胎鼠成骨细胞骨保护素及其配体mRNA表达与阿胶强骨口服液含药血清的影响
태서성골세포골보호소급기배체mRNA표체여아효강골구복액함약혈청적영향
Effects of donkey-hide glue reinforcing bone oral solution medicated serum on osteoprotegerin and osteoprotegerin ligand mRNA expression of osteoblast in fetal rats
  • 万方数据
    中国临床康复 중국림상강복
    CHINESE JOURNAL OF CLINICAL REHABILITATION
    2005年 42期 146-148 ,共3页

    沈霖%武嘉林%夏远军%李蕾%高兰%谢晶%周丕琪%杨艳萍

    침림%무가림%하원군%리뢰%고란%사정%주비기%양염평

    骨质疏松%成骨细胞%细胞,培养的%骨钙素%口服液 骨質疏鬆%成骨細胞%細胞,培養的%骨鈣素%口服液
    골질소송%성골세포%세포,배양적%골개소%구복액
    背景:阿胶强骨口服液治疗骨质疏松疗效肯定,但其确切作用机制有待探讨.目的:观察阿胶强骨口服液含药血清对胎鼠成骨细胞中骨保护素及保护素配体表达的影响,探讨阿胶强骨胶囊治疗骨质疏松的分子水平作用.设计:完全随机分组,对照实验.材料:实验于2003-06/2004-10在华中科技大学同济医学院附属协和医院中西医结合骨代谢实验室完成.选用3月龄Wistar大鼠(雌雄各半)30只,随机分为3组,阿胶强骨口服液组和雌激素组及对照组,每组10只.选用清洁级新生SD大鼠12只用于成骨细胞的分离和培养.方法:①各组Wistar大鼠灌胃7 d后,制备各组的含药血清.②将清洁级新生SD大鼠分离的颅骨成骨细胞,制成单细胞悬液,消化传代,取二代培养的成骨细胞,制成细胞悬液.培养细胞被分为5组,分别加同体积药液.阿胶强骨口服液组分别加入用培养液稀释为100和500及1 000 g/L浓度的阿胶强骨口服液含药血清;雌激素组分别加入100及1 000 g/L替勃龙含药血清;对照组仅加培养液.同时各组再加入含100g/L小牛血清的培养液,继续培养.③采用四甲基偶氮唑盐比色分析法、3H-胸腺嘧啶核苷掺入法测定成骨细胞增殖,用放免法测定细胞内骨钙素含量,反转录聚合酶链反应测定胎鼠成骨细胞中骨保护素及保护素配体mRNA的表达.④组间比较采用单因素方差分析.主要观察指标:胎鼠成骨细胞经阿胶强骨口服液或利维爱含药血清干预后,细胞中骨保护素及保护素配体mRNA的表达.结果:①成骨细胞增殖四甲基偶氮唑盐比色分析法及3H-胸腺嘧啶核苷测定结果:阿胶强骨口服液组和替勃龙组各药物血清组明显高于对照组(P＜0.05～0.01),并在500g/L时达到最大效应(P＜0.01),当浓度大于500g/L时,其作用趋于饱和.各浓度阿胶强骨口服液及替勃龙含药血清均可增加成骨细胞骨钙素含量(P均＜0.05).②骨保护素基因mRNA表达:阿胶强骨口服液组含药血清1000g/L时最强,明显高于100和500g/L(P＜0 05),阿胶强骨口服液组含药血清1000g/L及雌激素组含药血清100和1 000g/L明显高于对照组(P＜0 01).③RANKL基因表达:阿胶强骨口服液组含药血清1 000g/L时最强,明显低于100和500 g/L(P＜O 05),阿胶强骨口服液组含药血清1000g/L及雌激素组含药血清100和1 000g/L明显低于对照组(P＜0 01).结论:①阿胶强骨口服液对成骨细胞的增殖有明显的促进作用,并且这种作用呈现剂量依赖性和可饱和性,与替勃龙相近.②阿胶强骨口服液治疗骨质疏松疗效机制之一可能与其促进成骨细胞的增殖,调节OPG/RANKL表达有关.
    배경:아효강골구복액치료골질소송료효긍정,단기학절작용궤제유대탐토.목적:관찰아효강골구복액함약혈청대태서성골세포중골보호소급보호소배체표체적영향,탐토아효강골효낭치료골질소송적분자수평작용.설계:완전수궤분조,대조실험.재료:실험우2003-06/2004-10재화중과기대학동제의학원부속협화의원중서의결합골대사실험실완성.선용3월령Wistar대서(자웅각반)30지,수궤분위3조,아효강골구복액조화자격소조급대조조,매조10지.선용청길급신생SD대서12지용우성골세포적분리화배양.방법:①각조Wistar대서관위7 d후,제비각조적함약혈청.②장청길급신생SD대서분리적로골성골세포,제성단세포현액,소화전대,취이대배양적성골세포,제성세포현액.배양세포피분위5조,분별가동체적약액.아효강골구복액조분별가입용배양액희석위100화500급1 000 g/L농도적아효강골구복액함약혈청;자격소조분별가입100급1 000 g/L체발룡함약혈청;대조조부가배양액.동시각조재가입함100g/L소우혈청적배양액,계속배양.③채용사갑기우담서염비색분석법、3H-흉선밀정핵감참입법측정성골세포증식,용방면법측정세포내골개소함량,반전록취합매련반응측정태서성골세포중골보호소급보호소배체mRNA적표체.④조간비교채용단인소방차분석.주요관찰지표:태서성골세포경아효강골구복액혹리유애함약혈청간예후,세포중골보호소급보호소배체mRNA적표체.결과:①성골세포증식사갑기우담서염비색분석법급3H-흉선밀정핵감측정결과:아효강골구복액조화체발룡조각약물혈청조명현고우대조조(P＜0.05～0.01),병재500g/L시체도최대효응(P＜0.01),당농도대우500g/L시,기작용추우포화.각농도아효강골구복액급체발룡함약혈청균가증가성골세포골개소함량(P균＜0.05).②골보호소기인mRNA표체:아효강골구복액조함약혈청1000g/L시최강,명현고우100화500g/L(P＜0 05),아효강골구복액조함약혈청1000g/L급자격소조함약혈청100화1 000g/L명현고우대조조(P＜0 01).③RANKL기인표체:아효강골구복액조함약혈청1 000g/L시최강,명현저우100화500 g/L(P＜O 05),아효강골구복액조함약혈청1000g/L급자격소조함약혈청100화1 000g/L명현저우대조조(P＜0 01).결론:①아효강골구복액대성골세포적증식유명현적촉진작용,병차저충작용정현제량의뢰성화가포화성,여체발룡상근.②아효강골구복액치료골질소송료효궤제지일가능여기촉진성골세포적증식,조절OPG/RANKL표체유관.
    BACKGROUND:The therapeutic effects of donkey-hide glue reinforcing bone oral solution on osteoporosis have been determined, but the exact effective mechanism is to be approached. OBJECTIVE: To investigate the effects of donkey-hide glue reinforcing bone oral solution (DGRBOS) medicated serum on osteoprotegerin (OPG)and its ligand(OPGL)mRNAexpression of osteoblast in fetal rats and explore the molecular mechanism of treating osteoporosis with DGRBOS. DESIGN: A randomized controlled trial. MATERIALS: The experiment was carried out from June 2003 to October 2004 in Bone Metabolic Laboratory of Department of Integrative Chinese and Western Medicine, Affiliated Hospital of Tongji Medical College,Huazhong University of Technology and Science. Totally 30 3-month-old Wistar rats (15 males and 15 females) were randomly divided into 3 groups, I.e. DGRBOS group, estrogen group and control group, with 10 rats in every group. 12 clean newborn SD rats were selected to isolate and cul ture osteoblast. METHODS: ①After intragastric administration for 7 days, medicated serum was prepared respectively from the three groups. ②Skull osteoblast isolated from newborn SD rats was made into single cell suspension, then after digestion and passage, the subcultured osteoblast cell was made into cell suspension. The cultured osteoblasts were divided into 5 groups and given equal volumes of drug liquor. The DGRBOS group was given DGR BOS-medicated serum at the concentration of 100, 500 and 1 000 g/L which was diluted by nutrient solution; the estrogen group was given tibolone-medicated serum of 100 and 1 000 g/L; the control group was given only culture fluid. Meanwhile every group was given calf serum (100 g/L) for further culture. ③The osteoblast proliferation was measured by antigenic MTT colorimetric analysis and 3H-TdR penetration method. The in tra-cellular BGP contents were evaluated by radioimmunity .The mRNA expression of OPG and RANKL in osteoblast was analyzed by Rt-PCR. ④ One-way analysis of variance was applied to compare data among groups. MAIN OUTCOME MEASURES: mRNA expression of OPG and PAN KL in osteoblasts from fetal rats after intervention by medicated serum of DGRBOS or Livial. RESULTS: ①The osteoblast proliferation measured by antigenic MTT colorimetric analysis and 3H-TdR penetration method showed that the proliferation in the DGRBOS group and tibolone group was enhanced more significantly than that in the control group (P ＜ 0.05-0.01), and reached maximal effect at the concentration of 500 g/L (P ＜ 0.01), but when the concentration was over 500 g/L, the effect tended to saturate. The medicated serum with all concentrations from DGRBOS and estrogen groups could increase the contents of BGP in osteoblasts (P ＜ 0.05). ②The mRNA expression of OPG reached the peak when the DGRBOS medicated serum was 1 000 g/L, and was obviously higher than that at the concentration of 100 and 500 g/L (P ＜ 0.05). The expression in DGRBOS group at the concentration of 1 000 g/L and in the estrogen group at the concentration of 100 and 1 000 g/L was apparently higher than that of the control group (P ＜ 0.01). ③The mRNAexpression of RANKL was the highest in DGR BOS group with 1 000 g/L concentration, and was markedly lower than that of the concentration of 100 and 500 g/L (P ＜ 0.05). The expression in DGRBOS group at the concentration of 1 000 g/L and in the estrogen group at the concentration of 100 and 1 000 g/L was noticeably lower than that in the control group (P ＜ 0.01).CONCLUSION: ①The DGRBOS could remarkably enhance osteoblast proliferation in dose-dependent and a dose-saturable manner, and the effect was close to that of tibolone. ②Partial mechanism of DGRBOS in treating osteoporosis might be promoting osteoblast proliferation and regulating OPG/RANKL expression.
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