| ObjectiveTo induce rat osteoblasts apoptosis by different concentrations oflipopolysaccharide in vitro, and then select the appropriateconcentration of lipopolysaccharide to establish the model of ratosteoblasts apoptosis;To investigate the mechanism of osteoblastsapoptosis induced by lipopolysaccharide and provide experimentalbasis for the pathogenesis of osteonecrosis induced by infectivedisease.Methods1.Osteoblasts were isolated and cultured from the skull of SD rats24-hour-old by modified enzyme digestion; The third generation ofosteoblasts were used in this experiment.2.Identification of osteoblasts(1) Morphological change of the third generation of osteoblastsand the primary osteoblasts which were cultured1,7,14days wasobserved under the inverted phase contrast microscope.(2) The morphology of the third generation of osteoblasts whichwere cultured20days and stained by Alizarin red was observed underthe inverted phase contrast microscope.(3) The morphology of the third generation of osteoblasts whichwere cultured20days and stained by tetracycline was observed underthe fluorescence microscope.(4) The morphology of the third generation of osteoblasts which were cultured20days and alkaline phosphatase stained was observedunder the inverted phase contrast microscope.3.Establish the model of rat osteoblasts apoptosis induced bylipopolysaccharide.There were six groups which were induced by0.00,0.01,0.10，1.00,10.00,100.00μg/ml LPS in vitro experiment.The proliferation ofosteoblasts was evaluated by MTT assay at different time points.Theosteoblasts apoptosis was assessed by flow cytometry. And thenselect the appropriate concentration of lipopolysaccharide to establishthe model of rat osteoblasts apoptosis.4.Experimental groups:(1)The normal control group;Untreated osteoblasts.(2)LPS group: osteoblasts pretreated by10.00μg/ml LPS.(3)LPS+PMA group:osteoblasts pretreated by10.00μg/ml LPSand100nmol/LPMA.(4)LPS+Rottlerin group:osteoblasts pretreated by10.00μg/mlLPS and2μmol/L.5.The proliferation of osteoblasts was evaluated by MTT assay;The osteoblasts apoptosis of each group was assessed by flowcytometry and acridine orange/ethidium bromide fluorescence staining.The expression of apoptosis related proteins Caspase-3,Bcl-2,Baxand Phosphorylated PKC-δ were assayed by Western blot.Results1.Identification of rat osteoblastsThe isolated cells stick wall within24hours.Cell morphology werelong spindle,flake shape, triangle,etc.The mainly morphology werelong spindle. Cytoplasm has swelled, stretching outwards. Aftercultured7days after seeding, most cells were flake shape andbecome bigger. These cells have many pseudopodia,abundantcytoplasm and larger nucleus.The whole sample looks like pavingstone sample,Continue to grow, we found that many cells overlappinggrowth and black calcium nodule formation.Golden mineralized nodule was observed under fluorescent microscope stained by tetracycline.After stained by Alizarin red, red nodule was observed under aninverted microscope. Alkaline Phosphatase staining, the cytoplasmcolor was dark gray and the intercellular gaps were formed.2.Establish the model of rat osteoblasts apoptosis withappropriate concentration of lipopolysaccharide.The results of MTT assay showed that the OD value of LPSgroups, compared with the control group, was increased within0.01μg/ml to10.00μg/ml. And the cellular proliferation rate wassignificantly higher than the control group.The OD value of1.00μg/mlLPS group was the maximum，and the promoting effect of proliferationwas the maximum.The OD value of100.00μg/ml LPS group was lowerthan the control group,and the promoting effect of proliferation wasreduced. The results of flow cytometry showed that the meanapoptotic rates increased with the increase of concentration ofLPS.The apoptotic rates were respectively3.31%,5.25%,14.02%,25.61%,68.54%. And then we get the appropriate concentration oflipopolysaccharide, which was used to induce rat osteoblastsapoptosis, was10.00μg/ml.3.The results of MTT assay related with the cell proliferativeactivity of rat osteoblasts.The osteoblasts proliferative activity of LPS group,compared withthe control group, was reduced, the difference was statisticallysignificant(p<0.05);The osteoblasts proliferative activity of LPS andPMA group,campared with LPS,was reduced, the difference wasstatistically significant (p<0.05);The osteoblasts proliferative activityof LPS and Rottlerin group,campared with LPS,was increased, thedifference was statistically significant (p<0.05).4.The osteoblasts apoptosis rate of each group was assessed byflow cytometry.The results of flow cytometry showed that the cell apoptosis rateof the control group（%）3.51±0.46was lower than that of the group pretreated by Rottlerin（%）10.50±0.35, the difference was statisticallysignificant(t=6.421, p<0.05).The cell apoptosis rate of the grouppretreated by Rottlerin was lower than that of the group onlypretreated by LPS（%）19.03±0.54, the difference was statisticallysignificant(t=5.315,p<0.05).The cell apoptosis rate of the group onlypretreated by LPS was lower than that of the group pretreated by PMA（%）26.73±0.69, the difference was statistically significant(t=5.956,p<0.05).5.The osteoblasts apoptosis rate of each group was assessed byacridine orange/ethidium bromide(AO/EB)fluorescence staining.The results showed that the cell apoptosis rate of the controlgroup（%）3.8±1.03was lower than that of the group pretreated byLPS and Rottlerin（%）14.5±1.27; The cell apoptosis rate of the grouppretreated by LPS and Rottlerin was lower than that of the group onlypretreated by LPS（%）18.9±1.45; The cell apoptosis rate of the grouponly pretreated by LPS was lower than that of the group pretreated byLPS and PMA （%）31.4±1.71, the difference was statisticallysignificant(p<0.05).6.The expression of apoptosis related proteins andphosphorylated PKC-δ were assayed by Western blot.The results showed that the expression of Caspase-3Bax andphosphorylated PKC-δ of each experimental group, compared with thecontrol group, were increased,while the expression of Bcl-2wasreduced.The expression of Caspase-3Bax and phosphorylated PKC-δof the LPS and Rottlerin group were higer than that of the LPS group,while the expression of Bcl-2was lower. The expression ofCaspase-3、 Bax and phosphorylated PKC-δ of the LPS group werehigher than that of the LPS and PMA group, while the expression ofBcl-2was lower.Conclusions1.A low dose of LPS can enhance the cell proliferative activity. Among the experimental groups,the promoting effect of proliferation ofthe1.00μg/ml LPS group was the maximum.A higher dose of LPS canincrease the cell proliferative activity.2.The expression of Caspase-3,Bax and phosphorylated PKC-δ inrat osteoblasts induced by LPS were increased,while the expressionof Bcl-2reduced.3.The rat osteoblasts apoptosis can be induced by appropriateconcentration of lipopolysaccharide.The osteoblasts apoptosisinduced by LPS were related with phosphorylated PKC-δ. PMA couldincrease the osteoblasts apoptosis rate induced by LPS thoughenhance the phosphorylation of PKC-δ. while Rottlerin could reducethe osteoblasts apoptosis rate induced by LPS through inhibiting thephosphorylation of PKC-δ. |
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