Effect Of Glucose On The Differentiation And Activation Of Osteoclast Derived From Rat Bone Marrow Cells

PartⅠ:The culture of osteoclast-like cell obtained from rat bone marrow cells in vitroObjective:Establish the system of osteoclast-like cell(OLC)cultural in vitro and appraise the cultured cells were osteoclasts.Methods:Rat bone marrow mononuclear cells obtained from 4-6week-old Wistar rats were incubated with M-CSF and RANKL to induce the formation of OLC.Morphologic features by inverted phase contrast microscope,light microscope and electron microscope were observed to evaluate OLC.Result:Under inverted phase contrast microscope and light microscope,we can see the multinucleated OLC of typical and multiform,TRAP positive cells;under electron microscope OLC on dentin slice were different in size and feature,we can see bone absorptive lacuna below OLC,so we can indicate the cultured cells were OLC of bone resorptive activity.Conclusion:In the first day of OLC formation from bone marrow mononuclear cells obtained from rats induced by M-CSF and RANKL,this cultural method is convenient,potent and repetitive for OLC formation in vitro.PartⅡ:Effect of glucose on the differentiation and activation of osteoclast derived from rat bone marrow cellsObjective:To study the effect of glucose on the differentiation and activation of osteoclast derived from rat bone marrow cells,and probe the relationship between glucose and osteoclast.Methods:In the first day of OLC formation from bone marrow mononuclear cells obtained from rats induced by macrophage colony stimulating factor(M-CSF)and receptor activator of NF-κB ligand(RANKL),glucose hydrochloride with different concentration(final concentration is 0,5.5,15,25 mmol/L)were added.Effects of glucose on the differentiation of OLC was studied by the number of TRAP positive cells,the activity of TRAP and the mRNA expression of receptor activator of NF-κB(RANK)using semi-determine RT-PCR;Effects of glucose on the activation of OLC was studied by counting the number of bone absorptive lacuna and the ratio of its area on dentin slice and the expression of integrinαvβ3(CD61)using image analyzer and flow cytometry respectively.Result: 1.The effect on the differentiation of OLC:the glucose of different concentration at the 7 day of the culture induced a dose-dependent increase in TRAP positive OLC,the amount of TRAP-positive OLC in high glucose(25mmol/L)group were more than control(0mmol/L)group (P＜0.01);The amount of TRAP-positive OLC in high glucose group began more than control group at day 3(P＜0.01),we presumed the effect of the accelerate to OLC was beginning at nonage of differentiation.TRAP activities in high glucose group were higher than control group and glucose 5.5mmol/L group at day7(P＜0.01),no obviously changes in glucose 5.5mmol/L group and glucose 15mmol/L group compared with the control group,that indicated glucose accelerate the differentiation of OLC and that is relative to the concentration.2.The effect on the activation of OLC:the number of bone absorptive lacuna and the ratio of its area on dentin slice in high glucose group were increased and had significant difference than other 3 groups at day7(P＜0.01,P＜0.05).The expression and mean fluorescence intensity of CD61 were up-regulated in high glucose group than other 3 groups at day3(P＜0.01, P＜0.05),indicated that high glucose may accelerate the assembling and merging of the OLC at nonage of differentiation.The expression of CD61 were up-regulated in high glucose group and had significant difference than control group at day5,7(P＜0.05),combine with the number of bone absorptive lacuna and the ratio of its area on dentin slice,we presumed high glucose and enhance the activation of OLC.Conclusion:High glucose may accelerate the differentiation of OLC,and enhance the activation of OLC,that may be one of the key pathogen tic factor of diabetic osteoporosis under bone marrow conditions.