[1]林煜,张怡元,冯尔宥,等.人参皂甙Rg1与钛微粒对大鼠颅骨成骨细胞的影响[J].中医正骨,2013,25(07):8-15.
　LIN Yu*,ZHANG Yi-yuan,FENG Er-you,et al.Effect of Ginsenoside Rg1 and titanium particles on rat cranioaural osteoblasts[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2013,25(07):8-15.
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人参皂甙Rg1与钛微粒对大鼠颅骨成骨细胞的影响()
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《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第25卷
期数:
2013年07期
页码:
8-15
栏目:
基础研究
出版日期:
2013-07-31

文章信息/Info

Title:
Effect of Ginsenoside Rg1 and titanium particles on rat cranioaural osteoblasts
作者:
林煜1; 张怡元1; 冯尔宥1; 吴银生2; 林燕萍2
1.厦门大学附属福州第二医院,福建 福州 350007; 2.福建中医药大学中西医结合研究院,福建 福州 350108
Author(s):
LIN Yu*; ZHANG Yi-yuan; FENG Er-you; WU Yin-sheng; LIN Yan-ping.*
The second hospital of Fuzhou affiliated to Xiamen University,Fuzhou 350007,China
关键词:
成骨细胞 细胞培养技术 人参皂甙 细胞因子类 环氧化酶2 钛 人工关节
Keywords:
Osteoblasts;  Cell culture techniques;  Ginsenoside;  Cytokines;  Cyclooxygenase 2;  Titanium;  Joint prosthesis
摘要:
目的:探讨人参皂甙Rg1与钛微粒对大鼠颅骨成骨细胞的影响,为人工关节假体松动的防治提供新思路。方法:收集、纯化新生SD大鼠颅骨成骨细胞,筛选钛微粒,配制钛微粒浸提液,进行内毒素检测后,将培养的第3代成骨细胞以1×105个·mL-1的密度传代接种于5组培养液中,即正常组(含10%胎牛血清的DMEM培养基)、钛微粒组(体积比为0.1%的钛微粒混悬液+含10%胎牛血清的DMEM培养基)、钛+高Rg1组(体积比为0.1%的钛微粒混悬液+终浓度为100 μg·m-1的人参皂苷Rg1+含10%胎牛血清的DMEM培养基)、钛+中Rg1组(体积比为0.1%的钛微粒混悬液+终浓度为50 μg·m-1的人参皂苷Rg1+含10%胎牛血清的DMEM培养基)、钛+低Rg1组(体积比为0.1%的钛微粒混悬液+终浓度为25 μg·m-1的人参皂苷Rg1+含10%胎牛血清的DMEM培养基)。连续培养24 h后,观察成骨细胞形态; 采用酶联免疫吸附法检测细胞培养液中前列腺素E2、肿瘤坏死因子α、白细胞介素6、白细胞介素1及白细胞介素1受体拮抗剂的浓度; 采用实时荧光定量法检测成骨细胞中环氧化酶2mRNA、肿瘤坏死因子αmRNA的表达; 采用Western Blotting法检测成骨细胞中环氧化酶2蛋白的表达。结果:5组成骨细胞培养液前列腺素E2光密度值的差异有统计学意义(F=244.895,P=0.000); 钛微粒组、钛+高Rg1组、钛+中Rg1组均高于正常组[(53.362±0.307),(41.048±0.431),(38.998±0.234),(31.687±0.466),P=0.000,P=0.000,P=0.000]; 钛+高Rg1组、钛+中Rg1组、钛+低Rg1组(32.501±0.124)均低于钛微粒组(P=0.000,P=0.000,P=0.000); 钛+低Rg1组低于钛+高Rg1组、钛+中Rg1组(P=0.000,P=0.000); 钛+高Rg1组高于钛+中Rg1组(P=0.000); 正常组与钛+低Rg1组比较,差异无统计学意义(P=0.168)。5组成骨细胞培养液肿瘤坏死因子α光密度值的差异有统计学意义(F=72.340,P=0.000); 钛微粒组、钛+高Rg1组、钛+中Rg1组、钛+低Rg1组均高于正常组[(50.121±0.532),(49.675±0.336),(46.431±0.245),(42.521±0.513),(40.055±0.471),P=0.000,P=0.000,P=0.000,P=0.001]; 钛+中Rg1组、钛+低Rg1组低于钛微粒组(P=0.000,P=0.000); 钛+低Rg1组低于钛+高Rg1组、钛+中Rg1组(P=0.000,P=0.000); 钛+高Rg1组高于钛+中Rg1组(P=0.000); 钛微粒组与钛+高Rg1组比较,差异无统计学意义(P=0.230)。5组成骨细胞培养液白细胞介素6光密度值的差异有统计学意义(F=80.449,P=0.000); 钛微粒组、钛+高Rg1组、钛+中Rg1组、钛+低Rg1组均高于正常组[(80.537±0.883),(70.975±0.945),(68.154±0.745),(63.335±0.845),(57.550±0.610),P=0.000,P=0.000,P=0.000,P=0.000]; 钛+高、中、低Rg1组均低于钛微粒组(P=0.000,P=0.000,P=0.000); 钛+低Rg1组低于钛+高、中Rg1组(P=0.001,P=0.000); 钛+高Rg1组高于钛+中Rg1组(P=0.039)。5组成骨细胞培养液白细胞介素1光密度值的差异有统计学意义(F=38.483,P=0.000); 钛微粒组、钛+高Rg1组、钛+中Rg1组、钛+低Rg1组均高于正常组[(83.106±4.413),(59.506±1.294),(56.881±3.561),(45.081±3.459),(37.579±3.526),P=0.000,P=0.000,P=0.000,P=0.000]; 钛+高、中、低Rg1组均低于钛微粒组(P=0.000,P=0.000,P=0.000),钛+低Rg1组低于钛+高、中Rg1组(P=0.000,P=0.000),钛+中Rg1组与钛+高Rg1组相比,差异无统计学意义(P=0.187)。5组成骨细胞培养液白细胞介素1受体拮抗剂光密度值的差异有统计学意义(F=492.724,P=0.000); 钛微粒组、钛+高Rg1组、钛+中Rg1组、钛+低Rg1组均低于正常组[(64.111±1.364),(116.351±5.432),(229.768±3.545),(207.203±2.436),(268.019±3.871),P=0.000,P=0.000,P=0.000,P=0.000]; 钛+高、中、低Rg1组均高于钛微粒组(P=0.000,P=0.000,P=0.000),钛+中Rg1组高于钛+高、低Rg1组(P=0.000,P=0.000),钛+高Rg1组低于钛+低Rg1组(P=0.000)。5组成骨细胞中β-actin、环氧化酶2、肿瘤坏死因子α荧光定量RT-PCR部分扩增曲线和各基因的溶解曲线表现为单一的溶解峰,均为特异性扩增。5组成骨细胞环氧化酶2mRNA表达的差异有统计学意义(F=886.930,P=0.000); 与钛微粒组相比,钛+高Rg1组、钛+中Rg1组、钛+低Rg1组及正常组环氧化酶2mRNA的表达较弱[(0.734±0.065),(0.621±0.032),(0.517±0.042),(0.386±0.015),P=0.000,P=0.000,P=0.000,P=0.000]; 钛+高、中、低Rg1组的表达均较正常组强(P=0.000,P=0.000,P=0.000); 钛+低Rg1组较钛+高、中Rg1组弱(P=0.000,P=0.000),钛+中Rg1组较钛+高Rg1组弱(P=0.000)。5组成骨细胞肿瘤坏死因子αmRNA表达的差异有统计学意义(F=1635.878,P=0.000); 与钛微粒组(1.000±0.000)相比,钛+高Rg1组、钛+中Rg1组、钛+低Rg1组及正常组肿瘤坏死因子αmRNA的表达较弱[(0.823±0.078),(0.764±0.107),(0.543±0.042),(0.399±0.047),P=0.000,P=0.000,P=0.000,P=0.000)]; 钛+高、中、低Rg1组均较正常组强(P=0.000,P=0.000,P=0.000); 钛+低Rg1组较钛+高、中Rg1组弱(P=0.000,P=0.000); 钛+高Rg1与钛+中Rg1组比较,差异无统计学意义(P=0.057)。5组成骨细胞中环氧化酶2蛋白表达的差异有统计学意义(F=886.930,P=0.000); 与钛微粒组(0.854±0.067)相比,钛+高Rg1组、钛+中Rg1组、钛+低Rg1组及正常组环氧化酶2蛋白的表达较弱[(0.774±0.045),(0.634±0.054),(0.433±0.032),(0.304±0.058),P=0.000,P=0.000,P=0.000,P=0.000]; 钛+高、中、低Rg1组均较正常组强(P=0.000,P=0.000,P=0.000); 钛+高Rg1组较钛+中Rg1组、钛+低Rg1组强(P=0.010,P=0.000); 钛+中Rg1组较钛+低Rg1组强(P=0.002)。结论:成骨细胞与钛微粒共培养后,钛微粒能促进成骨细胞分泌炎症因子,人参皂甙Rgl干预可减弱钛微粒对成骨细胞的刺激、抑制炎症因子的表达,这可能是人参皂甙Rgl抑制假体周围骨吸收,防治人工关节假体松动的作用机制。但人参皂甙Rg1对成骨细胞产生影响的具体作用机制及药物剂量效应关系还有待进一步研究。
Abstract:
Objective:To observe the effect of ginsenoside Rg1 and titanium(Ti)particles on rat cranioaural osteoblasts,so as to provide new preventive treatment of joint prosthesis loosening.Methods:The cranioaural osteoblasts were collected from the newborn SD rats and were purified.The Ti particles were screened and the leaching liquor of Ti were prepared.After the endotoxin detection,the third-generation osteoblasts were cultured in Dulbecco's Modified Eagle Medium(DMEM)supplemented with 10% fetal bovine serum(normal group),DMEM supplemented with 10% fetal bovine serum and 0.1%(volume ratio)Ti particles suspension(Ti particles group),DMEM supplemented with 10% fetal bovine serum and 0.1% Ti particles suspension and Ginsenoside Rg1 with final concentration of 100 μg/mL(Ti+high Rg1 group),DMEM supplemented with 10% fetal bovine serum and 0.1% Ti particles suspension and Ginsenoside Rg1 with final concentration of 50 μg/mL(Ti+middle Rg1 group),DMEM supplemented with 10% fetal bovine serum and 0.1% Ti particles suspension and Ginsenoside Rg1 with final concentration of 25 μg/mL(Ti+low Rg1 group),one hundred thousand osteoblasts in one milliliter of culture solutions.The osteoblasts were cultured for 24 hours continuously and then the shape of them were observed.The concentration of prostaglandin E2(PGE-2),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1(IL-1)and interleukin 1 receptor antagonist protein(IL-1ra)in the culture solution were detected through enzyme-linked immunoadsordent assay(ELISA).The expression of cyclooxygenase 2(COX-2)mRNA and TNF-α mRNA in the osteoblasts were detected through real-time fluorescence quantitative polymerase chain reaction,and the expression of COX-2 protein in the osteoblasts were detected through Western Blotting.Results:There were statistical differences in the optical density(OD)values of PGE-2 among the 5 groups(F=244.895,P=0.000).The OD values of PGE-2 were higher in Ti particles group(53.362±0.307),Ti+high Rg1 group(41.048±0.431)and Ti+middle Rg1 group(38.998±0.234)compared to normal group(31.687±0.466),there were statistical differences between them(P=0.000,P=0.000,P=0.000).The OD values of PGE-2 were lower in Ti+high Rg1 group,Ti+middle Rg1 group and Ti+low Rg1 group(32.501±0.124)compared to Titanium particles group(P=0.000,P=0.000,P=0.000).The OD values of PGE-2 were lower in Ti+low Rg1 group compared to Ti+high Rg1 group and Ti+middle Rg1 group respectively(P=0.000,P=0.000).The OD values of PGE-2 were higher in Ti+high Rg1 group compared to Ti+middle Rg1 group(P=0.000).There were no statistical differences in the OD values of PGE-2 between normal group and Ti+low Rg1 group(P=0.168).There were statistical differences in the OD values of TNF-α among the 5 groups(F=72.340,P=0.000).The OD values of TNF-α were higher in Ti particles group,Ti+high Rg1 group,Ti+middle Rg1 group and Ti+low Rg1 group compared to normal group((50.121±0.532),(49.675±0.336),(46.431±0.245),(42.521±0.513),(40.055±0.471),P=0.000,P=0.000,P=0.000,P=0.001).The OD values of TNF-α were lower in Ti+middle Rg1 group and Ti+low Rg1 group compared to Ti particles group(P=0.000,P=0.000).The OD values of TNF-α were lower in Ti+low Rg1 group compared to Ti + high Rg1 group and Ti+middle Rg1 group respectively(P=0.000,P=0.000).The OD values of TNF-α were higher in Ti + high Rg1 group compared to Ti+middle Rg1 group(P=0.000).There were no statistical differences in the OD values of TNF-α between Ti particles group and Ti+high Rg1 group(P=0.230).There were statistical differences in the OD values of IL-6 among the 5 groups(F=80.449,P=0.000).The OD values of IL-6 were higher in Ti particles group,Ti+high Rg1 group,Ti+middle Rg1 group and Ti+low Rg1 group compared to normal group((80.537±0.883),(70.975±0.945),(68.154±0.745),(63.335±0.845),(57.550±0.610),P=0.000,P=0.000,P=0.000,P=0.000).The OD values of IL-6 were lower in Ti+high Rg1 group,Ti+middle Rg1 group and Ti+low Rg1 group compared to Ti particles group(P=0.000,P=0.000,P=0.000).The OD values of IL-6 were lower in Ti+low Rg1 group compared to Ti+high Rg1 group and Ti+middle Rg1 group respectively(P=0.001,P=0.000).The OD values of IL-6 were higher in Ti+high Rg1 group compared to Ti+middle Rg1 group(P=0.039).There were statistical difference in the OD values of IL-1 among the 5 groups(F=38.483,P=0.000).The OD values of IL-1 were higher in Ti particles group,Ti+high Rg1 group,Ti+middle Rg1 group and Ti+low Rg1 group compared to normal group((83.106±4.413),(59.506±1.294),(56.881±3.561),(45.081±3.459),(37.579±3.526),P=0.000,P=0.000,P=0.000,P=0.000).The OD values of IL-1 were lower in Ti+high Rg1 group,Ti+middle Rg1 group and Ti+low Rg1 group compared to Ti particles group(P=0.000,P=0.000,P=0.000).The OD values of IL-1 were lower in Ti+low Rg1 group compared to Ti+high Rg1 group and Ti+middle Rg1 group respectively(P=0.000,P=0.000).There were no statistical differences in the OD values of IL-1 between Ti+middle Rg1 group and Ti+high Rg1 group(P=0.187).There were statistical differences in the OD values of IL-1ra among the 5 groups(F=492.724,P=0.000).The OD values of IL-1ra were lower in Ti particles group,Ti+high Rg1 group,Ti+middle Rg1 group and Ti + low Rg1 group compared to normal group((64.111±1.364),(116.351±5.432),(229.768±3.545),(207.203±2.436),(268.019±3.871),P=0.000,P=0.000,P=0.000,P=0.000).The OD values of IL-1ra were higher in Ti+high Rg1 group,Ti+middle Rg1 group and Ti+low Rg1 group compared to Ti particles group(P=0.000,P=0.000,P=0.000).The OD values of IL-1ra were higher in Ti+middle Rg1 group compared to Ti+high Rg1 group and Ti+low Rg1 group respectively(P=0.000,P=0.000).The OD values of IL-1ra were lower in Ti+high Rg1 group compared to Ti+low Rg1 group(P=0.000).The amplification curve of β-actin,COX-2 and TNF-α in fluorescent quantitation RT-PCR were shown as specific amplification and the genetic-solubility curve were shown as single peak.There were statistical differences in the expression of COX-2 mRNA among the 5 groups(F=886.930,P=0.000).Compared to Ti particles group,the expression of COX-2 mRNA were weaker in Ti+high Rg1 group,Ti+middle Rg1 group,Ti+low Rg1 group and normal group((0.734±0.065),(0.621±0.032),(0.517±0.042),(0.386±0.015),P=0.000,P=0.000,P=0.000,P=0.000).The expression of COX-2 mRNA were stronger in Ti+high Rg1 group,Ti+middle Rg1 group,Ti+low Rg1 group compared to normal group respectively(P=0.000,P=0.000,P=0.000).The expression of COX-2 mRNA were weaker in Ti+low Rg1 group compared to Ti+high Rg1 group and Ti+middle Rg1 group respectively(P=0.000,P=0.000).The expression of COX-2 mRNA were weaker in Ti+middle Rg1 group compared to Ti+high Rg1 group(P=0.000).There were statistical differences in the expression of TNF-α mRNA among the 5 groups(F=1 635.878,P=0.000).Compared to Ti particles group(1.000±0.000),the expression of TNF-α mRNA were weaker in Ti+high Rg1 group,Ti+middle Rg1 group,Ti+low Rg1 group and normal group((0.823±0.078),(0.764±0.107),(0.543±0.042),(0.399±0.047),P=0.000,P=0.000,P=0.000,P=0.000)).The expression of TNF-α mRNA were stronger in Ti+high Rg1 group,Ti+middle Rg1 group,Ti+low Rg1 group compared to normal group respectively(P=0.000,P=0.000,P=0.000).The expression of TNF-α mRNA were weaker in Ti+low Rg1 group compared to Ti+high Rg1 group and Ti+middle Rg1 group respectively(P=0.000,P=0.000).There were no statistical differences in the expression of TNF-α mRNA between Ti+high Rg1 group and Ti+middle Rg1 group(P=0.057).There were statistical differences in the expression of COX-2 protein among the 5 groups(F=886.930,P=0.000).Compared to Ti particles group(0.854±0.067),the expression of COX-2 protein were weaker in Ti+high Rg1 group,Ti+middle Rg1 group,Ti+low Rg1 group and normal group((0.774±0.045),(0.634±0.054),(0.433±0.032),(0.304±0.058),P=0.000,P=0.000,P=0.000,P=0.000).Compared to Ti+high Rg1 group,Ti+middle Rg1 group and Ti+low Rg1 group respectively,the expression of COX-2 protein was weaker in normal group(P=0.000,P=0.000,P=0.000).The expression of COX-2 protein were stronger in Ti+high Rg1 group compared to Ti+middle Rg1 group and Ti+low Rg1 group respectively(P=0.010,P=0.000).Ti+middle Rg1 group surpassed Ti+low Rg1 group(P=0.002).Conclusion:After co-cultured with Ti particles,the osteoblasts can secrete more inflammatory factors promoted by Ti particles.Ginsenoside Rgl can weaken the impact of Ti particles on osteoblasts and suppress the expression of inflammation factors,which may be considered as the mechanism of action for ginsenoside Rgl to suppress bone resorption around the prosthesis and to prevent joint prosthesis loosening.However,further studies are needed to define the concrete mechanisms of influence of ginsenoside Rg1 on the osteoblasts and to demonstrate the concrete dose-effect relationship.

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备注/Memo

备注/Memo:
通讯作者:林煜 E-mail:linyu980945@163.com 基金项目:国家自然科学基金资助项目(81173282); 福建省科技厅重点项目(2009Y0028); 教育部博士点基金项目(200803930001)
更新日期/Last Update: 1900-01-01