【作者】 吴菲菲；

【导师】 徐永平；

【作者基本信息】 大连理工大学 ， 生物化工， 2013， 博士

【摘要】 骨质疏松症是一种以骨量减少、骨组织显微结构退行性改变,骨的脆性增加,易于发生骨折为特征的全身代谢性骨骼疾病,多发于绝经后妇女及老年人。骨质疏松性骨折发病率高,严重威胁着人们的生命健康,并给社会和国家带来了沉重的经济负担。目前,用于治疗骨质疏松症的西药,如鲑鱼降钙素、双膦酸盐类和重组人甲状旁腺素等,具有明显的副作用且价格昂贵。因此,研究开发安全、高效、经济的抗骨质疏松症药物已成为目前的研究热点。据《神农本草经》记载,鹿角盘具有温补肝肾、强筋健骨、活血消肿等作用。经我们课题组前期的体内实验研究发现,鹿角盘微切助粉对去卵巢大鼠绝经后骨质疏松症模型有明显且良好的治疗效果。但是,关于鹿角盘防治骨质疏松症的作用机制至今仍未有报道。因此,本论文旨在系统地研究鹿角盘提取物对体外培养的成骨细胞骨形成功能和破骨细胞骨吸收活性的影响,并进一步探讨其防治骨质疏松症的作用机制。具体研究内容和结果如下：(1)采用微切变-助剂互作技术加工制备鹿角盘微切助粉。然后经粒径分析及扫描电镜观察分析细胞的破碎程度,并检测其中与骨质疏松症相关的活性物质的含量。粒径分析及扫描电镜观察结果显示,经微切变-助剂互作技术处理后,鹿角盘微切助粉在粒径1-30μm范围内的颗粒数占82.73%,且细胞已被充分破碎,细胞内有效成分被暴露出来,呈释放的状态。活性物质含量分析结果显示,鹿角盘微切助粉中含有328.79士34.21pmol/L雌二醇、25.38士4.48nmol/L睾酮、17.56士3.45nmol/L孕酮,0.750±0.033μg/g类胰岛素生长因子-1,16.17±0.52mg/L柠檬酸钙和133.6±13.55mg/L钙,而不含有延胡索酸钙。且这些活性物质的含量均高于其粗粉,说明微切变-助剂互作技术增加了目标活性物质的溶出量,有利于鹿角盘中活性成分的释放,体现了该技术的优越性。(2)以人类成骨样细胞MG-63为研究对象,系统地研究了鹿角盘提取物(水提物和醇提物)对成骨细胞生长、分化和矿化的影响及其防治骨质疏松症的可能作用机制。LDH活性检测和细胞形态观察结果表明,鹿角盘提取物对成骨细胞没有细胞毒性作用。采用结晶紫试验、中性红吸收试验、台盼蓝排斥试验、MTT法和ALP活性检测考察细胞的生长和分化情况。结果表明,鹿角盘提取物不仅刺激了成骨细胞的生长和增殖,而且还促进了成骨细胞的分化。此外,茜素红-S染色和4-羟脯氨酸含量的检测结果显示,鹿角盘提取物是通过促进钙的沉积和增加Ⅰ型胶原蛋白的合成和分泌,促进矿化骨基质的形成,从而发挥促进骨形成的作用。采用ELISA法和RT-PCR分别检测了OPG和RANKL的蛋白含量和mRNA的表达量,结果显示,鹿角盘提取物极显著地增加了成骨细胞中OPG蛋白含量和OPG mRNA的表达量,而降低了RANKL蛋白含量和RANKL mRNA的表达量,提高了成骨细胞中OPG/RANKL的比值,间接地抑制破骨细胞的分化和成熟,从而起防治骨质疏松症的作用。(3)以RAW264.7细胞作为一种破骨前体细胞模型,诱导其分化成破骨细胞,并研究鹿角盘提取物对破骨细胞的形成和骨吸收活性的影响。TRAP染色和破骨细胞计数结果显示,用50ng/mL RANKL成功地诱导RAW264.7细胞分化成为成熟的破骨细胞。而且鹿角盘提取物可剂量依赖性地减少破骨细胞的数量,降低破骨细胞中TRAP的活性,从而抑制了RANKL诱导RAW264.7细胞分化形成成熟的破骨细胞。LDH活性检验、PI染色和DNA碎片定量分析结果一致表明,鹿角盘提取物是通过诱导成熟的破骨细胞发生凋亡而抑制其活性。而且,RT-PCR检测结果表明鹿角盘提取物是通过下调破骨细胞表型基因TRAP mRNA和MMP-9mRNA的表达,来抑制成熟破骨细胞的骨吸收活性,从而起防治骨质疏松症的作用。(4)采用p38特异性抑制剂SB203580干预处理MG-63成骨细胞,进一步验证鹿角盘提取物的抗骨质疏松作用机制。用p38特异性抑制剂SB203580干预处理成骨细胞,然后采用MTT法、ALP法、茜素红-S染色和4-羟脯氨酸含量的检测分别评价SB203580对成骨细胞的增殖、分化和矿化的影响。结果表明,SB203580可以有效地阻断鹿角盘提取物对成骨细胞增殖、分化和矿化骨基质形成的促进作用。而且,用ELISA法和RT-PCR检测OPG和RANKL蛋白含量和基因的表达,结果显示鹿角盘提取物上调了OPG蛋白和mRNA的表达,下调了RANKL蛋白和mRNA的表达,且通过调控成骨细胞中OPG和RANKL的表达,间接地抑制破骨细胞的分化和成熟。而SB203580能有效地阻断鹿角盘提取物对破骨细胞的抑制作用,提示鹿角盘提取物是通过调控p38MAPK信号通路,双向影响成骨细胞和破骨细胞之间的动态平衡,从而起抗骨质疏松的作用。综上所述,本研究证实了鹿角盘提取物是通过调控p38MAPK信号通路来促进成骨细胞的骨形成和抑制破骨细胞的骨吸收,从而起抗骨质疏松的作用,为鹿角盘用于治疗骨质疏松症提供了理论依据。更多还原

【Abstract】 Osteoporosis is a systematic skeleton disease characterized by low bone mineral density and structural deterioration of bone tissue leading to bone fragility and increased cases of fractures particularly of the hip, spine and wrist. It is a disease common in post-menopausal women and the elderly. The incidence of osteoporosis-related fractures is very high and has enormous impact on public health and on the quality of life of the patient. Nowadays, many medicines for osteoporosis have some disadvantages such as adverse side effects or a high cost, which including salmon calcitonin, bisphosphonates and human parathyroid hormone. Thus, alternative drugs of efficacy, safety and low cost, should be developed for the prevention and treatment of osteoporosis. Deer antler base (Cervus) has been recorded in the Chinese medical classics Shen Nong Ben Cao Jing2000years ago and is believed to tonify the kidney, invigorate the spleen, strengthen bones and muscles, and promote blood flow.Our previous study has showed that deer antler base has a good therapeutic effect on ovariectomy-induced osteoporosis in rats. But since now no studies have been carried out to evaluate the mechanisms by which deer antler base exerts its anti-osteoporotic effect. Therefore, the present study aimed to systematically investigate the effects of deer antler base extracts on the bone formation of osteoblasts and bone resorption of osteoclasts in vitro and its mechanisms of action. The main results are summarized as following.(1) The raw material of deer antler base was processed into PAI powders by the press-shear assisted interaction technology (PAI) in an energy-intensive vibrational mill. The particle size and the content of main functional ingredients related to osteoporosis were detected.To see what damage had been done to the cells, the PAI powders were examined using a laser diffraction particle analyzer and scanning electron microscopy. The results showed that the percentage of particle size ranged from1-30μm accounted for82.73%after PAI treatment, and the cells of deer antler base PAI powder were basically broken. The active ingredients were probably released upon mixing with cellular fragments. The tests for active ingredients showed that deer antler base contains328.79±34.21pmol/L estradiol,25.38±4.48nmol/L testosterone,17.56±3.45nmol/L progesterone,0.750±0.033μg/g IGF-1,16.17±0.52mg/L calcium citrate and133.61±3.55mg/L free calcium, but no calcium fumarate. Moreover, the contents of active ingredients were larger than those of coarse powder, indicating press-shear assisted interaction technology is not only conducive to the release of active ingredients, but also to raise the dissolution rate of active ingredients.(2) Influences of deer antler base extracts (aqueous extract and ethanolic extract) on the proliferation, osteogenic differentiation and mineralization of human osteoblast-like MG-63cells in vitro.The results of lactate dehydrogenase (LDH) activity and cell morphology indicated that deer antler base extracts did not show cytotoxicity. Cell growth and viability was assessed by crystal violet test, neutral red absorption test, trypan blue exclusion test and MTT test. And the cell differentiation was evaluated by alkaline phosphatase (ALP) activity. The results showed that deer antler base extracts not only stimulated the growth and proliferation of MG-63cells, but also promoted the differentiation of MG-63cells. The results of alizarin red-S staining and4-hydroxyproline test showed that deer antler base extracts promoted the bone matrix mineralization by stimulating the deposition of calcium and increasing the synthesis and secretion of type I collagen. In addition, the OPG and RANKL protein and mRNA levels were detected using an enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). The results indicated that deer antler base extracts can significantly increase the OPG protein and mRNA level, whereas decrease the RANKL protein and mRNA level, thereby increasing the ratio of OPG/RANKL and indirectly inhibit osteoclast differentiation and maturation. The findings suggest that deer antler base extracts promote bone formation of osteoblasts and play a role on prevention and treatment of osteoporosis.(3) The effects of deer antler base extracts on osteoclastogenesis and bone resorption activity were investigated using the macrophage cell line RAW264.7as a model of osteoclast precursors.The results of TRAP staining and cell counting showed that the RAW264.7cells can differentiate into osteoclast-like cells when stimulated by50ng/mL of recombinant mouse soluble RANKL. Moreover, deer antler base extracts could reduce the number of osteoclasts and the TRAP activity in a dose-dependent manner, indicating suppresses the RANKL-induced differentiation of RAW264.7cells. The results of LDH activity, PI staining and quantitative analysis of DNA fragmentation consistently indicated that deer antler base extracts could induce the apoptosis of mature osteoclasts. Furthermore, the RT-PCR results indicated that deer antler base extracts could down-regulate the osteoclastic phenotypic genes, TRAP and MMP-9mRNA expression levels. These results suggest that deer antler base extracts inhibit bone resorption of mature osteoclasts and play a role on prevention and treatment of osteoporosis. (4) The anti-osteoporotic mechanisms of deer antler base extracts were further investigated using MG-63cells treated with p38specific inhibitor SB203580.After treatment with p38specific inhibitor SB203580, MTT assay, ALP activity, alizarin red-S staining and4-hydroxyproline test were respectively used to evaluate the effects of SB203580on proliferation, differentiation and mineralization of MG-63cells. The results showed that deer antler base extracts can effectively promote the proliferation, differentiation and mineralization of osteoblasts, while the effects can be blocked by the p38specific inhibitor SB203580. Further, the up-regulation of OPG and down-regulation of RANKL at the protein and mRNA level were observed in deer antler base extracts treated MG-63cells using an enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). The results implied that deer antler base extracts can indirectly inhibit osteoclast differentiation and maturation by regulating the expression of OPG and RANKL in MG-63cells, while the inhibitory effect can be effectively blocked by the p38specific inhibitor SB203580. The findings suggest that deer antler base extracts could affect the dynamic balance of osteoblasts and osteoclasts, which may be mediated via the p38MAPK signal pathway.Taken together, our results confirmed that deer antler base extracts could promote bone formation of osteoblasts and inhibit bone resorption of mature osteoclasts through the p38MAPK signal pathway, which providing a theoretical basis for the prevention and treatment of osteoporosis.更多还原