| Objective:Using mouse Monocytes RAW264.7clone culture systemto study the effect of Rhein on formation and differentiation of osteoclastand at the same time, to discuss whether the inhibitive effect of Rhein onosteoclast is by apoptotic pathway, in order to provide evidence to explorefurther relationship between Rhein and osteoclastMethord:Mouse Monocytes RAW264.7clone culture system isinduced and cultured to osteoclasts in viro, to observe the effect of Rheinon formation and differentiation of osteoclast,and at the same time,toanalyse the effect of Rhein on the apoptosis in osteoclast by FCM(flowcytometry).1Cell culture and medicine administration:1.1Cells recovery and culture: Take the frozen mouse MonocytesRAW264.7clone to one tube and recover cells quickly into37℃constanttemperature water bath tin, and then transfer the liquid into centrifugewhich consists of the α-MEM containing10%fetal bovine serum. Extractthe cells under1000turn/min centrifugalization for5minutes and discardthe supernatant liquid,then add α-MEM containing10%fetal bovineserum, blow and mix enough to form cell suspension, transfer it intoculture bottle,put and culture it into CO2incubator under the37℃,5%CO2condition. When the cell density is up to90%,incubat withTrypsin/EDTA, then harvest cells, extract the cells under1000turn/mincentrifugalization for5minutes and discard the supernatant liquid,thenadd the α-MEM containing10%fetal bovine serum, blow and mixenough to form cell suspension, prepair the4.5×104cells/ml cellsuspension after count cell number by FACS and calculate theconcentration of this original cell liquid, Add sRANKL50ng/ml,M-CSF 20ng/ml,penicillin100u/ml and streptomycin100μg/ml. Seed cells in96–well plate(4rows6arranges),4.5×104cells/ml cell suspension150μlwas added to each well, Put this cell plate into CO2incubator after seeding,culture cells under the37℃,5%CO2condition. And the totally cultureprocedure is5days.1.2The medicine adds: After seeding,the first arrange is the controlgroup and Rhein was added from the second arrange to the sixtharrange in96–well plate,the concentration is10-3,10-4,10-5,10-6,10-7mol/L respectively.2Tartrate Resistant Acid Phosphatase (TRAP) stainings:Mouse Monocytes RAW264.7clone culture system were stained byTRAP after being cultured for5days,TRAP positive cells were observedand calculated under the microscope.3FCM(flow cytometry):3.1cell culture: Using passage cells continues to be cultured, when thecell density is up to90%of the diapire, proceed into digestion,then harvestcells. Extract the cells under1000turn/min centrifugalization for5minutes and discard the supernatant liquid,then add the α-MEMcontaining10%fetal bovine serum, blow and mix enough to form cellsuspension, prepair the4.5×104cells/ml cell suspension after count cellnumber by FACS and calculate the concentration of this original cellliquid. Add sRANKL50ng/ml,M-CSF20ng/ml,penicillin100u/ml andstreptomycin100μg/ml. Seed cells in2wells of24–well plate,4.5×104cells/ml cell suspension500μl was added to each well.3.2The medicine adds: The first well is the control group,no medicine isadded, the second well is added the Rhein concentration10-3mol/L inwhich it can extremely inhibit the formation of osteoclasts.3.3Detection cell apoptosis by FCM: When cells are cultured to the fifthday,proceed into digestion,then harvest cells. Extract the cells under1000turn/min centrifugalization for5minutes and discard the supernatantliquid.Blow and mix enough to form cell suspension by adding4℃ phosphate buffer solution(PBS), extract the cells under1000turn/mincentrifugalization for5minutes once again and discard the supernatantliquid. Blow and mix enough to form cell suspension by adding1xPBS300μl,standing30min after adding Annexin V5ul and PI10ul to eachtube,and then add1xPBS200μl,take the tubes to detect cell apoptosis byFCM at once. This procedure is required to be repeated three times.4The statistics analysis: The data processing was performed by analysisof variance of non-parametric test and S-N-K test,the data of FCM wasperformed by t-tests with SPSS13.0software.Result:1Morphology characteristic of osteoclasts:On the first day the cultured cells in each group did not formosteoclasts; From the second day on, there was a few osteoclasts in thecontrol group, nothing is changed in the experimental groups;On the thirdday, there was a few osteoclasts in the experimental groups;On the fifthday mature osteoclasts were observed in each group. A great many ofmature osteoclasts were found under microscopy after the TRAP staining,its morphology characteristic: its volume is bigger, funnel form, oval form,fry in oil egg form, linear form or irregular form. Osteoclast containsplenty of red cytoplasm with clear vacuole structure sometimes, several toeven more than10cell nucleuses with significant nucleoli.2The inhibitive effect of Rhein on formation and differentiation ofosteoclasts was observed. On the same condition,compared to the lowerconcentration,there was fewer osteoclasts in higher concentration. In themouse Monocytes RAW264.7clone culture system, the first arrange inthe TCPS was the control group,no medicine was added,the most quantityof osteoclasts was formed at last; in the second arrange, the least quantityof osteoclasts was formed; from the third arrange to the sixth,Rhein wasadded,the concentration was10-4,10-5,10-6,10-7mol/L respectively.Osteoclasts were formed in each group in the end, furthermore,thequantities were more than it in the second arrange and increased progressively with Gradient Change. Compared to the first and secondarrange, a remarkable difference in statistics was found (p<0.05),whichexplained the inhibitive effect of Rhein on formation and differentiation ofosteoclasts.3The effect of Rhein on formation and differentiation of osteoclasts wascarried out by cell apoptosis,compared to the control group,through FCMscatter diagram,which in the experimental group distributed increasinglyin Q2and Q4quadrant,and what is more,there was a statisticalsignificance between the control and experimental group,which explainedthe effect of Rhein on formation and differentiation of osteoclasts wascarried out by cell apoptosis,and mainly effected the late apoptosis ofcells.Conclusion:1The Rhein could inhibit the formation and differentiation of osteoclasts.2The effect of Rhein on the formation and differentiation of osteoclastswas carried out by cell apoptosis.3The Rhein mainly influences the middle and late apoptosis of cells. |
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