【作者】 杨丽；

【导师】 张荣华；

【作者基本信息】 暨南大学 ， 中西医结合临床， 2007， 博士

【摘要】 目的：通过比较正常与去卵巢骨质疏松（Osteoporosis，OP）大鼠骨髓间充质干细胞（Mesenchymal stem cells，MSCs）的生物学特性，探讨去卵巢对大鼠MSCs的影响；研究骨碎补及淫羊藿水提液、淫羊藿苷及柚皮苷、益骨胶囊含药血清及细胞因子转化生长因子β₁（Transforming growth factor-betal，TGF-β₁）、骨形成蛋白-2（Bone morphogenetic protein-2，BMP-2）对MSCs增殖和骨向分化能力的影响，并观察确有防治OP疗效的单味中药、中药单体、中药复方对Smads蛋白通路中上游细胞因子TGF-β₁、BMP-2的影响，分析它们可能的作用途径，为阐释其防治OP的作用机理提供实验依据，并为以TGF-β₁、BMP-2作为平台筛选防治OP药物提供可行性实验探索。方法：1．运用全骨髓贴壁法分离培养MSCs；2．通过形态学、流式细胞仪检测表面标志物及多向分化能力鉴定MSCs；3．选用10月龄Sprague-Dawley（SD）雌性大鼠双侧卵巢切除增龄3月法复制OP模型；4．通过比较正常与OP大鼠MSCs形态学、生长曲线、生长周期及骨向、脂向软骨向分化能力探讨去卵巢对大鼠MSCs的影响；5．运用MTT法检测不同浓度各干预药物对大鼠MSCs增殖的影响，以确定其最佳促增殖剂量；6．以碱性磷酸酶（Alkaline phosphatase，ALP）确定各干预药物不同浓度梯度中最佳促MSCs骨向分化剂量；7．实验分为空白组、经典组(10^-8地塞米松+10 mmol／Lβ-甘油磷酸钠+50μmol／L维生素C)、药物组（药物最佳诱导骨向分化剂量）和药物+经典组(药物最佳诱导骨向分化剂量+10^-8M地塞米松+10 mmol／Lβ-甘油磷酸钠+50μmol／L维生素C)，通过检测诱导分化过程中ALP、Ⅰ型胶原（TypeⅠcollagen，ColⅠ）、骨钙素（Bone gla protein，BGP）和钙化结节等指标的表达，来观察其对MSCs骨向分化能力的影响；8．ELISA法检测各药物骨向分化过程中TGF-β₁、BMP-2的表达，阐释其促MSCs骨向分化可能的作用机制；9．Real-time PCR法检测rhBMP-2、rhTGF-β₁对Smad4及其下游细胞因子Cbfα1 mRNA，进一步阐释各药物诱导MSCs骨向分化可能的作用机制。结果：1．流式细胞仪检测大鼠MSCs表面标志抗原CD44、CD29表达阳性，CD45及CD34表达阴性，MSCs能成功诱导为成骨细胞、脂肪细胞和软骨细胞；2．与正常组相比，OP大鼠MSCs体外增殖能力下降，其骨向分化能力及对成骨诱导剂反应力降低，脂向分化能力及对成脂诱导剂反应力增强，对软骨诱导剂反应力下降；3．骨碎补和淫羊藿水提液最佳促MSCs增殖浓度均为5μg／ml，最佳促MSCs骨向分化浓度分别为50μg／ml、500μg／ml。骨碎补和淫羊藿水提液诱导各组均能促进成骨指标的表达，且伴随TGF-β₁和BMP-2分泌增加；4．低灌胃剂量益骨胶囊灌胃1 h后采血所得含药血清以25％的浓度干预时具有最佳的促MSCs增殖作用；最佳益骨胶囊含药血清促MSCs骨向分化浓度是高灌胃剂量灌胃1h后采血所得含药血清25％-30％添加浓度；益骨胶囊含药血清诱导各组均能促进成骨指标的表达，且伴随TGF-β₁和BMP-2分泌增加；5．前期研究证实确有促MSCs骨向分化能力的中药单体淫羊藿苷和柚皮苷各诱导组均伴随TGF-β₁和BMP-2分泌增加；6．rhTGF-β₁和rhBMP-2最佳促MSCs增殖浓度分别为10 ng／ml和80 ng／ml，最佳促MSCs骨向分化浓度分别为5 ng／ml和40 ng／ml，rhTGF-β₁和rhBMP-2诱导各组均能促进成骨指标的表达，且伴随TGF-β₁和BMP-2分泌增加；7．rhTGF-β₁和rhBMP-2诱导各组均能提高Smad4和核心结合因α1（Core binding factor alpha1，Cbfα1）mRNA的表达量。结论：1．采用全骨髓贴壁法可获得稳定、均质性良好的大鼠MSCs；2．OP大鼠MSCs骨向及软骨向分化能力下降，脂向分化能力增强：3．中药骨碎补和淫羊藿水提液、益骨胶囊含药血清及细胞因子TGF-β₁、BMP-2均能促进MSCs骨向分化；4．在MSCs骨向分化过程中，上调TGF-β₁、BMP-2的表达量可能是各干预药物促进MSCs骨向分化的作用机制之一；5．各诱导药物可能通过增加TGF-β₁和BMP-2的分泌量，进而上调Smad4和Cbfα1mRNA的表达，来刺激MSCs骨向分化。更多还原

【Abstract】 Objective: The research was to investigate the effects of ovariectomy on SD rats by compairing the biological characteristics of the mesenchymal stem cells（MSCs） derived from the osteoporosis （OP） rats and normal rats. To study the effects of traditional chinese medicine such as Drynaria Rhizome, epimedium, Benefiting-bone capsule （BBC） and cytokines such as transforming growth factor-betal （TGF-β₁）、bone morphogenetic protein-2 （BMP-2） on the proliferation and differentiation into osteoblast of MSCs to further confirm the best proliferation and differentiation concentration of them on MSCs. To observe the expression of TGF-β₁ and BMP-2 in the induced systems of the herbs above which were confirmed to have the accurate effects of preventing and treating OP to further explain the mechanism of them in inducing MSCs differenting into osteoblast, to explain the mechanism of them in preventing and treating OP and to provide the experimental exploration in establish a platform by TGF-β₁、BMP-2 to screen the medicine of preventing and treating OEMethods:1. MSCs was isolated and purified by differential time adherent method;2. MSCs was identified by morphologic characteristics, surface antigens detected by flow cytometer and differentiation into osteoblasts, adipocytes and chondrocytes;3. The osteoporotic animal model was established by ovariectomy on female Sprague-Dawley （SD） rats aged 10 month;4. To investigate the effects of ovariectomy by compairing the morphology, growth curve and cell cycle and differentiation capacity into osteoblasts, adipocytes and chondrocytes of the MSCs derived from the OP rats and normal rats;5. MTT method was used to confirm the most effective concentration of the traditional chinese medicine and cytokines on the proliferation of MSCs;6. The most effective concentration of the traditional chinese medicine and cytokines on the differentiation of MSCs into osteoblast was jurged by alkaline phosphatase （ALP）;7. According to the different induced condition, MSCs were divided into 4 groups: control group, classic group （induced by the classic osteoblast-induced system）, drugs group （induced by the most effective concentration of drugs on differentiation into osteoblast） and drugs+classic group （induced by the combination of classic osteoblast-induced system and the most effective concentration of drugs on differentiation into osteoblast）. ALP、typeⅠcollagen （ColⅠ）、bone gla protein （BGP）and calcium nodes in each group were detected and compaired to indicate the osteoblast-formation function of different group;8. ELISA was used to detect the expression of TGF-β₁、BMP-2 of each induced group in the progress of MSCs differenting into osteoblast to explain the mechanism of the drugs in inducing MSCs differenting into osteoblast;9. Real-time PCR was used to detect the expression of Smad4 and core binding factor alphal （Cbfα1） mRNA in the induced groups of TGF-β₁ and BMP-2 to further explain the mechanism of the drugs in inducing MSCs differenting into osteoblast.Results:1. The results of the surface antigens of MSCs detected by flow cytometer showed the positive expressions of CD29 and CD44 and the negative expressions of CD34 and CD45; MSCs isolated from SD rats could be induced successfully into osteoblasts, adipocytes and chondrocytes;2. The proliferation in vitro of MSCs from OP rats decreased compaired with that form normal rats; the ability of differentiation into osteoblast and the reaction to the classic osteoblast-induced agent of MSCs from OP rats was lower than that form normal rats; the ability of differentiation into adipocytes and the reaction to the classic adipocytes-induced agent of MSCs from OP rats was higher than that form normal rats; the reaction to the classic chondrocytes-induced agent of MSCs from OP rats decreased compaired with that form normal rats; 3. The most effective concentration of the Rhizoma Drynariae and epimedium water-extraction on the proliferation of MSCs both was 5μg/ml; and the most effective concentration of them on the differentiations of MSCs into osteoblast were 50μg/ml and 500μg/ml; the Rhizoma Drynariae and epimedium water-extraction could both increase the expression of osteoblast-indices which follwed the increase of the secretion of TGF-β₁ and BMP-2;4. The 25% concentration of the drug-containing sera of BBC collected form SD rats one hour later after intragastric infusion with low dose BBC was proved to be the most effective concentration on the proliferation of MSCs; the 25%-30% concentration of the drug-containing sera of BBC collected form SD rats one hour later after intragastric infusion with high dose BBC was proved to be the most effective concentration on the differentiations of MSCs into osteoblast; the induced groups of BBC were confirmed to increase the expression of osteoblast-indices which follwed the increase of the secretion of TGF-β₁ and BMP-2;5. Naringin and Icariin which had been proved to possess the ability to induce MSCs differenting into osteoblast in former study could both increase the expression of TGF-β₁ and BMP-2;6. The most effective concentrations of rhTGF-β₁ and rhBMP-2 water-extraction on the proliferation of MSCs were 10ng/ml and 80ng/ml; and the most effective concentration of them on the differentiations of MSCs into osteoblast were 5 ng/ml and 40ng/ml; rhTGF-β₁ and rhBMP-2 could both increase the expression of osteoblast-indices which follwed the increase of the secretion of TGF-β₁ and BMP-2;7. The induced groups of rhTGF-β₁ and rhBMP-2 could improve the expression of Smad4 and Cbfα1 mRNA.Conclusion:1. The stable and uniformal MSCs could be obtained by differential time adherent method;2. The MSCs from OP rats’ ability of differentiation into adipocytes and chondrocytes decreased and the ability of differentiation into adipocytes increased;3. The Chinese herbs of Rhizoma Drynariae, epimedium and BBC and the cytokines of rhTGF-β₁ and rhBMP-2 could all promote the differentiation of MSCs into osteoblast;4. In the osteoblast-differentiation progress, the induced groups of the drugs in this expriment all could increase the expression of TGF-β₁ and BMP-2, which may he the mechanism of them on improving the differentiation of MSC into osteoblast;5. The increase of the expression of Smad4 and Cbfα1 mRNA may be the further mechanism of the drugs on improving the differentiation of MSC into osteoblast after they stimulated the secretion of TGF-β₁ and BMP-2.更多还原