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[1]林煜,張怡元,馮爾宥,等.人參皂甙Rg1與鈦微粒對大鼠顱骨成骨細(xì)胞的影響[J].中醫(yī)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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人參皂甙Rg1與鈦微粒對大鼠顱骨成骨細(xì)胞的影響()
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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07期
頁碼:
8-15
欄目:
基礎(chǔ)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; 張怡元1; 馮爾宥1; 吳銀生2; 林燕萍2
1.廈門大學(xué)附屬福州第二醫(yī)院,福建 福州 350007; 2.福建中醫(yī)藥大學(xué)中西醫(yī)結(jié)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
關(guān)鍵詞:
成骨細(xì)胞 細(xì)胞培養(yǎng)技術(shù) 人參皂甙 細(xì)胞因子類 環(huán)氧化酶2 鈦 人工關(guān)節(jié)
Keywords:
Osteoblasts;  Cell culture techniques;  Ginsenoside;  Cytokines;  Cyclooxygenase 2;  Titanium;  Joint prosthesis
摘要:
目的:探討人參皂甙Rg1與鈦微粒對大鼠顱骨成骨細(xì)胞的影響,為人工關(guān)節(jié)假體松動的防治提供新思路。方法:收集、純化新生SD大鼠顱骨成骨細(xì)胞,篩選鈦微粒,配制鈦微粒浸提液,進(jìn)行內(nèi)毒素檢測后,將培養(yǎng)的第3代成骨細(xì)胞以1×105個·mL-1的密度傳代接種于5組培養(yǎng)液中,即正常組(含10%胎牛血清的DMEM培養(yǎng)基)、鈦微粒組(體積比為0.1%的鈦微粒混懸液+含10%胎牛血清的DMEM培養(yǎng)基)、鈦+高Rg1組(體積比為0.1%的鈦微粒混懸液+終濃度為100 μg·m-1的人參皂苷Rg1+含10%胎牛血清的DMEM培養(yǎng)基)、鈦+中Rg1組(體積比為0.1%的鈦微粒混懸液+終濃度為50 μg·m-1的人參皂苷Rg1+含10%胎牛血清的DMEM培養(yǎng)基)、鈦+低Rg1組(體積比為0.1%的鈦微粒混懸液+終濃度為25 μg·m-1的人參皂苷Rg1+含10%胎牛血清的DMEM培養(yǎng)基)。連續(xù)培養(yǎng)24 h后,觀察成骨細(xì)胞形態(tài); 采用酶聯(lián)免疫吸附法檢測細(xì)胞培養(yǎng)液中前列腺素E2、腫瘤壞死因子α、白細(xì)胞介素6、白細(xì)胞介素1及白細(xì)胞介素1受體拮抗劑的濃度; 采用實時熒光定量法檢測成骨細(xì)胞中環(huán)氧化酶2mRNA、腫瘤壞死因子αmRNA的表達(dá); 采用Western Blotting法檢測成骨細(xì)胞中環(huán)氧化酶2蛋白的表達(dá)。結(jié)果:5組成骨細(xì)胞培養(yǎng)液前列腺素E2光密度值的差異有統(tǒng)計學(xué)意義(F=244.895,P=0.000); 鈦微粒組、鈦+高Rg1組、鈦+中Rg1組均高于正常組[(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]; 鈦+高Rg1組、鈦+中Rg1組、鈦+低Rg1組(32.501±0.124)均低于鈦微粒組(P=0.000,P=0.000,P=0.000); 鈦+低Rg1組低于鈦+高Rg1組、鈦+中Rg1組(P=0.000,P=0.000); 鈦+高Rg1組高于鈦+中Rg1組(P=0.000); 正常組與鈦+低Rg1組比較,差異無統(tǒng)計學(xué)意義(P=0.168)。5組成骨細(xì)胞培養(yǎng)液腫瘤壞死因子α光密度值的差異有統(tǒng)計學(xué)意義(F=72.340,P=0.000); 鈦微粒組、鈦+高Rg1組、鈦+中Rg1組、鈦+低Rg1組均高于正常組[(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]; 鈦+中Rg1組、鈦+低Rg1組低于鈦微粒組(P=0.000,P=0.000); 鈦+低Rg1組低于鈦+高Rg1組、鈦+中Rg1組(P=0.000,P=0.000); 鈦+高Rg1組高于鈦+中Rg1組(P=0.000); 鈦微粒組與鈦+高Rg1組比較,差異無統(tǒng)計學(xué)意義(P=0.230)。5組成骨細(xì)胞培養(yǎng)液白細(xì)胞介素6光密度值的差異有統(tǒng)計學(xué)意義(F=80.449,P=0.000); 鈦微粒組、鈦+高Rg1組、鈦+中Rg1組、鈦+低Rg1組均高于正常組[(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]; 鈦+高、中、低Rg1組均低于鈦微粒組(P=0.000,P=0.000,P=0.000); 鈦+低Rg1組低于鈦+高、中Rg1組(P=0.001,P=0.000); 鈦+高Rg1組高于鈦+中Rg1組(P=0.039)。5組成骨細(xì)胞培養(yǎng)液白細(xì)胞介素1光密度值的差異有統(tǒng)計學(xué)意義(F=38.483,P=0.000); 鈦微粒組、鈦+高Rg1組、鈦+中Rg1組、鈦+低Rg1組均高于正常組[(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]; 鈦+高、中、低Rg1組均低于鈦微粒組(P=0.000,P=0.000,P=0.000),鈦+低Rg1組低于鈦+高、中Rg1組(P=0.000,P=0.000),鈦+中Rg1組與鈦+高Rg1組相比,差異無統(tǒng)計學(xué)意義(P=0.187)。5組成骨細(xì)胞培養(yǎng)液白細(xì)胞介素1受體拮抗劑光密度值的差異有統(tǒng)計學(xué)意義(F=492.724,P=0.000); 鈦微粒組、鈦+高Rg1組、鈦+中Rg1組、鈦+低Rg1組均低于正常組[(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]; 鈦+高、中、低Rg1組均高于鈦微粒組(P=0.000,P=0.000,P=0.000),鈦+中Rg1組高于鈦+高、低Rg1組(P=0.000,P=0.000),鈦+高Rg1組低于鈦+低Rg1組(P=0.000)。5組成骨細(xì)胞中β-actin、環(huán)氧化酶2、腫瘤壞死因子α熒光定量RT-PCR部分?jǐn)U增曲線和各基因的溶解曲線表現(xiàn)為單一的溶解峰,均為特異性擴(kuò)增。5組成骨細(xì)胞環(huán)氧化酶2mRNA表達(dá)的差異有統(tǒng)計學(xué)意義(F=886.930,P=0.000); 與鈦微粒組相比,鈦+高Rg1組、鈦+中Rg1組、鈦+低Rg1組及正常組環(huán)氧化酶2mRNA的表達(dá)較弱[(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]; 鈦+高、中、低Rg1組的表達(dá)均較正常組強(P=0.000,P=0.000,P=0.000); 鈦+低Rg1組較鈦+高、中Rg1組弱(P=0.000,P=0.000),鈦+中Rg1組較鈦+高Rg1組弱(P=0.000)。5組成骨細(xì)胞腫瘤壞死因子αmRNA表達(dá)的差異有統(tǒng)計學(xué)意義(F=1635.878,P=0.000); 與鈦微粒組(1.000±0.000)相比,鈦+高Rg1組、鈦+中Rg1組、鈦+低Rg1組及正常組腫瘤壞死因子αmRNA的表達(dá)較弱[(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)]; 鈦+高、中、低Rg1組均較正常組強(P=0.000,P=0.000,P=0.000); 鈦+低Rg1組較鈦+高、中Rg1組弱(P=0.000,P=0.000); 鈦+高Rg1與鈦+中Rg1組比較,差異無統(tǒng)計學(xué)意義(P=0.057)。5組成骨細(xì)胞中環(huán)氧化酶2蛋白表達(dá)的差異有統(tǒng)計學(xué)意義(F=886.930,P=0.000); 與鈦微粒組(0.854±0.067)相比,鈦+高Rg1組、鈦+中Rg1組、鈦+低Rg1組及正常組環(huán)氧化酶2蛋白的表達(dá)較弱[(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]; 鈦+高、中、低Rg1組均較正常組強(P=0.000,P=0.000,P=0.000); 鈦+高Rg1組較鈦+中Rg1組、鈦+低Rg1組強(P=0.010,P=0.000); 鈦+中Rg1組較鈦+低Rg1組強(P=0.002)。結(jié)論:成骨細(xì)胞與鈦微粒共培養(yǎng)后,鈦微粒能促進(jìn)成骨細(xì)胞分泌炎癥因子,人參皂甙Rgl干預(yù)可減弱鈦微粒對成骨細(xì)胞的刺激、抑制炎癥因子的表達(dá),這可能是人參皂甙Rgl抑制假體周圍骨吸收,防治人工關(guān)節(jié)假體松動的作用機(jī)制。但人參皂甙Rg1對成骨細(xì)胞產(chǎn)生影響的具體作用機(jī)制及藥物劑量效應(yīng)關(guān)系還有待進(jìn)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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備注/Memo

備注/Memo:
通訊作者:林煜 E-mail:[email protected] 基金項目:國家自然科學(xué)基金資助項目(81173282); 福建省科技廳重點項目(2009Y0028); 教育部博士點基金項目(200803930001)
更新日期/Last Update: 1900-01-01