【作者】 郭敏；

【导师】 王建华；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2008， 硕士

【摘要】 目的:随着老龄化社会的到来,很多与增龄有关的老年病越来越受到人们的重视。骨质疏松症是一种增龄性病变,目前全世界约有2亿患者,其发病率跃居常见病、多发病的第7位。据2000年普查数据显示,我国约有骨质疏松症患者8800万,占总人口的6.6%。60~69岁老年妇女骨质疏松发生率高达50%~70%,老年男性发生率为30%左右。每年的医疗费用按最保守估计也需要50亿人民币。骨质疏松引起的骨折及其并发症严重影响着中老年人的生活质量,成为全球性的公共卫生问题之一。因此,有效预防与治疗骨质疏松症已成为当务之急。现代医学用于骨质疏松症防治的药物主要为骨吸收抑制剂和骨形成促进剂2类。目前最常用的是“激素替代疗法”,此种疗法虽然可以防治绝经后妇女骨量丢失,提高骨密度,但长期应用有促进子宫内膜增生、肝功损害、静脉栓塞、血管疾病以及增加乳腺癌的发生率等不良后果。植物雌激素(phytoestrogen, PE)是存在于植物、水果和蔬菜中的一类天然的、结构及生物活性均类似于雌激素的非甾体类化合物,具有弱雌激素活性,为杂环多酚类化合物。PE可与雌激素受体结合,对内源性雌激素起双向调节作用,其在改善围绝经期症状、降低血脂水平、防治骨质疏松、预防尿失禁等方面有着有益作用,且没有因服用雌激素而出现的各种不良后果。所以植物雌激素在防治骨质疏松症方面代替雌激素将是一个很好选择。中药在补骨方面有很好的疗效。中药补骨脂为豆科植物补骨脂Psoralea corylifolia L.的干燥成熟果实,有补肾壮阳,温脾止泻之功效。从补骨脂的现代药理研究来看,补骨脂水提物对骨转换的影响可能包括抑制骨吸收和促进骨形成两方面。并对雌激素依赖性骨丢失具有防治作用。补骨脂丙酮提取物能增加大鼠骨强度,提高血中磷酸盐水平,用补骨脂复方制剂给模型大鼠灌胃,能使其血清碱性磷酸酶降至正常水平,显著降低尿中Ca/cr和HOP/cr水平,并显著抑制骨吸收,使大鼠骨代谢处于骨形成大于骨吸收的平衡状态。补骨脂的主要化学成分是香豆素类和黄酮类化合物。补骨脂素是补骨脂的主要活性成分之一。已经证明补骨脂素在体外促进成骨细胞增殖、分化及合成1型胶原,但其对成骨细胞内信号系统的作用机制尚不清楚。本研究在前人研究基础上对补骨脂素防治骨质疏松的分子生物学机制进行探讨,以期为临床防治骨质疏松提供可参考的理论依据,并为研制防治骨质疏松药物提供了有效的作用靶点。本研究探讨香豆素类植物雌激素防治骨质疏松的分子生物学机制。方法:1采用改良的组织块培养法,分离培养新生(24h)SD大鼠颅骨成骨细胞。将不同浓度的异欧前胡素(10-9 mol/L~10-5 mol/L)加入到培养成骨细胞的培养基中,用倒置显微镜观察细胞形态和数量的改变。2采用噻唑蓝比色(MTT)法,以雌二醇(estradiol,E2)为阳性对照,考察加药后,异欧前胡素24h,48h,72h时对成骨细胞增殖的影响。3采用对硝基二钠基质动力学法(pNPP),以雌激素为阳性对照,检测加药后,异欧前胡素在24h,48h,72h时对成骨细胞碱性磷酸酶活性的影响。4采用逆转录聚合酶链反应(reverse transcriptase PCR, RT-PCR)方法,以雌激素为阳性对照,检测含有补骨脂素培养基的成骨细胞在72小时骨保护素(OPG)和核因子κB受体激活因子配体(RANKL)mRNA的表达。5采用免疫组织化学(immunohistochemistry)方法,雌激素为阳性对照组,在倒置相差显微镜下观察含补骨脂素的培养基中骨形态发生蛋白2(BMP-2)的表达。结果:1阳性对照雌激素组对成骨细胞增殖与分化的影响1.1阳性对照雌激素组细胞形态学观察刚接种的成骨细胞成球形,悬浮于培养液中,逐渐沉降贴壁,24 h细胞贴壁展开。展开细胞形态不规则,呈多角形,培养72 h~96 h,细胞为长梭形、条索状,融合成片,分界较为模糊。培养120 h,细胞呈铺路石状。如果继续培养,细胞逐渐形成细胞小结,随后胶原堆积,钙盐沉积,形成不透光的矿化结节。连续培养20代,可见细胞体积增大,胞质稀薄,细胞分裂速度大大下降等细胞衰老的表现。雌激素处理组与空白对照组相比,在72 h~96 h时,细胞更加密集,细胞间隙少,形态无明显差异。1.2阳性对照雌激素组对大鼠成骨细胞增殖的影响大鼠成骨细胞在含有不同浓度雌激素培养基中培养24 h ,在浓度为1×10-5 mol/L,1×10-7 mol/L和1×10-8 mol/L有促增殖作用(P<0.05);培养48 h,浓度为1×10-6 mol/L, 1×10-8 mol/L和1×10-9 mol/L有促增殖作用(P<0.05);培养72 h ,浓度为1×10-5 mol/L~1×10-7 mol/L与空白对照组比较有显著的促增殖作用(P<0.01)。1.3阳性对照雌激素组对大鼠成骨细胞碱性磷酸酶活性的影响培养48 h ,与空白对照组比较,雌激素组浓度为1×10-5 mol/L~1×10-6 mol/L有明显的增强碱性磷酸酶活性的作用(P<0.05);培养72 h ,浓度为1×10-6 mol/L~1×10-9 mol/L有增强碱性磷酸酶活性的作用(P<0.05)。2异欧前胡素对成骨细胞增殖和分化的影响2.1异欧前胡素组细胞形态学观察与阳性对照雌激素组类似。2.2异欧前胡素对大鼠成骨细胞增殖的影响大鼠成骨细胞在含有不同浓度异欧前胡素培养基中培养48h,浓度为1×10-9 mol/L~1×10-8 mol/L有促增殖作用(P<0.05);72h时,浓度为1×10-8 mol/L~1×10-7 mol/L有促增殖作用(P<0.05)。2.3异欧前胡素对大鼠成骨细胞碱性磷酸酶活性的影响培养48小时,与空白对照组比较,异欧前胡素各浓度组均没有明显差别(P>0.05)。72小时,与空白对照组比较浓度1×10-6mol/L~1×10-5mol/L的异欧前胡素能够提高碱性磷酸酶的活性,促进成骨细胞的分化。3补骨脂素对大鼠成骨细胞分泌的OPG mRNA ,RANKLmRNA表达的影响3.1补骨脂素对大鼠成骨细胞分泌的OPG mRNA表达的影响培养72小时后,空白对照组的OPG mRNA /GAPDH mRNA的相对表达量为0.6084,阳性对照组雌二醇组的OPG mRNA /GAPDH mRNA的相对表达量为1.3395,与空白对照组比较促进了OPGmRNA的表达,差异有显著性(P<0.05);补骨脂素组的OPGmRNA /GAPDHmRNA的相对表达量为1.0794,与空白对照组比较促进了OPGmRNA的表达,差异有显著性(P<0.05)。3.2补骨脂素对大鼠成骨细胞分泌的RANKL mRNA表达的影响培养72小时后,空白对照组的RANKL mRNA /GAPDH mRNA的相对表达量为1.0203;阳性对照组雌二醇组的RANKL mRNA /GAPDH mRNA的相对表达量为0.8907,与空白对照组比较抑制了RANKLmRNA的表达,差异有显著性(P<0.05);补骨脂素组的RANKLmRNA /GAPDHmRNA的相对表达量为0.8374,与空白对照组比较抑制了RANKLmRNA的表达,差异有显著性(P<0.05),与雌激素组比较无明显差别。各组OPG/RANKL比值的比较结果是:补骨脂素组与空白对照组比较OPG/RANKL值增加,差别有统计学意义(P<0.05)。4补骨脂素对大鼠成骨细胞分泌骨形态发生蛋白(BMP-2)的影响通过免疫组化方法,在倒置相差显微镜下观察,每个低倍镜下(200×)数阳性细胞数,补骨脂素组平均每个低倍镜视野下细胞阳性率为86.67%,雌激素组为93.33%,空白对照组为33.33%,经统计学分析,补骨脂素组与空白对照组比较,BMP-2的表达明显升高,与雌激素组比较无明显差别。结论1异欧前胡素作用48小时后,浓度为1×10-9 mol/L~1×10-8 mol/L有促增殖作用(P<0.05);72h时,浓度为1×10-8 mol/L~1×10-7 mol/L有促增殖作用(P<0.05)。2异欧前胡素作用72小时,浓度1×10-6mol/L~1×10-5mol/L的异欧前胡素能够提高碱性磷酸酶的活性,促进成骨细胞的分化。3补骨脂素能够上调OPGmRNA的表达,下调RANKLmRNA的表达并使得OPG/RANKL比值增大,这说明香豆素类化合物有可能是通过促进骨形成抑制骨吸收达到防治骨质疏松目的。4补骨脂素与空白对照组比较能促进成骨细胞分泌骨形态发生蛋白,有统计学意义。说明补骨脂素有可能是通过促进细胞分泌骨形态发生蛋白来发挥其促进骨形成作用。5从分子生物学角度为香豆素类化合物防治骨质疏松提供一个理论支持。更多还原

【Abstract】 Objective: With the arrival of the aging society, more and more attention is drawn to the aging problems. Osteoporosis was an important disease affecting human health,which is related to aging. Till now about 2 billion people are infected with Osteoporosis in the world, and its incidence rate is high. A primary analysis of the main statistics of 2000 census, in China about 8800 million people(about occupide 6.6% of population) suffering from Osteoporosis,especially thoses people whose age is in 60~69.it is estimated that the medical care expenses on the treatment of Osteoporosis is about 50 billion rmb at least every year. Osteoporosis, along with it’s searious complication and fractures, is severly threatening the wrinkly’s health. So it is urgent affairs to find out effective drugs and methods to prevent and treat osteoporosis. Bone absorptioninhibitor drugs and bone formationacceleration drugs are two kinds of anti-osteoporosis drugs. Hormone Replacement Therapy is more commonly used , which could decrease bone mineral loss in Post-menopause Women and improve the density of bone, but promote endometrial hyperplasia, liver damage, venous embolization,vessels diseases and breast cancer.Phytoestrogen (PE) is a group of natural non steroid compound which displays estrogen like activity because of their structural similarity to human estrogens and exhibits high affinity binding to estrogen receptor beta. PE could improvement perimenopause syndrome,decrease the blood lipid level, prevent osteoprosis and urinary incontinence. Substitute for estrogen in preventing osteoprosis is a hopeful application field for PE. Traditional Chinese drugs Psoralea fruit can improve Yangqi of spleen and kidney. Modern pharmacological research shows the water soluble extractive of Psoralea has the effect on the bone turnover.The acetone-extract from Psoralea fruits can increase bone strength,improve the phosphate in blood. The model of rats osteoprosis were orally injected into the stomach by psoralea compound preparation, decrease alkaline phosphatase(ALP) leavel to normal, decrese Ca/cr and HOP/cr leavels in urine, inhibit absorbtion of bone, the rate of bone formation is eventually greater than bone absorption. Psoralen was one of active component in psoralea. Psoralen has the effects on stimulating the proliferation and maturation of cultured osteoblasts in vitro and promoting typeⅠcollagen (COLI)synthesis. The intracellular signal transduction mechanism of the psoralen to promote the growth of the osteoblasts is not clear. Based on the previous research, we explore the molecular mechanisms biology for psoralen preventing osteoprosis. In order to provide theoretical basis for clinical preventing osteoprosis. Methods:1 Cell culture: Improved tissue block culture, to demesh and cultivate osteoblast from cranial bone of newly born SD rats.By ading different concentrations of isoimperatorin (1×10-5 mol/L ~1×10-9 mol/L ) to the nutrient medium of osteoblast it can be observed the alteration of the shape and quantity of cells using inverted phase contrast microscope.2 Cell proliferation: It can be inspected that the effect of isoimperatorin on the proliferation of osteoblast in 24 h ,48 h and 72 h by MTT methods with the contrast of the group of dealing with estrogen and control group.3 Cell differentiation: It can be detected that the change of the activity of ALP in osteoblast of the group of dealing with isoimperatorin in24h,48h,72h byρ-nitrophenyl phosphate(рNPP) disodium matrix dynamics methods with the contrast of the group of dealing with estrogen.4 Cell factors: It can be detected that the expression of Osteoprotegerin (OPG) mRNA, and receptor activator of nuclear factor–κB(RANKL)mRNA of the group of dealing with psoralen in 72 h via RT-PCR with the contrast of the group of dealing with estrogen and control group.5 Cell protein: It can be detected that the expression of Bone morphogenetic protein (BMP-2) of the group of dealing with psoralen in 72h via immunohistochemistry with the contrast of the group of dealing with estrogen and control group. Results:1 The effect of estrogen,as positive control ,on the proliferation and differentiation of the osteoblast1.1 Cell morphology observation of estrogen group,as positive controlThe osteoblast cells which is inoculated is globular at first and then floats in culture fluid . Subsequently it is adherent,it will expand in 24 h.The shape of expanded cell is irregular with a appearance of triangle. After 48 h~96 h,osteoblast shows on long fusiform and trabs shape and mixes together and the boundary is vague between cells. Osteoblast shows slabstone shape after 120 h. If you keep on culturing cell, cells will form cell nodule and opaque mineral nodus with accumulating of collagen and calcium salts. When you culture the 20th generation, the volume of the cell is larger than normal, periplast is more subtile than normal,the speed of cleavage is slower and other phenomena which are signs of aging. Osteoblasts are more intensive in estrogen than the blank group. Also its intercellular space is smaller than those of the blank.1.2 The effect of estrogen,as positive control on the proliferation of osteoblastAfter being cultivated 24 h ,contrasted with control group, the concentrations of 1×10-5mol/L, 1×10-7 mol/L, 1×10-8 mol/L can promote the proliferation of osteoblast (P<0.05); After being cultivated 48 h, the concentrations of 1×10-6mol/L,1×10-8 mol/L,1×10-9mol/L can urge the proliferation of osteoblast (P<0.05); After being cultivated 72 h, the concentratons of 1×10-5 mol/L~1×10-7 mol/L estrogen can promote the proliferation of osteoblast (P<0.05) .1.3 The effect of estrogen on the ALP of osteoblastAfter being cultivated 48 h, contrasted with control group, the concentratons of 1×10-5 mol/L~1×10-6 mol/L can promote the differentiation of osteoblast (P<0.05); after being cultivated 72 h, 1×10-5mol/L~1×10-6mol/L concentrations can urge the differentiation of osteobalst(P<0.05)2 The effect of isoimperatorin ,as one of the phytoestrogen ,on the proliferation and differentiation of the osteoblast2.1 Cell morphology observation of the isopsoralen group The phenomenon is similar to estrogen group.2.2 The effect of isoimperatorin on the proliferation of osteoblast After cultivated 24 h ,contrasted with control group, none of the concentration can promote the proliferation of osteoblast (P>0.05);After cultivated 48h,1×10-9mol/L~1×10-8mol/L concentrations can urge the proliferation of osteoblast(P<0.05);after cultivated 72h, 1×10-8mol/L~1×10-7mol/L concentration can urge the proliferation of osteoblast(P<0.05).2.3 The effect of isopsoralen on the ALP of osteoblast Contrasted with control group, after cultivated 72h 1×10-6 mol/L~1×10-5mol/L can urge the differentiation of osteobalst(P<0.05).3 The effect of psoralen on the expression of OPG mRNA in osteobalst After cultivated 72 h ,the relative quantity of OPG mRNA/GAPDH mRNA expressions in the blank group, in the psoralen group and in the estrogen group are 0.6048, 1.0794 and 1.3395. There is significant difference between psoralen group and the blank group(P<0.05). The expression of OPG mRNA in estrogen group is higher than that in black group(P<0.05). There is no significant difference between estrogen group and psoralen group.4 The effect of paoralen on the expression of RANKL mRNA in osteobalstAfter cultivated 72 h ,the relative quantity of RANKLmRNA/GAPDHmRNA expressions in the blank group ,in the psoralen group and in the estrogen group are 1.0203, 0.8374 and 0.8907. There is significant difference between psoralen group and the blank group(P<0.05). The expression of RANKLmRNA in estrogen group is higher than that in black group(P<0.05). There is no significant difference between estrogen group and psoralen group.5 The effect of paoralen on the relative quantity of OPG/RANKL in osteobalstAfter cultivated 72 h, the relative quantity of VOPG / VRANKL in the blank group ,in the psoralen group and in the estrogen group are 0.599, 1.282 and 1.513. There is significant difference between psoralen group and the blank group(P<0.05). psoralen increased VOPG / VRANKL.6 Effects of psoralen on the expression of bone morphogenetic protein 2 of osteoblastthe expression of bone morphogenetic protein 2 was tested by immunohistochemistry method.The posotive rates of osteoblast cells in psoralen group is 86.67%, 93.33% in E2 and 33.33% in black group. to compare the black group , psoralen can significantly enhance the expression of BMP-2.Conclusion:1 Certain dose of isopsoralen as phytoestrogen, can promote the proliferation of osteoblast.2 Certain dose of isopsoralen urge the differentiation of osteoblast.3 The influence of psoralen on bone may be achieved through the pathway of promoting bone formation and inhibiting bone absorption.4 The influence of psoralen on bone maybe achieved through increasing the expression of OPG mRNA , decreasing the expression of RANKL mRNA,and increasing the rate of VOPG / VRANKL.5 BMP may be the critical protein in the effection of Phytoestrogen on preventing osteoprosis. The influence of psoralen on bone maybe achieved through increasing the expression of BMP-2更多还原