Effects Of Different Concentrations Of Salmon Calcitonin On Cementoblast Proliferation,differentiation And Mineralization In Vitro

Objective:To study the effect of different concentrations of salmon calcitonin on the proliferation,differentiation,mineralization,apoptosis and cell cycle of cementoblast OCCM-30 at different time point,and provide a preliminary experimental research basis for whether salmon calcitonin can interfere in root resorption and its possible intervention mechanism.Method:Cementoblast cell line OCCM-30 was divided into 6 groups(five experimental groups and one control group);different concentrations of salmon calcitonin(0.1IU/L?1IU/L?10IU/L?100IU/L?1000IU/L)were prepared by culture medium and the salmon calcitonin was added into OCCM-30 as experimental group,the OCCM-30 only added to the culture medium was used as the control group.In this experiment,the following items are tested:cell proliferation at different time(24h,48 h,72h)by MTT was detected;alkaline phosphatase(ALP)activity in different time points(48h,72h)by Micro ELISA was detected;the content of mineralized nodules by alizarin red method in Fourteenth days was detected;cell apoptosis and cell cycle of OCCM-30 by flow cytometry was detected.The experimental data are analyzed by SPSS 18.Results:1.MTT method result:the effect of salmon calcitonin to OCCM-30 of different concentrations at different time(24h,48 h,72h),the experimental group and the control group absorbance value analysis showed that differences in cell proliferation effect of salmon calcitonin 24 h is not significant,but only significant in 100IU/L group(P < 0.05);48h,10IU/,100IU/L,L 1000IU/L salmon calcitonin has a promoting effect on OCCM-30;after 72 h,only 0.1IU/L of salmon calcitonin on theproliferation of OCCM-30 effect is not significant,the remaining concentrations(1IU/L,10IU/L,100IU/L,1000IU/L)significantly promoted the proliferation of OCCM-30,and the 100IU/L effect is most obvious,the difference was statistically significant,and salmon calcitonin to promote the proliferation of OCCM-30 as time increases.2.Micro ELISA method for detection of alkaline phosphatase(ALP)activity results showed that at different time points(48h,72h),salmon calcitonin showed promoting effect on OCCM-30 cell ALP activity,but no significant at low concentration,but high concentration(10-1000IU/L)increased,the 100IU/L salmon calcitonin to OCCM-30 is most obvious.The difference was also statistically significant(P < 0.01).And the ALP activity of OCCM-30 in 72 h was greater than that of 48 h,indicating that the activity of ALP in OCCM-30 increased with time.3.Determination of mineralization by alizarin red staining showed that OCCM-30 can generate mineralized nodules under mineralization induction,but the promotion effect is not obvious in low concentration(0.1IU/L,1IU/L,10IU/L),the difference was not statistically significant;high concentration of salmon calcitonin(100IU/L,1000IU/L)with statistical significance for promoting mineralization(P <0.01),and reached the peak in the 100IU/L group,which showed that 100IU/L salmon calcitonin is the optimal concentration of promoting mineralization of OCCM-30.4.The experimental results of apoptosis showed that the serum starvation can induce the apoptosis of OCCM-30,and adding different concentrations of salmon calcitonin can inhibit the apoptosis of OCCM-30,and the inhibitory effect increased with salmon calcitonin concentration raised,the effect of 1000IU/L salmon calcitonin is the most obvious,the difference was statistically significant(P < 0.01).5.The cell cycle experiments showed that different concentrations of salmon calcitonin(1IU/L,10IU/L,100IU/L,1000IU/L)can improve the cell proliferation index(PI)while in 100IU/L salmon calcitonin on OCCM-30 PI is the most obvious,the difference was statistically significant(P < 0.01).Conclusion:1.A certain concentration of salmon calcitonin could promote the proliferation and differentiation of cementoblast OCCM-30,and with the increase of time,the effect of salmon calcitonin in promoting proliferation and differentiation is gradually increased.2.Mineralization induction can stimulate OCCM-30 to produce mineralized nodules,and a certain concentration of salmon calcitonin can promote the formation of mineralized nodules in OCCM-30.3.Serum starvation can induce the apoptosis of OCCM-30,and the inhibitory effect of salmon calcitonin on OCCM-30 apoptosis is concentration dependent.4.Different concentrations of salmon calcitonin can affect OCCM-30 cell cycle and increase cell proliferation index.