【摘要】 目的:观察人工关节假体磨损颗粒对巨噬细胞游走抑制因子表达的影响。方法:配制钛颗粒悬液,并培养小鼠巨噬细胞。将培养的第3代小鼠巨噬细胞分别进行以下实验:①将细胞分为4组,A组不作任何处理,B组加入浓度为0.01%的钛颗粒;C组加入浓度为0.05%的钛颗粒,D组加入浓度为0.1%的钛颗粒。培养24 h后分别用酶联免疫吸附剂测定法和聚合酶链式反应法检测各组的巨噬细胞游走抑制因子蛋白和巨噬细胞游走抑制因子mRNA的含量。②将细胞分2组,对照组不作任何处理,观察组加入浓度为0.1%的钛颗粒。分别在实验开始后0 h、6 h、12 h、24 h和36 h对2组的不同培养孔的细胞用酶联免疫吸附剂测定法和聚合酶链式反应法检测各组的巨噬细胞游走抑制因子蛋白和巨噬细胞游走抑制因子mRNA的含量。③将细胞分为4组,Ⅰ组不作任何处理,Ⅱ组加入浓度为0.1%的钛颗粒,Ⅲ组加入浓度为100μmol.mL-1的吡咯烷二硫代氨基甲酸盐,Ⅳ组先加入浓度为100μmol.mL-1的吡咯烷二硫代氨基甲酸盐,1 h后再加入浓度为0.1%的钛颗粒。培养24 h后用酶联免疫吸附剂测定法检测各组的巨噬细胞游走抑制因子蛋白含量。④将细胞分为2组,a组不作任何处理,b组加入浓度为0.1%的钛颗粒。分别在实验开始后0 h、0.5 h、1 h、3 h和6 h对2组的不同培养孔的细胞用酶联免疫吸附剂测定法测定磷酸化p65的含量。结果:①钛颗粒浓度对巨噬细胞游走抑制因子表达的影响:各组小鼠巨噬细胞巨噬细胞游走抑制因子蛋白和巨噬细胞游走抑制因子mRNA含量的组间比较,差异均有统计学意义(F=207.158,P=0.000;F=64.955,P=0.000)。A组巨噬细胞游走抑制因子蛋白和巨噬细胞游走抑制因子mRNA的含量[(3.93±0.11)ng.mL-1,(0.03±0.01)]与B组[(4.21±0.27)ng.mL-1,(0.10±0.01)]比较,差异无统计学意义(P=0.167;P=0.223);A组小于C组[(6.56±0.27)ng.mL-1,(0.25±0.09)],差异有统计学意义(P=0.000;P=0.004);A组小于D组[(7.82±0.21)ng.mL-1,(0.70±0.09)],差异有统计学意义(P=0.000;P=0.000);B组小于C组(P=0.000;P=0.025)和D组(P=0.000;P=0.000);C组小于D组(P=0.000;P=0.000)。②钛颗粒刺激时间对巨噬细胞游走抑制因子的影响:对照组巨噬细胞游走抑制因子蛋白和巨噬细胞游走抑制因子mRNA含量各时点间比较,差异无统计学意义(F=0.310,P=0.865;F=0.065,P=0.991)。观察组巨噬细胞游走抑制因子蛋白和巨噬细胞游走抑制因子mRNA含量各时点间比较,差异有统计学意义(F=32.857,P=0.000;F=15.621,P=0.000),各时点间两两比较:6 h时的巨噬细胞游走抑制因子蛋白和巨噬细胞游走抑制因子mRNA表达量[(4.14±0.24)ng.mL-1,(0.08±0.04)]与0 h[(3.60±0.40)ng.mL-1,(0.03±0.01)]比较,差异无统计学意义(P=0.133;P=0.621);12 h的表达量[(5.61±0.47)ng.mL-1,(0.36±0.21)]大于0 h,差异有统计学意义(P=0.000;P=0.004);24 h的表达量[(7.15±0.10)ng.mL-1,(0.71±0.12)]大于12 h,差异有统计学意义(P=0.000;P=0.003);36 h的表达量[(5.22±0.85)ng.mL-1,(0.31±0.18)]小于24 h,差异有统计学意义(P=0.000;P=0.004),但高于0 h的表达量(P=0.000;P=0.003)。③钛颗粒和吡咯烷二硫代氨基甲酸盐对巨噬细胞表达巨噬细胞游走抑制因子蛋白的影响:实验结束后4组小鼠巨噬细胞的巨噬细胞游走抑制因子蛋白量分别为,Ⅰ组(3.77±0.42)ng.mL-1,Ⅱ组(7.59±0.28)ng.mL-1,Ⅲ组(4.23±0.42)ng.mL-1,Ⅳ组(6.48±0.5)ng.mL-1。析因方差分析结果提示,单独使用0.1%钛颗粒能促进巨噬细胞表达巨噬细胞游走抑制因子蛋白(F=153.363,P=0.000);单独使用100μmol.mL-1的吡咯烷二硫代氨基甲酸盐对巨噬细胞表达巨噬细胞游走抑制因子蛋白无影响(F=1.762,P=0.221);100μmol.mL-1的吡咯烷二硫代氨基甲酸盐对0.1%钛颗粒促进巨噬细胞表达巨噬细胞游走抑制因子蛋白具有抑制效应(F=10.325,P=0.012)。④钛颗粒浓度对巨噬细胞NF-κB信号通路的影响:a组各时点磷酸化p65含量比较,差异无统计学意义(F=0.248,P=0.904)。b组各时点磷酸化p65含量比较,差异有统计学意义(F=30.217,P=0.000),各时点间两两比较:0.5 h磷酸化p65含量[(17.68±0.55)pg.mg-1]高于0 h[(10.38±3.18)pg.mg-1],差异有统计学意义(P=0.005);1 h磷酸化p65含量[(23.31±2.05)pg.mg-1]高于0.5 h,差异有统计学意义(P=0.020);3 h磷酸化p65含量[(31.80±1.84)pg.mg-1]高于1 h,差异有统计学意义(P=0.002);6 h磷酸化p65含量[(18.42±3.61)pg.mg-1]低于3 h(P=0.000),但高于0 h(P=0.003)。结论:人工关节假体磨损颗粒可通过NF-κB信号通路上调巨噬细胞游走抑制因子的表达,促进假体周围炎症反应,导致假体周围骨吸收、溶解,导致假体无菌性松动。更多还原

【Abstract】 Objective:To observe the effects of wear particles from joint prosthesis on the expression of macrophage migration inhibiting factors(MMIF).Methods:Suspension with titanium particles was prepared and mice macrophages were cultured.The following experimentations were respectively carried on the third generation of mice macrophages.①The cells were divided into 4 groups,cells in group A were added with nothing,while cells in group B,C and D were added with titanium particles suspension(0.01%,0.05% and 0.1%,respectively).The contents of MMIF protein and MMIF mRNA of the 4 groups were respectively detected through enzyme linked immunosorbent assay(ELISA)and polymerase chain reaction(PCR)after culturing for 24 hrs.②The cells were divided into 2 groups,cells in control group were added with nothing,while cells in observation group were added with 0.1% titanium particles suspension.The contents of MMIF protein and MMIF mRNA of the 2 groups were respectively detected through ELISA and PCR at 0,6,12,24 and 36 hrs from the beginning of the experimentation.③The cells were divided into 4 groups,cells in groupⅠwere added with nothing,and cells in groupⅡwere added with 0.1% titanium particles suspension and groupⅢwere added with pyrrolidine dithiocarbamate(PDTC)(100 μmol/ml),while cells in groupⅣwere firstly added with PDTC(100 μmol/ml)and titanium particles suspension(0.1%)one hr later.The contents of MMIF protein of the 4 groups were detected through ELISA after culturing for 24 hrs.④The cells were divided into 2 groups,cells in group a were added with nothing,while cells in group b were added with 0.1% titanium particles suspension.The contents of phosphorylation p65 of the 2 groups were respectively detected through ELISA at 0,0.5,1,3 and 6 hrs from the beginning of the experimentation.Results:①Effect of titanium particles suspension concentration on MMIF expression:There were statistical differences in the contents of MMIF protein and MMIF mRNA of macrophages among the 4 groups(F=207.158,P=0.000;F=64.955,P=0.000).There were no statistical differences in the contents of MMIF protein and MMIF mRNA between group A((3.93±0.11) ng/ml,(0.03±0.01))and group B((4.21±0.27) ng/ml,(0.10±0.01)(P=0.167;P=0.223);the contents of MMIF protein and MMIF mRNA of group A were lower than those of group C((6.56±0.27) ng/ml,(0.25±0.09))and there were statistical differences(P=0.000;P=0.004);the contents of MMIF protein and MMIF mRNA of group A were lower than those of group D((7.82±0.21) ng/ml,(0.70±0.09)),and there were statistical differences(P=0.000;P=0.000);the contents of MMIF protein and MMIF mRNA of group B were lower than those of group C(P=0.000;P=0.025)and group D(P=0.000;P=0.000);the contents of MMIF protein and MMIF mRNA of group C were lower than those of group D(P=0.000;P=0.000).②Effect of titanium particles stimulation time on MMIF expression:There were no statistical differences in the contents of MMIF protein and MMIF mRNA of control group among different time points(F=0.310,P=0.865;F=0.065,P=0.991).There were statistical differences in the contents of MMIF protein and MMIF mRNA of observation group among different time points(F=32.857,P=0.000;F=15.621,P=0.000).Further comparison between any two time points showed that there were no statistical differences in the contents of MMIF protein and MMIF mRNA between 6 hrs((4.14±0.24) ng/ml,(0.08±0.04))and 0 hr((3.60±0.40) ng/ml,(0.03±0.01))(P=0.133,P=0.621);the contents of MMIF protein and MMIF mRNA at 12 hrs((5.61±0.47) ng/ml,(0.36±0.21))were larger than those at 0 hr,and there were statistical differences(P=0.000;P=0.004);the contents of MMIF protein and MMIF mRNA at 24 hrs((7.15±0.10) ng/ml,(0.71±0.12))were larger than those at 12 hrs,and there were statistical differences(P=0.000;P=0.003);the contents of MMIF protein and MMIF mRNA at 36 hrs((5.22±0.85) ng/ml,(0.31±0.18))were lower than those at 24 hrs(P=0.000;P=0.004)and larger than those at 0 hr(P=0.000;P=0.003).③Effect of titanium particles and PDTC on the expression of MMIF protein:After experimentations,the MMIF protein contents were(3.77±0.42) ng/ml(groupⅠ),(7.59±0.28) ng/ml(groupⅡ),(4.23±0.42) ng/ml(groupⅢ)and(6.48±0.5) ng/ml(groupⅣ),respectively.The results of analysis of variance of factorial design showed that single use of 0.1% titanium particles suspension could promote the expression of MMIF protein(F=153.363,P=0.000),while single use of PDTC(100 μmol/ml)had no influence on the expression of MMIF protein(F=1.762,P=0.221).PDTC(100 μmol/ml)could depress the promotive effect of 0.1% titanium particles suspension on expression of MMIF protein(F=10.325,P=0.012).④Effect of titanium particles suspension concentration on NF-κB signal pathway in macrophages:There were no statistical differences in phosphorylation p65 contents among different time points in group a(F=0.248,P=0.904).There were statistical differences in phosphorylation p65 contents among different time points in group b(F=30.217,P=0.000).Further comparison showed that phosphorylation p65 contents at 0.5 hr((17.68±0.55) pg/mg)was higher than that at 0 hr((10.38±3.18) pg/mg)(P=0.005);phosphorylation p65 contents at 1 hr((23.31±2.05) pg/mg)was higher than that at 0.5 hr(P=0.020);phosphorylation p65 contents at 3 hrs((31.80±1.84) pg/mg)was higher than that at 1 hr(P=0.002);phosphorylation p65 contents at 6 hrs((18.42±3.61) pg/mg)was lower than that at 3 hrs(P=0.000)and larger than that at 0 hr(P=0.003).Conclusion:Wear particles from joint prosthesis can up-regulate the expression of MMIF through NF-κB signal pathway and promote inflammatory reactions around prosthesis,which leads to bone absorption and osteolysis,and leads to aseptic prosthesis loosening as a further result.更多还原