The Effects Of Tnf-α On Proliferation, Apoptosis, RANKL/OPG MRNA Expression Of Cementoblast In Vitro

Objective:The study aims to investigate the influence of different concentration of tumor necrosis factor alpha(TNF-α) on proliferation, apoptosis and expression of RANKL, OPG mRNA of cementoblast in vitro, providing evidence for the biological mechanism of TNF-α involved in the root resorption. Method:Murine cementoblast cell line OCCM-30 were divided into six groups(five experimental group and one control group). Different concentrations of 5、10、25、50、100 ng/ml TNF- α were respectively added into the experimental groups, and without drug as control group. MTT was performed to detect cell proliferation after exposed to TNF- α for 24、48、72 hours; Flow Cytometry(FCM) was performed to detect cell apoptosis after exposed to TNF- α for 48 hours; RT-PCR was performed to test the expression of RANKL/OPG mRNA after exposed to TNF- α for 24 hours. The results were analyzed with SPSS statistics V 19.0 software. Results:1. MTT results shows that: after exposed to different concentration of TNF- α for 24 hours, the OD value of 5ng/ml group is higher than the control group, the OD value of 25ng/ml、50ng/ml、100ng/ml group is lower than control group, and the difference has statistical significance(P<0.05). After exposed to TNF-α for 48 hours and 72 hours, the OD value of all experimental groups gradually decrease compared with the control group, and the difference has great statistical significance(P<0.01).2. Flow Cytometry results shows that: after exposed to different concentration of TNF- α for 48 hours, the late apoptosis rate of OCCM-30 in experimantal groups increases gradually with the rising of concentration, the difference between groups has obvious statistical significance(P<0.01).3. RT-PCR results shows that: the expression of RANKL mRNA of OCCM-30 in 5 ng/ml group is lower than control group, the 50ng/ml、100ng/ml group is higher than control group, and the difference has statistical significance(P<0.05). Low concentration of TNF- α(5ng/ml、10ng/ml) has little influence in the OPG mRNA expression and RANKL/OPG ratio(P>0.05). Higher concentration(25ng/ml 、50ng/ml 、 100ng/ml) can inhibit the expression of OPG mRNA and greatly up-regulates the RANKL/OPG ratio(P<0.01). The effect is most significant in the concentration of 100 ng/ml. Conclusion:1. The effect of TNF-α on the proliferation of cementoblast is biphasic: presenting low concentration promote cell proliferation, high concentration inhibit cell proliferation, and there is significant concentration and time dependence.2. TNF-α can promote apoptosis of cementoblasts, and the effect is dose--dependent.3. High concentration of TNF-α can promote the expression of RANKL, inhibit the expression of OPG, up-regulate the ratio of RANKL/OPG, stimulate the differentiation and activation of osteoclast, and participate in the root resorption indirectly.