【作者】 徐贤柱；

【导师】 王曼莹；

【作者基本信息】 江西师范大学 ， 运动人体科学， 2007， 硕士

【摘要】 目的:为竞技运动员提供安全有效的护骨类功能食品与研制高效防治运动性骨质疏松症的杜仲类中成药提供理论基础。从细胞水平与分子水平研究杜仲叶提取物对大鼠成骨细胞生长的影响和对肾细胞分泌护骨素(OPG)的影响,探讨药食两用的杜仲叶多靶位防治骨质疏松症的分子机理。方法:①分析比较竞技运动员骨组织健康状况与运动性骨质疏松症的发生;②杜仲叶提取物——从新鲜杜仲叶获得提取液,制备杜仲叶浸膏;③细胞培养——在体外培养的大鼠成骨细胞和肾细胞中加入不同浓度的杜仲提取物作用96h,采用双抗体夹心ABC-ELISA法对肾细胞分泌的OPG和成骨细胞分泌的破骨细胞分化因子(RANKL)、骨钙素(OC)、Ⅰ型胶原(COLL-Ⅰ)的浓度进行测定;对体外培养的成骨细胞和肾细胞进行1:1和1:2的混合培养,加入不同浓度的杜仲提取物,采用MTT法测定细胞的增殖情况。④分子水平研究——分别对加入最佳浓度杜仲叶提取物作用的成骨细胞和肾细胞OPG mRNA进行RT-PCR,检测mRNA的转录量。结果:体外培养成骨细胞在不同浓度杜仲叶提取物的作用下,分泌RANKL、OC、COLL-Ⅰ都有区别,和空白组进行对照有显著性差异。利用RT-PCR检测OPG的mRNA转录量,加药组比空白组的转录量更高;体外培养肾细胞在杜仲叶提取物不同浓度的作用下,OPG的分泌量不同,和空白组相比有显著性差异。混合培养的最佳比例是成骨细胞:肾细胞为2:1,这样更能促进成骨细胞的生长。MTT的检测说明不同浓度杜仲叶提取物对细胞生长的作用不同,在10-4g/ml、10-5g/ml、10-6g/ml这三个浓度中,表现出作用效果的剂量依赖性。对成骨细胞作用最明显的剂量是10-5g/ml。对肾细胞作用最明显的剂量是10-4g/ml,而10-3g/ml反而会抑制细胞的生长。结论:杜仲叶提取物对成骨细胞和肾细胞具有明显的作用,可协同调节骨代谢过程。是一种多靶点发挥成骨作用的药食两用的道地中药材。它降低破骨细胞的活性,增加骨密度,促进骨重建,有效防治骨质疏松症。是研制竞技运动员的护骨性功能食品与防治运动性骨质疏松症的药物的理想原料之一。更多还原

【Abstract】 Objective: The impacts of Eucommia leaves extract on the growth of rat’s osteoblast and OPG secreting from kidney cell were study, and the function of Eucommia as an functional food and the mechanism in preventing osteoporosis were discussed. The mechanism of exercise-osteoporosis and health care were studied to provide the theory basis for athletics movement and develop a kind of functional food being able to prevent exercise-osteoporosis.Method: Firstly, the Eucommia leaves extract was extracted from the fresh Eucommia leaves; the Eucommia extract in different density were joined into rat’s osteoblast and the kidney cell in vitro and interacted for 96h, then the concentrations of kidney cell secretion OPG, the osteoblast secretion osteoclast differentiation factor (RANKL), the osteocalcin (OC) and I collagen (COLL- I) were determined by ABC-ELISA; osteoblast and the kidney cell in vitro were mixed by 1:1 and 1:2, joining with the Eucommia extract in different density, and the cell multiplication was determined by MTT. RT-PCR was used for transcription OPG mRNA which was stimulated by Eucommia leaves extract in the best density, to examine the transcription quantity of mRNA. Results: Under the stimulation of Eucommia leaves extract in different density, osteoblast in vitro secreted RANKL, OC, COLL- I differently, appearing the obvious difference compared with the blank contrast group. Examining OPG mRNA by RT-PCR, the transcription quantity of drug group was higher than the blank contrast group. Under the stimulation of Eucommia leaves extract in different density, the OPG secretion from kidney cell in vitro also appeared differently. There was an obvious difference compared with the blank contrast group. The osteoblast mixed with the kidney cell best in 2:1, which would promote osteoblast to grow better; The MTT testing indicated that cells grew differently in different density Eucommia extract. Among 10-4g/ml, 10-5g/ml and 10-6g/ml, 10-5g/ml affected the osteoblast obviously, and 10-4g/ml affected the kidney cell remarkably, but 10-3g/ml suppressed the cell’s growth. There was no significant difference between 10-7g/ml and the blank contrast group. Conclusion: The Eucommia leaves extract has the obvious function to the kidney cell and the osteoblast. It promoted the osteoblast and the kidney cell to grow, and was a kind of multi-targets functional Chinese medicine. It can increase the bone reconstruction and the bone density,and reduced the osteoclast activeness, to prevent osteoporosis.更多还原