The Effect Of Taxol On Formation, Differentiation And Apoptosis Of Osteoclast

Objective: To research the effect of Taxol on the formation anddifferentiation preosteoclast in RAW264.7mouse macrophage cell cultureline（preosteoclast lines） in vitro. And to explore whether the formation anddifferentiation of preosteoclast were mediated by Taxol-induced apoptosis byFlowcytometry.Methord: Observe the effect of Taxol on formation and differentiationin the RAW264.7mouse macrophage cell culture line （preosteoclast lines） invitro. Meanwhile, analyse the effects of the taxol on the apoptosis byFlowcytometry.1Cell culture and medicine administration:1.1cells recovery and culture: Take two tube frozen RAW264.7mousemacrophage cell from-70℃liquid nitrogen tank into37℃constanttemperature water bath box to recover rapidly（the recovery time less than1minute）, then transfer the recovered two tube cells into centrifuge whichconsists of the-MEM containing10%fetal bovine serum.Aftercentrifugation for5minutes with1000turn/min, discard the supernatant liquid.Then add-MEM containing10%fetal bovine serum10ml. Mix the cellswell by blowing to reform Single cell suspension, transfer them into flask toculture for48-72huors in CO₂incubator under the37℃,5%CO₂condition.When the cell density in the flask bottom is almost up to90%,collection thesuspending cell with1%of the pancreatic enzyme+EDTA under1000turn/min centrifugalization for5minutes and discard the supernatant then addα-MEM containing10%fetal bovine serum5ml. Mix cells well by blowing toreform single cell suspension, calculate the cells with hemacytometer anddilute them to4.5×10⁴cells/ml. Add sRANKL50ng/ml, penicillin100u/mland streptomycin100μg/ml. Seed cells in96–well plate（4rows6arranges）, with4.5×10⁴cells/ml cell suspension150μl in each well. In the end, put andculture the96–well plate into CO₂incubator under the37℃,5%CO₂condition for5days.1.2The medicine administration: After seeding desired density cells in thesterilizated96well culture plate （4row and6column）, add media without anytaxol to the first arrange as control,add10^-5,10^-6,10^-7,10^-8,10^-9mol/L taxolinto the second arrange to the sixth arrange,respectively.2Tartrate Resistant Acid Phosphatase （TRAP） stainings:After cultured for5days,the RAW264.7mouse macrophage cell werestained by TRAP（According to the kit instructions operation）, then observeand calculate TRAP positive cells under the microscope.3FCM（flow cytometry）:3.1cell culture: Recovery and culture the cells for flow cytometry. When thecell density in the flask bottom is almost up to90%, harvest the cells with1%trypsin+EDTA, then add-MEM containing10%fetal bovine serum8ml.Mix the cells by blowing to reform single cell suspension, collect thesuspending cell into centrifuge tube under1000turn/min centrifugalization for5minutes and discard the supernatant. Then add-MEM containing10%fetalbovine serum5ml again. Mix the cells by blowing to reform single cellsuspension, calculate the cell with hemacytometer and dilute them into4.5×10⁴cells/ml. Add sRANKL50ng/ml, penicillin100u/ml and streptomycin100μg/ml. Seed the500μl cell suspension with cytokines and two antibioticsin2wells of24–well plate.3.2The medicine administration: The first well is the control group withnothing added, the second well is added the10^-5mol/L Taxol in which it canextremely inhibit the formation of osteoclasts.3.3Detection cell apoptosis by FCM: When the cells are cultured for5days,harvest cells with1%trypsin+EDTA then collect the cells to two conical, oneis the control group, the other is treatment group. After centrifuging the twoconicals under1000turn/min for5minutes, discard the supernatant liquid.Add8ml4℃phosphate buffer solution（PBS） to reform single cell suspension by blowing and mixing enough, give the same centrifugalizationwhich is under1000turn/min for5minutes once again and discard thesupernatant. Mix the cells well by blowing to form cell suspension by adding1xPBS300μl, add Annexin V5μl and Pi10μl to each tube in30min, and thenadd1xPBS200μl, take the tubes to detect cell apoptosis by FCM after addingthe medicine5days at once.4The statistics analysis: The data processing was performed by analysis ofvariance of non-parametric test and S-N-K test with SPSS13.0software.Result:1morphology characteristic of preosteoclasts:On the first day there is no osteoclasts in each cell culture well. On thesecond and third day, a few osteoclasts can be observed in the control group,On the fifth day preosteoclasts can be observed in each group obviously. A lotof positive cells can be found under microscopy through the TRAP staining,the morphology characteristic is: size of cells are small, oval form, linear formor irregular form; plenty of red cytoplasm, nucleolus is smaller whichnumber is one to two, sometimes three.2Taxol has an inhibitory effect against the formation and differentiationof osteoclasts. Under the same conditions, the lower drug concentration, themore numbers of osteoclast to form. There is no medicine in the first arrangeas the control group, the most of osteoclasts formation in the end; the secondarrange adds Taxol10^-5mol/L concentration, the least of osteoclasts formation;from the third arrange to the sixth, the Taxol concentration was10^-6、10^-7、10^-8、10^-9mol/L respectively. The number of osteoclast formation is increasewith the increasing of concentrations. Compared with the first arrange, aremarkable difference in statistics was showed （p<0.05）, which descriptionthe Taxol has an inhibitive effect on formation and differentiation ofosteoclasts.3The Taxol-inhibited formation and differentiation of osteoclasts ismediated through the cell apoptosis way. The results of FCM with3times show that the most scatter of the control group is distributed in Q3 quadrant, which are survival cells; while the scatter of the experimentalgroup（the dosing group） are concentrated in Q2，Q4quadrant clearly, canpromote the middle-late apoptosis after treatment. From the test results wecan prove that Taxol influence the formation and differentiation of osteoclastsis through the cell apoptosis way.Conclusion:1Taxol could inhibit the formation and differentiation of osteoclasts.2The suppressive effect of Taxol on the formation and differentiation ofosteoclasts was mediated by cell apoptosis.