温度对过氧化氢抑制前成骨细胞MC3T3-E1细胞增殖和成骨分化的影响

Effect of temperature on the inhibitory effect induced by hydrogen peroxide on cell proliferation and osteogenic differentiation in preosteoblast MC3T3-E1 cells

目的探讨温度对过氧化氢(H_(2)O_(2))抑制前成骨细胞增殖和成骨分化的影响。方法取对数生长期MC3T3-E1细胞,随机分为0、450、500、550、600、650μmol·L^(-1)H_(2)O_(2)干预组,分别给予0、450、500、550、600、650μmol·L^(-1)H_(2)O_(2)溶液干预2 h。另取对数生长期MC3T3-E1细胞,随机分为对照组、模型组、低温组和高温组。对照组细胞置于37℃、含体积分数5%CO_(2)培养箱中孵育24 h;模型组细胞置于37℃、含体积分数5%CO_(2)培养箱中孵育24 h,并给予H_(2)O_(2)刺激2 h;低温组细胞置于32℃、含体积分数5%CO_(2)培养箱中孵育24 h,并给予H_(2)O_(2)刺激2 h;高温组细胞置于40℃、含体积分数5%CO_(2)培养箱中孵育24 h,并给予H_(2)O_(2)刺激2 h。采用细胞计数试剂盒-8检测各组细胞增殖能力,实时荧光定量聚合酶链反应法检测细胞中Runt相关转录因子2(RUNX2)、骨桥蛋白(OPN)和骨钙素(OC)mRNA表达水平,Western blot法检测MC3T3-E1细胞中RUNX2、OPN和OC蛋白表达水平。结果0、450、500μmol·L^(-1)H_(2)O_(2)干预组细胞增殖率比较差异无统计学意义(P>0.05);550、600、650μmol·L^(-1)H_(2)O_(2)干预组细胞增殖率显著低于0、450、500μmol·L^(-1)H_(2)O_(2)干预组,且随H_(2)O_(2)浓度增加细胞增殖率显著降低(P<0.05)。为保证后续实验有足够的细胞,选择H_(2)O_(2)的干预浓度为550μmol·L^(-1)。模型组和低温组细胞增殖率显著低于对照组和高温组,低温组细胞增殖率显著低于模型组(P<0.05);对照组与高温组细胞增殖率比较差异无统计学意义(P>0.05)。模型组和高温组细胞中RUNX2 mRNA相对表达量显著高于对照组和低温组,低温组细胞中RUNX2 mRNA相对表达量显著低于对照组(P<0.05);模型组与高温组细胞中RUNX2 mRNA相对表达量比较差异无统计学意义(P>0.05)。模型组、低温组和高温组细胞中OPN mRNA相对表达量显著高于对照组,低温组和高温组细胞中OPN mRNA相对表达量显著高于模型组,低温组细胞中OPN mRNA相对表达量显著高于高温组(P<0.05)。模型组、低温组和高温组细胞中OC mRNA相对表达量显著高于对照组,低温组和高温组细胞中OC mRNA相对表达量显著高于模型组(P<0.05);低温组与高温组细胞中OC mRNA相对表达量比较差异无统计学意义(P>0.05)。模型组、低温组和高温组细胞中RUNX2、OPN和OC蛋白相对表达量显著低于对照组(P<0.05)。低温组细胞中RUNX2、OPN蛋白相对表达量显著低于模型组和高温组,OC蛋白相对表达量显著低于高温组(P<0.05);低温组与模型组细胞中OC蛋白相对表达量比差异无统计学意义(P>0.05)。高温组细胞中RUNX2、OPN和OC蛋白相对表达量显著高于模型组(P<0.05)。结论H_(2)O_(2)可抑制MC3T3-E1细胞增殖和成骨分化,低温可增强H_(2)O_(2)对MC3T3-E1细胞增殖和成骨分化的抑制作用,高温可缓解H_(2)O_(2)对细胞增殖和成骨分化的抑制作用。RUNX2、OPN和OC蛋白可能在温度调控细胞增殖和成骨分化的过程中发挥重要作用。

Objective To investigate the effect of temperature on cell proliferation and osteogenic differentiation inhibition of preosteoblast induced by hydrogen peroxide(H_(2)O_(2)).Methods The MC3T3-E1 cells in the logarithmic phase were randomly divided into 0,450,500,550,600,650μmol·L^(-1) H_(2)O_(2) intervention groups and incubated with 0,450,500,550,600,650μmol·L^(-1) H_(2)O_(2) for 2 h,respectively.Other MC3T3-E1 cells in the logarithmic phase were selected and randomly divided into the control group,model group,low-temperature group,and high-temperature group.Cells in the control group were cultured in an incubator with 5%CO_(2) for 24 h at 37℃;cells in the model group were incubated with H_(2)O_(2) for 2 h and cultured in an incubator with 5%CO 2 for 24 h at 37℃;cells in the low-temperature group were incubated with H_(2)O_(2) for 2 h and cultured in an incubator with 5%CO_(2) for 24 h at 32℃;cells in the high-temperature group were incubated with H_(2)O_(2) for 2 h and cultured in an incubator with 5%CO 2 for 24 h at 40℃.The cell proliferation in all groups was detected by cell counting kit-8.The expression levels of Runt-related transcription factor 2(RUNX2),osteopontin(OPN)and osteocalcin(OC)mRNA were detected by real-time fluorescence quantitave polymerase chain reaction;and the expression levels of RUNX2,OPN and OC protein were detected by Western blot.Results There was no statistically significant difference in cell proliferation among the 0,450 and 500μmol·L^(-1) H_(2)O_(2) intervention groups(P>0.05);the cell proliferation rate in the 550,600 and 650μmol·L-1 H_(2)O_(2) intervention groups was significantly lower than that in the 0,450 and 500μmol·L^(-1) H_(2)O_(2) intervention groups,showing a significant decrease in cell proliferation with the increase of H_(2)O_(2) concentrations(P<0.05).In order to ensure that there were enough cells to perform the following experiments,550μmol·L^(-1) H_(2)O_(2) was chosen.The cell proliferation rate in the model group and the low-temperature group was significantly lower than that in the control group and high-temperature group(P<0.05);there was no significant difference in the cell proliferation rate between the control group and high-temperature group(P>0.05).The relative expression of RUNX2 mRNA in the model group and high-temperature group were significantly higher than that in the control group and low-temperature group(P<0.05);the relative expression of RUNX2 mRNA in the low-temperature group was significantly lower than that in the control group(P<0.05);there was no significant difference in the relative expression of RUNX2 mRNA between the model group and high-temperature group(P>0.05).The relative expression of OPN mRNA in the model group,low-temperature group and high-temperature group was significantly higher than that in the control group(P<0.05);the relative expression of OPN mRNA in the low-temperature group and high-temperature group was significantly higher than that in the model group(P<0.05);the relative expression of OPN mRNA in the low-temperature group was significantly higher than that in the high-temperature group(P<0.05).The relative expression of OC mRNA in the model group,low-temperature group and high-temperature group was significantly than that in the control group(P<0.05);the relative expression of OC mRNA in the low-temperature group and high-temperature group was significantly higher than that in the model group(P<0.05);there was no significant difference in the relative expression of OC mRNA between the low-temperature group and high-temperature group(P>0.05).The relative expressions of RUNX2,OPN and OC protein the model group,low-temperature group and high-temperature group were significantly lower than those in the control group(P<0.05);the relative expressions of RUNX2 and OPN protein in the low-temperature group were significantly lower than those in the model group and high-temperature group(P<0.05);the relative expression of OC protein was significantly lower than that in the high-temperature group(P<0.05);and there was no siqnificantly difference in the relatiwe experesson of OC protein between the low-temperature group and model group(P>0.05);the relative expressions of RUNX2,OPN and OC protein in the high-temperature group were significantly higher than those in the model group(P<0.05).Conclusion The inhibitory effects of H_(2)O_(2) on cell proliferation and osteogenic differentiation are observed in MC3T3-E1 cells;low-temperature incubation can enhance the inhibition of H_(2)O_(2) on cell proliferation and osteogenic differentiation in MC3T3-E1 cells,while high-temperature incubation can relieve its inhibitory effect on cell proliferation and osteogenic differentiation.RUNX2,OPN and OC protein might play an important role in cell proliferation and osteogenic differentiation mediated by temperature.