【作者】 程龙；

【导师】 何巍；

【作者基本信息】 郑州大学 ， 口腔医学（专业学位）， 2022， 硕士

【摘要】 背景及目的骨质疏松（Osteoprosis,OP）是临床常见的全身骨系统疾病,其特点是持续性的骨量减少,骨组织内部广泛微结构破坏,对全身骨骼影响巨大。OP的发生与发展十分复杂,涉及到遗传,环境,营养,内分泌等多个方面。颌面部骨作为全身骨系统的一部分,其健康也深受OP的影响。临床上常观察到OP患者的牙槽骨高度或有降低,骨质显著下降,甚至直接影响患者口内余留牙的数量及种植体骨结合的质量。此外牙周组织及颞下颌关节的健康也与全身OP有着密切联系。在我国“健康中国2030”规划纲要中明确提出开展“健康口腔,健康骨骼”专项行动,也倡导我们将口腔健康与全身骨骼健康更加紧密的联系起来,为健康中国贡献力量。植物激素在我国传统医药体系中占据重要地位,近几年,对于植物激素的研究主要集中在抗氧化,抗癌、抗炎、治疗糖尿病等方向,例如通过抑制COX-2相关酶来预防和治疗新冠病毒引起的肾损伤,通过肝细胞GPER1受体抑制肝癌的发生与发展。芒柄花黄素（Formononetin,FMN）作为植物雌激素的一种,最初来源于红车轴草,常富集于黄芪,甘草等植物的根茎组织内部,是我国传统医药体系的常用药物。作为天然来源的异黄酮类物质,其分子结构上有一个效能官能团:甲氧基。相较于其他双甲氧基甚至三甲氧基类的植物雌激素,芒柄花黄素的类雌激素效果相对较弱,但其毒副作用更小,药效温和持久,是一种非常稳定的植物雌激素。此外,芒柄花黄素的结构更加趋向于二维,比其他三维结构的植物雌激素更易吸收利用。加上其生产与保存容易,本研究选择芒柄花黄素作为实验药物。目前,对OP的预防和治疗主要通过抑制破骨细胞的增殖与分化以及补充钙离子,减缓钙质流失两种途径。直接作用于成骨细胞的治疗方式或药物不多。临床上寻找一种可以增加成骨细胞活性并促进其分化成骨的药物,成为当下的热点方向。芒柄花黄素作为植物激素,其对破骨细胞的抑制效应先前已有充分研究。但是对成骨细胞的影响有待深入论证。本实验用芒柄花黄素对前成骨细胞进行干预,探讨其对前成骨细胞增殖及分化的影响。并初步探究其作用机制和相关通路,希望为缓解或治疗骨质疏松症提供一个新思路。方法1)芒柄花黄素（FMN）干预小鼠成骨前体细胞（MC3T3-E1细胞系）体外培养,观察其增殖情况。2)CCK-8法检测芒柄花黄素对MC3T3-E1细胞系增殖活动的影响。3)碱性磷酸酶（ALP）染色法检测MC3T3-E1细胞系的成骨分化活动。4)茜素红S染色法检测MC3T3-E1细胞系的钙化情况。5)芒柄花黄素干预下,MC3T3-E1细胞系碱性磷酸酶（ALP）表达量的测定。6)MC3T3-E1细胞系总RNA的提取,Real-time PCR检测Wnt/β-catenin通路及成骨分化相关基因的m RNA表达。7)MC3T3-E1细胞系总蛋白的提取,Western-Blot法检测Wnt/β-catenin通路与成骨分化相关蛋白的表达。结果1)芒柄花黄素（FMN）干预下,小鼠成骨前体细胞（MC3T3-E1细胞系）生长情况良好,形态稳定,细胞正常。2)CCK-8法检测结果表明,培养第五天时,和对照组相比,当FMN终浓度在1×10^-6 mol/L时,小鼠MC3T3-E1细胞的增殖活力最强。3)茜素红-S染色结果表明,与对照组相比,1×10^-6 mol/L浓度FMN干预的MC3T3-E1细胞钙化情况较好,矿化结节形成明显。4)碱性磷酸酶（ALP）染色及表达量测定结果表明,与对照组相比,FMN浓度在1×10^-6 mol/L时,MC3T3-E1细胞的ALP表达活跃。第七天ALP的表达量提高（P<0.05）,第十四天ALP表达量明显提高（P<0.01）。5)Real-time PCR及Western-Blot结果显示,FMN浓度在1×10^-6 mol/L时,MC3T3-E1细胞Wnt/β-catenin通路及成骨分化相关m RNA表达量明显升高（P<0.05）,相关蛋白表达量明显升高（P<0.05）。结论体外环境培养下,10^-6mol/L的芒柄花黄素（FMN）可以促进小鼠MC3T3-E1细胞的增殖,并通过上调Wnt/β-catenin通路,促进MC3T3-E1细胞成骨分化。更多还原

【Abstract】 Background and objective Osteoporosis(OP)is a common clinical disease of the systemic skeletal system.It is characterized by persistent osteopenia and extensive microstructural damage within the bone tissue,which has a considerable impact on the skeleton of the whole body.The occurrence and development of OP are very complex,involving genetics,environment,nutrition,endocrine and other aspects.The health of the maxillofacial bones,as part of the skeletal system of the whole body,is also greatly affected by OP.In clinical practice,the height of the alveolar bone in OP patients is significantly reduced,and the bone quality is significantly reduced,which even directly affects the number of remaining teeth in the patient’s mouth and the quality of the osseointegration of the implant.In addition,the health of periodontal tissue and the temporomandibular joint is also closely related to the systemic OP.In the outline of our country’s "Healthy China 2030" plan,it is proposed to carry out the particular action of "healthy mouth,healthy bones",and it is also advocated that we will more closely link oral health and whole-body bone health,and contribute to a healthy China.Phytohormones occupy an important position in our country’s traditional medicine system and have been widely used for medicinal value since ancient times.In modern medicine,phytohormones still maintain a very high use value and research enthusiasm.In recent years,research on phytohormones has mainly focused on anti-oxidation,anti-cancer,anti-inflammatory,treatment of diabetes.For example,by inhibiting COX-2-related enzymes from preventing and treating kidney damage caused by the new coronavirus,GPER1 in liver cells is affected by GPER1.Inhibit the occurrence and development of liver cancer.Formononetin(FMN),a type of phytoestrogen derived initially from red clover,is often enriched in the rhizomes of astragalus,liquorice and other plants and is a commonly used drug in my country’s traditional medicine system.As a natural source of isoflavones,its molecular structure has a functional group: methoxy.Compared with other dimethoxy or even trimethoxy phytoestrogens,the estrogen-like effect of formononetin is relatively weak,but has less toxic side effects,mild and long-lasting efficacy,and is a very stable plant.In addition,the structure of formononetin tends to be two-dimensional,which is easier to absorb and utilize than other three-dimensional phytoestrogens.In addition to its easy production and preservation,formononetin was selected as the experimental drug in this study.At present,the prevention and treatment of OP mainly slow down the loss of calcium by inhibiting the proliferation and differentiation of osteoclasts and supplementing calcium ions.There are not many therapeutic modalities or drugs that act directly on osteoblasts.The clinical search for a drug that can increase the activity of osteoblasts and promote their differentiation into bone has become a hot topic at present.Formononetin,a phytohormone,has previously been well studied for its inhibitory effect on osteoclasts.However,the effect on osteoblasts remains to be further demonstrated.In this experiment,formononetin was used to intervene in osteoblasts to explore its effect on osteoblasts..Methods 1)Formononetin intervened in the culture of mouse osteogenic precursor cells(MC3T3-E1 cell)to observe their proliferation.2)The effect of formononetin on the proliferation activity of the MC3T3-E1 cell was detected by the CCK-8 method.3)Alkaline phosphatase(ALP)staining was used to detect the osteogenic differentiation activity of the MC3T3-E1 cell.4)Alizarin red S staining was used to detect the calcification of the MC3T3-E1 cell.5)Determination of alkaline phosphatase(ALP)expression in MC3T3-E1 cell under the intervention of formononetin 6)Total RNA was extracted from the MC3T3-E1 cell line,and Real-time PCR was used to detect the m RNA expression of the Wnt/β-catenin pathway and osteogenic differentiation-related genes.7)Extraction of total protein from MC3T3-E1 cell and Western-Blot method to detect the expression of Wnt/β-catenin pathway and osteogenic differentiation related proteins.Result 1)Formononetin intervened in the culture of mouse osteogenic precursor cells(MC3T3-E1 cell);the cells grew well,the shape was stable,and the cells were normal.2)The results of the CCK-8 assay showed that compared with the control group when the final concentration of formononetin(FMN)was 1×10-6 mol/L,the proliferation activity of mouse MC3T3-E1 cells was the strongest.3)Alizarin red-S staining results showed that the MC3T3-E1 cells treated with FMN had better calcification compared with the control group,and the formation of mineralized nodules was prominent.4)The alkaline phosphatase(ALP)staining and expression assay showed that compared with the control group when the concentration of FMN was 1×10-6 mol/L,the expression of ALP in MC3T3-E1 cells was active.The expression of ALP was increased on the seventh day(P<0.05),and the expression of ALP was significantly increased on the fourteenth day(P<0.01).5)Western-Blot and Real-time PCR results showed that when the concentration of FMN was 1×10-6 mol/L,the expression of Wnt/β-catenin pathway and osteogenic differentiation-related proteins in MC3T3-E1 cells increased significantly high(P<0.05),the m RNA expression of related genes was significantly increased(P<0.05).Conclusion In vitro culture,10-6mol/L formononetin can promote the proliferation of mouse MC3T3-E1 cells and promote the osteogenic differentiation of MC3T3-E1 cells by up-regulating the Wnt/β-catenin pathway.更多还原