast

Objective: Using stromal cell-depleted bone marrow cell culture system(former osteoclast formation system) and mouse mononuclear cell RAW264.7clone to induce and culture preosteoclasts and mature osteoclasts respectively, toexplore the effect of Rhein on formation and differentiation of two osteoclast celllines in viro. Observing morphology variations of early stage and mature stage ofosteoclasts, in order to provide a new meditation which can prevent and treatdestructiveness paroxysm diseases due to osteoclast activity.Methord: Two osteoclast cell lines, stromal cell-depleted bone marrow cellculture system (former osteoclast formation system) and mouse mononuclear cellRAW264.7clone are induced and cultured to osteoclasts in viro. The effects ofRhein on formation and differentiation of preosteoclasts and mature osteoclasts areobserved respectively.1Cell culture and medicine administration:1.1Former osteoclast formation system: Under ether anaesthesia, the tibia andfemur were gained from a4-week-old SD male rat(80-120g) with sterilization.epiphysis were cut away and bone cavity were exposed, then flush the cavity by10ml a-MEM. Extract the whole marrow cells under1500turn/mincentrifugalization for5minutes, and discard the supernatant liquid. Then add20mlthe a-MEM containing15%(FBS; fetal bovine serum), blow and mix enough toform cell suspension. This is the whole marrow cell culture fluid. The wholemarrow media was condensed into2ml after withdrawing, mesenchymal cell wasremoved by Sephadex G10. Then collect cell original liquid10-12ml, prepare the2×106cells/ml cell suspension13ml after count cell number by FACS andcalculate the concentration of this original cell liquid. Add1α,25(OH)2D3andosteoclast differentiation factor (sRANKL), and make sure it’s eventuallyconcentration are10-8Mol/ml and40ng/ml respectively. Then seed with24-wellcell plate,0.5ml cell suspension with density of2×106cells/ml (1×106cells) were added to each well. Put this cell plate into CO2incubator after seeding, culturecells under the37℃,5%CO2condition. Change media on the4th day at one time,and the totally culture procedure is7days.1.2mouse mononuclear cell RAW264.7clone culture system: Take RAW264.7clone out of liquid nitrogen tin and then recover cells quickly into37℃water bathtin,transfer the liquid into15ml centrifuge tube with sterile suction tube,suck upnormal saline and flush the frozen tube twice,and transfer the cleaning mixture intoabove centrifuge tube, Extract the cells under1000turn/min centrifugalization for5minutes and discard the supernatant liquid, then add10ml the1640containing10%fetal bovine serum, blow and mix enough to form cell suspension and thenculture the cells according to the requirement of study.When the cells grow upwell in certain condition and the quantity is up to90%of the diapire, proceed intodigestion,then harvest cells, Extract the cells under1000turn/mincentrifugalization for5minutes and discard the supernatant liquid, then add10mlthe1640containing10%fetal bovine serum, blow and mix enough to form cellsuspension, prepair the4.5×104cells/ml cell suspension5ml after count cellnumber by FACS and calculate the concentration of this original cell liquid. Addosteoclast differentiation factor (sRANKL)50ng/ml. Then choose96–wellplate,seed24wells(4rows6arranges),4.5×104cells/ml cell suspension150μl wasadded to each well, Put this cell plate into CO2incubator after seeding, culturecells under the37℃,5%CO2condition. and the totally culture procedure is4~5days.1.3The medicine adds: Rhein was added in24-wellcell plate，make sure the finalconcentrations are0、10-3、10-4、10-5、10-6、10-7mol/L respectively.4rows6arrangeswere concluded, each row contains6kinds of concentration, and eachconcentration contains4wells (n=4). The first row0is the control group. Rheinwas added in24well（s4rows6arranges）of96-well cell plate，make sure the finalconcentrations are0、10-3、10-4、10-5、10-6、10-7mol/L respectively. each rowcontains6kinds of concentration, and each concentration contains4wells (n=4).The first row0is the control group.2Tartrate Resistant Acid Phosphatase (TRAP) stainings: Stromal cell-depleted bone marrow cell culture system were stained by TRAPafter being cultured for7days，mouse mononuclear cell RAW264.7clone culturesystem were stained by TRAP after being cultured for5days, TRAP positive cellswere observed and calculated under the microscope and then recorded.3The statistics analysis: The data processing was performed by mean±SDexamination analysis, each concentration was adopted by student’s t-test andnon-parametric analysis of variance with SPSS13.0software. All differentmedicine concentrations were compared with the control group.Result:1Former osteoclast formation system:1.1The Morphology of preosteoclasts A great many of preosteoclasts werefound after the TRAP staining, which characteristic is similar to maturityosteoclast, but only its volume is smaller. Its morphology is diversified to ovalform、class round form、quadrilateral form、linear form and irregular form andso on (Fig.1). Most preosteoclast has only one nucleus, but some have twonucleus.1.2The inhibitive effect of Rhein on formation of preosteoclast In the formerosteoclast formation system: the numbers of TRAP positive cells in10-3、10-4、10-5、10-6、10-7mol/L groups are4.98%、12.27%、18.57%、34.14%and78.57%of the control group respectively, statistics analysis showed the treatm-ent groups have the inhibitive effect on the preosteoclast formation, when themedicine concentration is10-3mol/L, comparing with control group, a remar-kable repression to the preosteoclast formation was showed (p﹤0.05)(Fig.3,Table1).2RAW264.7clone culture system:2.1The Morphology of osteoclastic-like cells (mature osteoclasts) A great dealof osteoclast-like cells were clearly found after the TRAP staining, whichcharacteristic is similar to mature osteoclast, its volume is bigger, its morphologyis diversified to starlike form、round form、class round form、fusiform form、linearform or irregular form(Fig.5). Most osteoclastic-like cells have more than onenucleus. 2.2The inhibitive effect of Rhein on formation of osteoclastic-like cells Inthe RAW264.7clone culture system, the numbers of TRAP positive cells(≥3nucleus) in10-3、10-4、10-5、10-6、10-7mol/L groups are4.36%、63.39%、72.73%、81.94%and92.73%of the control group respectively, statisticsanalysis showed the treatment groups have the inhibitive effect on theosteoclastic-like cell formation, when the medicine concentration is10-3mol/L, comparing with control group, a remarkable repression to theosteoclastic-like cell formation was showed (p﹤0.05)(Fig.6and Table2).Conclusion:1Rhein could inhibit the formation and differentiation of preosteoclasts andmature osteoclasts.2The inhibitive effects of Rhein on preosteoclasts and mature osteoclastsdepended on its concentration in certain medicine concentrations extent.