Comparative Study Of M-CSF, RANKL And 1,25-(OH)₂D₃ On Formation And Differention Of Osteoclast

Objective: To study the influence of M-CSF,RANKL and 1,25-(OH)₂D₃ on formation and differentiation of cultured osteoclast in the whole bone marrow cell culture system and explore the interaction and synergy of these three cytokines, which will provide the theory evidence for the futher study on osteoclast and screening of cytokines.Method:A 4-week-old SD male rat(weight 80-120g) was selected to obtain the whole marrow culture system.The influence of M-CSF,RANKL and 1,25-(OH)₂D₃ on the formation and differentiation of osteoclasts were observed and the effect of three cytokines were compared.1 Cell culture and cytokines adds;1.1 The whole marrow culture system:Under ether anaesthesia,the femur and tibia were gained from a 4-week-old SD male rat(weight 80-120g) with sterilization.Cut away epiphysis and expose bone cavity, then flush the cavity by 10ml a-MEM culture fluid to extract the whole marrow cells,Placed in centrifuge 5 minutes (1500 turn/min)after blow and mix enough to form cell suspension.Discarded the supernatant liquid,then added the a-MEM culture fluid containing 15% FBS(fetal bovine serum), blow and mix enough to form cell suspenion. Prepare the 2×10⁶cells/ml cell suspension after compute a cell number by FACS and calculating the concentration of this original cell liquid. Added 100 units of Penicillin per milliliter and 100 micrograms of Streptomycin per milliliter. The cells were seeded in 24-well culture plates (4 line 6), each well by adding the cell suspension 0.5ml of 2×10⁶cells/ml.1.2 Cytokines adds: The first column of 24-well cell culture plate as the control group,without adding any drug;The second column by adding 1,25-(OH)2D(31×10^-8Mol/ml); The third column by adding 1,25-(OH)₂D₃ and RANKL(1,25-(OH)₂D₃ 1×10^-8Mol/ml and RANKL 20ng/ml);The fourth column by adding 1,25-(OH)₂D₃ and M-CSF(1,25-(OH)₂D₃ 1×10^-8Mol/ml and M-CSF 20ng/ml);The fifth column by adding 1,25-(OH)₂D₃,M-CSF and RANKL(1,25-(OH)₂D₃ 1×10^-8 Mol/ml,M-CSF 20ng/ml and RANKL 20ng/ml);The sixth column by adding RANKL and M-CSF(RANKL 20ng/ml and M-CSF 20ng/ml)。1.3 Cell culture: Put the 24-well cell plate into CO₂ incubator after adding cytokines,culture cells under the 37℃, 5% CO₂ condition.Changed culture fluid on the 4^th day for one time,and totally culture procedure is 7 days.2 Tartrate Resistant Acid Phosphatase (TRAP) stainings: The whole marrow culture system was stained by TRAP after being culture for 7 days respectively. TRAP positive cells were observed and calculated under the microscope (more than 3 nucleus in the whole marrow cell culture system).3 SPSS13.0 software used for statistical analysis. Data processing using non-parametric analysis of variance and S-N-K test.Result:1 Morphological characteristics of osteoclast:In the first three days culturing,no osteoclast was formed in each group,few osteoclast were formed in experimental group from the fifth day of culture.At the seventh day,the osteoclasts had been mature in the experimental groups, while in the control group,osteoclast formation was always not found.A large number of mature ostelclasts were found after the TRAP staining, which were characteristiced by larger cell size , oval form, funnel form, fry in oil egg form, linear form or irregular form.Osteoclast contained large number of red cytoplasm with clear vacuole struvture, several to two or three dozens of cell nucleuses with significant nucleoli, and reachs out many lamellipodia(Figure 1-5).2 M-CSF, RANKL and 1,25-(OH)₂D₃ played a facilitating role on osteoclast formation and differentiation.Under the same conditions, multi-factor formed more osteoclasts than single factor and two-factor.In the whole bone marrow cell culture system, the first column (control group)of culture plate did not add cytokines,finaly,there was no formation of osteoclasts;in the second column by adding 1,25-(OH)₂D₃, there was formation of osteoclasts,which proved that 1,25-(OH)₂D₃ has a promoting role on osteoclast formation and differentiation; In the third column and the fourth column,except adding 1,25-(OH)₂D₃, also added RANKL and M-CSF, Eventually had the formation of osteoclasts, and the number of osteoclasts was larger than the second column,and compared with the second column had statistically significant difference(P<0.05), RANKL and M-CSF also play a promoting role in ostelclast formation and differentiation, but the third and fourth column showed no significant difference(P>0.05); In the fifth column, the largest number of osteoclasts were formed after added M-CSF,RANKL and 1,25-(OH)₂D₃,which indicated that three cytokines had synergistic effects on osteoclast formation and differentiation, and compared with the second, third, fourth and sixth column there was significant difference (P<0.05). In the sixth column also formed osteoclasts after added RANKL and M-CSF, but the number of osteoclasts were similar compared with the third and fourth column (Table 1,histogram).Conclusion:1 M-CSF, RANKL and 1,25-(OH)₂D₃ have promoting role on osteoclast formation and differentiation.2 There is significant difference on osteoclast formation and differentiation between the single factor and two-factor.3 There is a synergistic effect on osteoclast formation and differentiation,when three cytokines are together exist.