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[1]潘孝云,許心弦,張先龍,等.人工關(guān)節(jié)假體磨損顆粒對巨噬細胞游走抑制因子表達的影響[J].中醫(yī)正骨,2013,25(01):14-18.
　PAN Xiao-yun*,XU Xin-xian,ZHANG Xian-long,et al.Effect of wear particles from joint prosthesis on the expression of macrophage migration inhibiting factor[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2013,25(01):14-18.
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人工關(guān)節(jié)假體磨損顆粒對巨噬細胞游走抑制因子表達的影響()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第25卷
期數(shù):
2013年01期
頁碼:
14-18
欄目:
關(guān)節(jié)置換
出版日期:
2013-01-20

文章信息/Info

Title:
Effect of wear particles from joint prosthesis on the expression of macrophage migration inhibiting factor
作者:
潘孝云1; 許心弦1; 張先龍2; 毛昕2; 溫宏1; 張宇1
1.溫州醫(yī)學(xué)院附屬第二醫(yī)院,浙江 溫州 325027;
2.上海市第六人民醫(yī)院,上海 200211
Author(s):
PAN Xiao-yun*; XU Xin-xian; ZHANG Xian-long; MAO Xin; WEN Hong; ZHANG Yu.
*The Second Affiliated Hospital of Wenzhou Medical College,Wenzhou 325027,Zhejiang,China
關(guān)鍵詞:
人工關(guān)節(jié) 巨噬細胞游走抑制因子 NF-κB 動物實驗
Keywords:
Joint prosthesis;  Macrophage migration-inhibitory factors;  NF-kappa B;  Animal experimentation
摘要:
目的:觀察人工關(guān)節(jié)假體磨損顆粒對巨噬細胞游走抑制因子表達的影響。方法:配制鈦顆粒懸液,并培養(yǎng)小鼠巨噬細胞。將培養(yǎng)的第3代小鼠巨噬細胞分別進行以下實驗:①將細胞分為4組,A組不作任何處理,B組加入濃度為0.01%的鈦顆粒; C組加入濃度為0.05%的鈦顆粒,D組加入濃度為0.1%的鈦顆粒。培養(yǎng)24 h后分別用酶聯(lián)免疫吸附劑測定法和聚合酶鏈式反應(yīng)法檢測各組的巨噬細胞游走抑制因子蛋白和巨噬細胞游走抑制因子mRNA的含量。②將細胞分2組,對照組不作任何處理,觀察組加入濃度為0.1%的鈦顆粒。分別在實驗開始后0 h、6 h、12 h、24 h和36 h對2組的不同培養(yǎng)孔的細胞用酶聯(lián)免疫吸附劑測定法和聚合酶鏈式反應(yīng)法檢測各組的巨噬細胞游走抑制因子蛋白和巨噬細胞游走抑制因子mRNA的含量。③將細胞分為4組,Ⅰ組不作任何處理,Ⅱ組加入濃度為0.1%的鈦顆粒,Ⅲ組加入濃度為100 μmol·mL-1的吡咯烷二硫代氨基甲酸鹽,Ⅳ組先加入濃度為100 μmol·mL-1的吡咯烷二硫代氨基甲酸鹽,1 h后再加入濃度為0.1%的鈦顆粒。培養(yǎng)24 h后用酶聯(lián)免疫吸附劑測定法檢測各組的巨噬細胞游走抑制因子蛋白含量。④將細胞分為2組,a組不作任何處理,b組加入濃度為0.1%的鈦顆粒。分別在實驗開始后0 h、0.5 h、1 h、3 h和6 h對2組的不同培養(yǎng)孔的細胞用酶聯(lián)免疫吸附劑測定法測定磷酸化p65的含量。結(jié)果:①鈦顆粒濃度對巨噬細胞游走抑制因子表達的影響:各組小鼠巨噬細胞巨噬細胞游走抑制因子蛋白和巨噬細胞游走抑制因子mRNA含量的組間比較,差異均有統(tǒng)計學(xué)意義(F=207.158,P=0.000; F=64.955,P=0.000)。A組巨噬細胞游走抑制因子蛋白和巨噬細胞游走抑制因子mRNA的含量[(3.93±0.11)ng·mL-1,(0.03±0.01)]與B組[(4.21±0.27)ng·mL-1,(0.10±0.01)]比較,差異無統(tǒng)計學(xué)意義(P=0.167; P=0.223); A組小于C組[(6.56±0.27)ng·mL-1,(0.25±0.09)],差異有統(tǒng)計學(xué)意義(P=0.000; P=0.004); A組小于D組[(7.82±0.21)ng·mL-1,(0.70±0.09)],差異有統(tǒng)計學(xué)意義(P=0.000; P=0.000); B組小于C組(P=0.000; P=0.025)和D組(P=0.000; P=0.000); C組小于D組(P=0.000; P=0.000)。②鈦顆粒刺激時間對巨噬細胞游走抑制因子的影響:對照組巨噬細胞游走抑制因子蛋白和巨噬細胞游走抑制因子mRNA含量各時點間比較,差異無統(tǒng)計學(xué)意義(F=0.310,P=0.865; F=0.065,P=0.991)。觀察組巨噬細胞游走抑制因子蛋白和巨噬細胞游走抑制因子mRNA含量各時點間比較,差異有統(tǒng)計學(xué)意義(F=32.857,P=0.000; F=15.621,P=0.000),各時點間兩兩比較:6 h時的巨噬細胞游走抑制因子蛋白和巨噬細胞游走抑制因子mRNA表達量[(4.14±0.24)ng·mL-1,(0.08±0.04)]與0 h[(3.60±0.40)ng·mL-1,(0.03±0.01)]比較,差異無統(tǒng)計學(xué)意義(P=0.133; P=0.621); 12 h的表達量[(5.61±0.47)ng·mL-1,(0.36±0.21)]大于0 h,差異有統(tǒng)計學(xué)意義(P=0.000; P=0.004); 24 h的表達量[(7.15±0.10)ng·mL-1,(0.71±0.12)]大于12 h,差異有統(tǒng)計學(xué)意義(P=0.000; P=0.003); 36 h的表達量[(5.22±0.85)ng·mL-1,(0.31±0.18)]小于24 h,差異有統(tǒng)計學(xué)意義(P=0.000; P=0.004),但高于0 h的表達量(P=0.000; P=0.003)。③鈦顆粒和吡咯烷二硫代氨基甲酸鹽對巨噬細胞表達巨噬細胞游走抑制因子蛋白的影響:實驗結(jié)束后4組小鼠巨噬細胞的巨噬細胞游走抑制因子蛋白量分別為,Ⅰ組(3.77±0.42)ng·mL-1,Ⅱ組(7.59±0.28)ng·mL-1,Ⅲ組(4.23±0.42)ng·mL-1,Ⅳ組(6.48±0.5)ng·mL-1。析因方差分析結(jié)果提示,單獨使用0.1%鈦顆粒能促進巨噬細胞表達巨噬細胞游走抑制因子蛋白(F=153.363,P=0.000); 單獨使用100 μmol·mL-1的吡咯烷二硫代氨基甲酸鹽對巨噬細胞表達巨噬細胞游走抑制因子蛋白無影響(F=1.762,P=0.221); 100 μmol·mL-1的吡咯烷二硫代氨基甲酸鹽對0.1%鈦顆粒促進巨噬細胞表達巨噬細胞游走抑制因子蛋白具有抑制效應(yīng)(F=10.325,P=0.012)。④鈦顆粒濃度對巨噬細胞NF-κB信號通路的影響:a組各時點磷酸化p65含量比較,差異無統(tǒng)計學(xué)意義(F=0.248,P=0.904)。b組各時點磷酸化p65含量比較,差異有統(tǒng)計學(xué)意義(F=30.217,P=0.000),各時點間兩兩比較: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],差異有統(tǒng)計學(xué)意義(P=0.005); 1 h磷酸化p65含量[(23.31±2.05)pg·mg-1]高于0.5 h,差異有統(tǒng)計學(xué)意義(P=0.020); 3 h磷酸化p65含量[(31.80±1.84)pg·mg-1]高于1 h,差異有統(tǒng)計學(xué)意義(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)。結(jié)論:人工關(guān)節(jié)假體磨損顆粒可通過NF-κB信號通路上調(diào)巨噬細胞游走抑制因子的表達,促進假體周圍炎癥反應(yīng),導(dǎo)致假體周圍骨吸收、溶解,導(dǎo)致假體無菌性松動。
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.

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備注/Memo

備注/Memo:
基金項目:浙江省溫州市科技計劃項目(Y20120027)
更新日期/Last Update: 2013-01-20