【作者】 孙楠；

【导师】 蔡德鸿； 李青南；

【作者基本信息】 南方医科大学 ， 临床医学（专业学位）， 2013， 博士

【摘要】 研究背景骨质疏松是一种以骨量减少,骨组织微结构破坏,导致骨强度损害,骨脆性增加,易发生骨折为特征的系统性骨骼疾病,随着年龄增长发病率逐渐升高。目前,中国正迅速进入老龄化社会,骨质疏松带来的严重并发症如骨折等将严重影响老年人的生活质量,并大大加重了社会负担。骨质疏松主要病理过程为破骨细胞(osteoclasts, OC)骨吸收和成骨细胞(osteoblasts,OB)骨形成平衡失调,但其具体机制十分复杂。近年来,多项临床研究发现骨质疏松患者血清过氧化物水平升高而抗氧化物水平降低,从而引起人们对氧化应激在骨质疏松病程中所起作用的关注。既往研究发现,在骨质疏松病程中,氧化应激主要通过耗竭抗氧化物质、激活破骨细胞、抑制成骨细胞及骨髓间充质干细胞(mesenchymal stem cells,MSCs)的增殖和分化成熟,抑制骨形成来影响骨代谢。氧化应激是指机体或细胞内氧自由基的消长失衡,引起活性氧(reactive oxygen species, ROS)在体内蓄积的一种状态。目前,常见的氧化应激标志物有蛋白羰基,脂质过氧化物和晚期糖基化终产物(advanced glycation end products, AGEs)等。由于蛋白质的氧化先于脂质过氧化,且羟自由基与蛋白质直接相互作用的可能性要比与DNA作用大20倍,因此目前普遍认为,蛋白质是体内ROS损伤的主要原初靶。所以,阐明蛋白质氧化修饰及其产物在人类疾病中的作用,将为研究氧化应激相关性疾病的发病学提供新的理论,并为其干预提供新的途径或靶标。在许多人类重要慢性疾病,如糖尿病、代谢综合征、慢性肾脏病、动脉粥样硬化等都存在蛋白质氧化修饰产物潴留,其中某些蛋白质修饰产物,如AGEs已被证实参与了许多慢性疾病的发病学如透析相关性淀粉样变、老年性痴呆、糖尿病微血管病变和糖尿病性骨质疏松等。这些证据表明,蛋白质氧化修饰产物蓄积不仅是氧化应激的后果(或标志物),其本身可能作为致病介质参与人类多种重要疾病的发生或发展。晚期蛋白氧化产物(advanced oxidation protein products,AOPPs)是体内血清白蛋白发生氯化氧化反应后生成的含双酪氨酸的蛋白交连物,由Witko等首次在尿毒症患者血浆中发现,是一种新型的氧化应激标志物。最近的研究认为AOPPs可能是一种促炎症活性的毒素,既是氧化应激的产物,又可正反馈诱导或加重氧化应激反应,呈现持续氧化应激状态。目前认为,AOPPs参与了肥胖、糖尿病、冠心病和肾脏纤维化等疾病病程,在衰老及老年相关性疾病如老年性白内障中也起到重要作用。骨质疏松是老年相关性疾病,且研究发现骨质疏松大鼠血浆AOPPs水平明显升高,氧化应激也能使小鼠成骨系细胞MC3T3-E1中AOPPs含量增加。这些提示我们AOPPs在骨质疏松的发病中可能起着促进作用,但其具体作用机制仍不清楚。AOPPs与AGEs一样能够和晚期糖基化终产物受体(receptor for advanced glycation end products,RAGE)结合,RAGE属于免疫球蛋白超家族多配体受体,在成熟的动物及人体中呈低水平表达。当机体处于应激状态如糖尿病、炎症时,受损细胞中RAGE表达急剧增加。RAGE基因敲除小鼠的骨量及骨生物力学强度明显增加,破骨细胞生成减少；慢病毒转染使成骨细胞内RAGE表达增加后能通过抑制Wnt, PI3K and ERK通路从而抑制成骨细胞增殖；RAGE与AGEs结合后能通过NADPH酶促使ROS生成增加,从而诱导OB/MSCs凋亡,并抑制OB/MSCs增殖和分化,以上均说明RAGE信号通路参与骨质疏松的发病。AOPPs与AGEs生物学结构及活性相似,结合AGEs的相关报道,我们猜想AOPPs能够抑制MSCs/OB增殖和成骨方向分化成熟,其相关机制可能为AOPPs与RAGE结合后,刺激细胞内ROS水平升高,同时增加RAGE表达,形成恶性循环,从而抑制MSCs/OB增殖和成骨方向分化成熟。由于骨形成主要受OB调节,而OB由MSCs分化而来,MSCs和OB的数量及功能直接影响了骨质疏松的发生发展。因此,本实验用不同浓度AOPPs干预大鼠骨髓间充质干细胞及乳鼠颅骨成骨细胞,研究AOPPs对其增殖、分化、氧化应激指标及RAGE表达的影响,旨在进一步明确AOPPs在骨质疏松病程中的作用。目的本课题拟建立AOPPs对大鼠MSCs及OB的损伤模型,通过观测AOPPs对大鼠MSCs及OB增殖及成骨分化相关指标,并检测AOPPs干预后细胞内ROS水平及相关受体RAGE的表达,初步探讨AOPPs对大鼠MSCs及OB的作用及分子机制。内容课题分以下两大部分：第一章晚期蛋白氧化产物对大鼠骨髓间充质干细胞增殖、分化的影响及其相关机制第一节晚期蛋白氧化产物对大鼠骨髓间充质干细胞增殖、分化的影响目的建立AOPPs对大鼠MSCs的损伤模型,观测AOPPs对大鼠MSCs增殖及成骨分化能力的影响。方法1.分离、培养、鉴定大鼠MSCs采用全骨髓培养法分离大鼠MSCs,并通过传代进行纯化,用高糖DMEM基础培养基,置于37℃、5%CO2饱和湿度孵箱中培养。运用流式细胞仪检测细胞表面标志物CD29和CD34,并检测细胞向脂肪细胞和成骨细胞分化的能力,对MSCs进行鉴定。2.体外制备、鉴定晚期蛋白氧化产物将牛血清白蛋白(BSA)和次氯酸溶于PBS,室温避光孵育30min制备AOPPs。 AOPPs的含量以氯胺T为标准曲线,通过测定酸性条件下340nm的吸光度值确定。3.四唑盐(MTT)比色法测定细胞增殖能力不同浓度的AOPPs(50、100、200、400μg/ml)作用大鼠MSCs72h(本论文以浓度或时间代表不同分组,下同)；200μg/ml AOPPs分别作用大鼠MSCs0.24、48、72h。将细胞接种于96孔板中,随机进行实验分组处理后,每孔加MTT溶液20μL,4h后吸除培养基,加入二甲亚枫(DMSO),选择570nm波长测吸光度以判断细胞活力。MTT比色法所测的吸光度反映细胞的损伤情况。4.对硝基苯磷酸盐法(PNPP)检测碱性磷酸酶活性(ALP)不同浓度的AOPPs(50.100.200.400μg/ml)作用大鼠MSCs72h;200μg/ml AOPPs干预MSCs0、24、48、72h。用0.1%TritonX-100溶解液破碎制成细胞悬液,PNPP法检测ALP量,并用BCA法检测样本蛋白浓度,根据公式计算ALP浓度。5.茜素红染色法检测钙化结节用成骨分化培养基诱导MSCs两周后,分别用DMEM、200μg/ml BSA和200μg/ml AOPPs干预细胞,后用茜素红染色法进行染色,检测钙化结节形成情况。6.实时荧光定量PCR (RT-PCR)检测MSCs中ALP. Collagen I mRNA的表达用成骨分化培养基诱导MSCs1周后,用不同浓度AOPPs(50、100、200、400μg/ml)干预MSCs72h并检测ALP、Collagen I mRNA的表达。Trizol法提取总RNA,分光光度计测mRNA浓度后进行RT-PCR反应,采用Delta-delta Ct法计算目的基因表达变化。统计学处理实验数据以x±s表示,采用SPSS18.0统计软件进行统计学分析,多样本比较用单因素方差分析,组间多重比较采用LSD检验。P<0.05为有统计学意义。结果1. MSCs的鉴定细胞呈三角形或长梭形,可成螺旋形集落样生长。流式细胞术检测细胞表面标记物,其中CD34表达阴性,CD29表达阳性。用成脂分化培养基和成骨分化培养基诱导MSCs,分别可见细胞内有脂滴及钙化结节形成。2.不同浓度AOPPs对MSCs增殖的影响50,100.200、400μg/ml AOPPs作用MSCs72h后,OD值分别为0.4722±0.0050,0.4563±0.0170,0.4170±0.0147,0.3770±0.0266,均显著低于正常对照组(0.5397±0.0080)和BSA对照组(0.5273±0.0297)(P=0.000)。3. AOPPs不同作用时间对MSCs增殖的影响200μg/ml AOPPs作用时间(24、48、72h)后的细胞OD值分别为0.5458±0.0193,0.4768±0.0261,0.4170±0.0147,均显著低于0h组的OD值(0.5955±0.0283)(P<0.01)。4.不同浓度AOPPs对MSCs ALP活性的影响50、100、200、400μg/ml AOPPs作用MSCs72h后,ALP活性(单位：U/g-protein)分别为92.9291±4.2248,87.4805±3.8420,85.2812±1.5028,81.6935±2.0550,均显著低于正常对照组(98.1610±2.0889)和BSA对照组(97.4307±3.5394)(P<0.05)。5.比较AOPPs不同作用时间对MSCs ALP活性的影响200μg/ml AOPPs作用时间(24、48、72h)后的细胞ALP活性(单位：U/g-protein)分别为93.2511±2.0402,90.2572±1.8179,85.2812±1.5028,均显著低于0h组值(98.3545±1.6831)(P=0.000)。6.AOPPs对MSCs钙化结节形成的影响经200μg/ml AOPPs干预后,正常对照组及BSA组内可见红色钙化结节形成,但AOPPs组未见明显钙化结节7.不同浓度AOPPs对MSCs中ALP、Collagen I mRNA表达的影响50、100、200、400μg/ml AOPPs作用MSCs72h后,RT-PCR检测细胞ALP、 Collagen I mRNA水平。各浓度AOPPs组ALP mRNA相对表达量分别为0.4557±0.0661,0.2945±0.0464,0.2182±0.0311,0.1632±0.0379,均显著低于BSA对照组(0.9058±0.0876)(P=0.000), Collagen I mRNA相对表达量分别为0.5575±0.0276,0.4755±0.0457,0.3827±0.0507,0.1893±0.0219,均显著低于BSA对照组(0.9123±0.0108)(P=0.000)。结论本实验所提取细胞鉴定为大鼠MSCs. AOPPs呈剂量、时间依赖性抑制MSCs增殖。AOPPs呈剂量、时间依赖性抑制MSCs ALP活性,并能剂量依赖性抑制MSCs ALP和Collagen I mRNA表达,且能够抑制钙化结节形成,提示AOPPs能够抑制MSCs成骨分化能力。第二节晚期蛋白氧化产物对大鼠骨髓间充质干细胞氧化应激及RAGE表达的影响目的观测AOPPs干预后细胞内ROS水平及相关受体RAGE的表达,初步探讨AOPPs对大鼠MSCs的作用机制。方法1.荧光显微镜及流式细胞术检测细胞活性氧(ROS)不同浓度的AOPPs (50、100、200、400μg/ml)干预大鼠MSCs2h。干预后加入终浓度20gM的DCFH-DA,孵育30min,荧光倒置显微镜下观察拍照,消化后用流式细胞仪检测细胞中DCF的平均荧光强度。2.实时荧光定量PCR(RT-PCR)检测MSCs中RAGE mRNA的表达直接用不同浓度AOPPs (50、100、200、400μg/ml)干预MSCs72h后检测RAGE mRNA表达。余同第一章第一节。3.Western Blot检测细胞RAGE蛋白表达水平收集各组细胞,分别提取各组细胞总蛋白,采用BCA法测定蛋白浓度。每组蛋白质样本25μg,以SDS-PAGE进行电泳,转膜后封闭液封闭4h,洗膜后加入抗RAGE单克隆抗体(1：500)、抗GAPDH单克隆抗体(1：500),室温孵育4h,洗膜后加入辣根过氧化物酶标记的二抗(1：5000、1：4000)杂交50min,洗膜后ECL超敏发光液进行显色,用BIO-RAD软件进行光密度积分值分析。统计学处理实验数据以X±s表示,采用SPSS18.0统计软件进行统计学分析,多样本比较用单因素方差分析,组间多重比较采用LSD检验。P<0.05为有统计学意义。结果1.不同浓度AOPPs对MSCs ROS水平的影响50、100、200、400μg/ml AOPPs作用MSCs2h后,以DCFH-DA为荧光探针检测细胞内ROS水平,荧光显微镜下观察各组细胞,可见AOPPs组荧光强度明显大于正常对照组及BSA组。将各组平均荧光强度与正常对照组相比得比值,分别为111.4910±4.3167,175.7478±18.0156,328.8604±15.8096,441.6609±17.5993,579.6729±26.2623,AOPPs组ROS水平明显高于正常对照组(P=0.000)。2.不同浓度AOPPs对MSCs中RAGE mRNA表达的影响各浓度AOPPs组RAGE mRNA相对表达量分别为1.6144±0.2747,3.0609μ0.4656,5.2401±0.3257,5.6851μ1.0159,除50μg/ml AOPPs组外,余均显著高于BSA对照组(1.2209±0.1104)(P=0.000)。3.不同浓度AOPPs对MSCs RAGE蛋白表达的影响50、100、200、400μg/ml AOPPs作用MSCs72h后,RAGE蛋白相对表达量分别为0.3980±0.0145,0.4566±0.0122,0.5398±0.0208,0.6249±0.0246,除50μg/mlAOPPs组外,余均显著高于正常对照组(0.3799±0.0249)及BSA对照组(0.3898±0.0209)(P<0.01)。结论AOPPs能够剂量依赖性促进MSCs内ROS水平升高,并上调细胞RAGE mRNA和蛋白水平。说明AOPPs可能与受体RAGE结合后,促进RAGE表达,刺激细胞ROS水平升高,从而抑制MSCs增殖及向成骨分化的能力。第二章晚期蛋白氧化产物对大鼠成骨细胞增殖、分化的影响及其相关机制第一节晚期蛋白氧化产物对大鼠成骨细胞增殖、分化的影响目的建立AOPPs对大鼠OB的损伤模型,观测AOPPs对大鼠OB增殖及分化成熟的影响。方法1.分离、培养、鉴定大鼠OB新生大鼠颅骨酶解法培养OB,用成骨分化培养基,置于37℃、5%CO2饱和湿度孵箱中培养。碱性磷酸酶染色法鉴定OB。2.MTT法测定细胞增殖能力不同浓度的AOPPs(50、100、200、400μg/ml)作用大鼠OB72h；200μg/ml AOPPs分别作用大鼠OB0、24、48、72h。将细胞接种于96孔板中,随机进行实验分组处理后,每孔加MTT溶液20gL,4h后吸除培养基,加入二甲亚枫(DMSO),选择570nm波长测吸光度以判断细胞活力。MTT比色法所测的吸光度反映细胞的损伤情况。3.对硝基苯磷酸盐法(PNPP)检测碱性磷酸酶活性(ALP)不同浓度AOPPs(50、100、200、400μ/ml)干预OB72h;200μg/ml AOPPs分别作用大鼠OB0、24、48、72h后用PNPP法检测细胞分泌的ALP活性。4.实时荧光定量PCR(RT-PCR)检测OB中ALP、Collagen I mRNA的表达不同浓度AOPPs(50、100、200、400μg/ml)干预OB72h后检测细胞ALP. Collagen I mRNA的表达,余同第一章第一节。统计学处理实验数据以x±s表示,采用SPSS18.0统计软件进行统计学分析,多样本比较用单因素方差分析,组间多重比较采用LSD检验。P<0.05为有统计学意义。结果1.OB的鉴定细胞为单核,呈梭形和多角形等多种形态。ALP染色阳性,胞浆中可见红棕色颗粒。2.不同浓度AOPPs对OB增殖的影响50.100、200、400μg/ml AOPPs作用OB72h后,OD值分别为0.5207±0.0431,0.4947±0.0164,0.4087±0.0170,0.2673±0.0031,均显著低于正常对照组(0.6290±0.0092)和BSA对照组(0.6170±0.0168)(P=0.000)。3. AOPPs不同作用时间对OB增殖的影响200μg/ml AOPPs作用时间(24、48、72h)后的细胞OD值分别为0.5480±0.0175,0.4923±0.0091,0.4087±0.0170,均显著低于0h组的OD值(0.6403±0.0075)(P<0.01)。4.不同浓度AOPPs对OB ALP活性的影响50、100、200、400μg/ml AOPPs作用OB72h后,ALP活性(单位：U/L)分别为40.1066±0.7870,37.0065±1.4205,31.2424±0.9305,20.5793±0.7350,均显著低于正常对照组(53.2817±3.2130)和BSA对照组(52.3129±3.7520)(P=0.000)。5.比较AOPPs不同作用时间对OB ALP活性的影响200μg/ml AOPPs作用时间(24、48、72h)后的细胞ALP活性(单位：U/L)分别为48.3454±3.0748,40.5973±3.2361,31.2424±0.9305,均显著低于0h组值(56.6586±4.3970)(P=0.000)。6.不同浓度AOPPs对OB中ALP、Collagen I mRNA表达的影响50.100.200,400μg/ml AOPPs作用OB72h后,RT-PCR检测细胞ALP. Collagen I mRNA水平。各浓度AOPPs组ALP mRNA相对表达量分别为0.8960±0.0187,0.7017±0.0438,0.6044±0.0299,0.4765±0.0323,均显著低于BSA对照组(0.9648±0.0366)(P<0.05),Collagen I mRNA相对表达量分别为0.8811±0.0007,0.8151±0.0075,0.6378±0.0309,0.5730±0.0061,均显著低于BSA对照组(0.9664±0.0113)(P=0.000)。结论本实验所提取细胞鉴定为大鼠OB。AOPPs呈剂量、时间依赖性抑制OB增殖。AOPPs呈剂量、时间依赖性抑制OB ALP活性,并能剂量依赖性抑制OB ALP和Collagen I mRNA表达,提示AOPPs能够抑制OB分化成熟。第二节晚期蛋白氧化产物对大鼠成骨细胞氧化应激及RAGE表达的影响目的观测AOPPs干预后细胞内ROS水平及相关受体RAGE的表达,初步探讨AOPPs对大鼠OB的作用机制。方法1.荧光显微镜及流式细胞术检测细胞活性氧(ROS)同第一章第二节。2.实时荧光定量PCR(RT-PCR)检测OB中RAGE mRNA的表达同第一章第二节。统计学处理实验数据以x±s表示,采用SPSS18.0统计软件进行统计学分析,多样本比较用单因素方差分析,组间多重比较采用LSD检验。P<0.05为有统计学意义。结果1.不同浓度AOPPs对OBROS水平的影响50.100.200.400μg/ml AOPPs作用MSCs2h后,以DCFH-DA为荧光探针检测细胞内ROS水平,荧光显微镜下观察各组细胞,可见AOPPs组荧光强度明显大于正常对照组及BSA组。将各组平均荧光强度与正常对照组相比得比值,分别为112.7752±6.7175,183.6783±14.1578,307.5650±13.8079,366.9337±20.0321,606.2912±46.2415,AOPPs组ROS水平明显高于正常对照组(P<0.01)。2.不同浓度AOPPs对OB中RAGE mRNA表达的影响各浓度AOPPs组RAGE mRNA相对表达量分别为1.3626±0.1090,2.9922±0.3198,5.1792±0.3936,5.8637±0.3561,除50μg/ml AOPPs组外,余均显著高于BSA对照组(1.0703±0.0557)(P=0.000)。结论AOPPs能够剂量依赖性促进OB内ROS水平升高,并上调细胞RAGE mRNA水平。说明AOPPs可能与受体RAGE结合后,促进RAGE表达,刺激细胞ROS水平升高,从而抑制OB增殖及分化成熟。更多还原

【Abstract】 BackgroundOsteoporosis is a skeletal disorder characterized by decreased bone mass and damage bone tissue microstructure, culminating in fragility fractures, pain and disability. As the incidence of osteoporosis has been increasing with age, it has significantly increased the social burden and affected quality of life of elders with aging society.The main pathological process of osteoporosis is an imbalance between bone resorption and bone formation, but mechanisms in detail are still unclear. Resent years there is growing evidence that the level of serum peroxide in patients with osteoporosis increased while antioxidant decreased compared with controls, which gets attention to the role of oxidative stress in pathogenesis of osteoporosis. Previous studies indicate that in the development of osteoporosis oxidative stress may affect bone metabolism through consuming antioxidant, activating osteoclasts and inhibiting osteoblasts and bone formation.Oxidative stress is an imbalance between the free radicals and antioxidant mechanism in biological systems and can make an accumulation of reactive oxygen species (ROS) in the body. At present, the most common markers of oxidative stress are protein carbonyl, lipid peroxide and advanced glycation end products (AGEs). Because protein carbonyl detection occurred earlier than lipid peroxidation and the possibility of direct interaction between hydroxyl radical and protein is20times higher than with DNA, it is generally believed that protein is the main original target of ROS. Therefore, clarifying the role of oxidative modification of protein and its products in human diseases will provide new theories for researches of oxidative stress related diseases, and new ways or targets for intervention.Oxidative protein products retention has involved in many important chronic diseases including diabetes, metabolic syndrome, chronic kidney disease and atherosclerosis. It has been demonstrated that some modificative protein products such as AGEs are related to many chronic diseases containing dialysis-related amyloidosis, Alzheimer disease, and diabetic microangiopathy and diabetic osteoporosis. All these studies have demonstrated that oxidative protein products retention is not only the result/marker of oxidative stress, but also disease-causing agents involved in the development of many important human deseases.Protein quantity in human body is huge, and albumin which is most susceptible to oxidative modification is also the main protein in the cycle. Advanced oxidation protein products are a kind of protein conjugate containing tyrosine by chlorination and oxidation reaction with serum albumin. They are first discovered in the plasma of patients with uremia. Recent studies suggest that AOPPs, a kind of uremic toxin causing inflammatory, may not only be the product of oxidative stress, but also could induce or aggravate oxidative stress. It has been demonstrated that AOPPs involve in obesity, renal fibrosis, aging, age-related cataract, and so on. Osteoporosis is also an age-related disease. It has been indicated that the level of AOPPs in patients with osteoporosis increases compared with control. Oxidative stress can also raise AOPPs in MC3T3-E1cells, but the mechanisms in detail between AOPPs and osteoporosis are still unclear.Receptor for advanced glycation end products (RAGE) is multiple ligand receptor in mmunoglobulin superfamily that can combine with advanced glycation end products (AGEs) and AOPPs. The level of AOPPs is usually low in human, but rises in oxidative stress such as inflammation and diabetes following RAGE expression increased dramatically. Experiments using RAGE knockout mice show an increased bone mass and bone biomechanical strength and a decreased number of osteoclasts in RAGE-/-mice compared to wild-type mice; RAGE overexpression by lenti virus transfection inhibits osteoblast proliferation via suppression of Wnt, PI3K and ERK pathways; AGEs-RAGE interaction can induce the generation of ROS through NADPH oxidase resulting in apoptosis of osteoblasts/MSCs, and inhibition of proliferation and differentiation of osteoblasts/MSCs. The studies above all indicate that RAGE plays a modulatory role in development of osteoporosis. The biological structure and activity of AOPPs are similar as AGEs. Combining previous researches about AGEs, we speculate that AOPPs might inhibit the proliferation and osteogenic differentiation of MSCs/OB by increasing ROS generation following an up-regulating of RAGE expression.As bone formation is regulated by osteoblasts which originate from MSCs, the negative effects on MSCs/OB osteogenic differentiation may aggravate the development of osteoporosis. So in this research we induced rat MSCs and OB with different concentrations of AOPPs, and detected indexes about proliferation, differentiation, ROS and expression of RAGE in MSCs/OB to determine the effect of AOPPs on osteoporosis.ObjectivesIn this project we aimed to establish the injury model by treating rat MSCs and osteoblasts with AOPPs. We detected the indexes about proliferation and osteogenic differentiation of rat MSCs and osteoblasts, and tested the levels of ROS and RAGE expression, to discuss the effects and mechanisms of AOPPs on MSCs and osteoblasts.ContentThe whole project includes two parts: Part I The effects and mechanisms of AOPPs on proliferation and osteogenic differentiation of rat MSCs Section I The effects of AOPPs on proliferation and osteogenic differentiation of rat MSCsObjectivesWe aimed to establish the injury model by treating rat MSCs with AOPPs, and to analyze the effects of AOPPs on proliferation and osteogenic differentiation of MSCs.Methods1. Isolation, culture and identification of cellsMSCs were isolated from SD rat’s bone marrow from femurs and were cultured with FBS-DMEM medium at37℃in a humidified atmosphere of5%CO2. These cells were identified for surface marker by flow cytometry and for the adipogenic and osteogenic differentiation capability by oil red O staining and alizarin red staining.2. Preparation of AOPPs in vitroBriefly, BSA was dissolved in phosphate-buffered saline with HOCl, and incubated for30min in the dark. AOPPs was identified using a spectrophotometer at340nm.3. Assessment of cell proliferation by colorimetric3-(4,5-dimethylthiazol-2-yl)-2,5-dihenyltetrazolium bromide (MTT) assayMSCs were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h (In this manuscript, we grouped according to different concentrations and time); cells were cultrured by200μg/ml AOPPs alone for0,24,48,72h respectively.The cells are planted in96-well plates. After each experimental treatment, add MTT solution to each hole, and add150μl DMSO to each hole. Absorption was measured at570nm with a microplate reader. The absorbance measured by MTT assay reflects cells injury.4. Assessment of ALP activity by P-nitrophenyl Phosphate (PNPP) assayMSCs were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h; cells were cultrured by200μg/ml AOPPs alone for0,24,48,72h respectively. Cells were lysed by0.1%TritonX-100, then detected ALP activity by PNPP assay, and tested protein concentrations by BCA assay.5. Detection of calcified nodules by alizarin red stainingCells were treated by AOPPs at concentrations of50,100,200,400μg/ml for1week after being cultured in osteogenic differentiation medium for2weeks. Then cells were stained using alizarin red to detect calcified nodules formation.6. Detection of ALP and collagen I mRNA levels by RT-PCR After being induced with osteogenic differentiation medium for1week, MSCs were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h. Then total RNA was extracted using Trizol, and was detected by RT-PCR.Statistical AnalysisAll analyses were carried out with SPSS18.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p <0.05.Results1. MSCs were isolated from rat femur bone marrow and formed elongated spindle or polygon. The surface marker CD34was negative while CD29was positive. After induced by adipogenic differentiation and osteogenic differentiation medium, lipid droplets in cells and bone nodules were observed respectively.2. After treatment with50,100,200,400∴g/ml AOPPs for72h, the OD value were0.4722±0.0050,0.4563±0.0170,0.4170±0.0147,0.3770±0.0266, respectively, and significantly lower than control group (0.5397±0.0080) and BSA group (0.5273±0.0297)(P=0.000).3. Comparison OD value of cells after being incubated with AOPPs for different time showed that a significant negative correlation exists between the OD value of MSCs and time (0,24,48,72h)(P=0.000).4. After treatment with50,100,200,400μg/ml AOPPs for72h, the ALP activities (U/g-protein) were92.9291±4.2248,87.4805±3.8420,85.2812±1.5028, 81.6935±2.0550, respectively, and significantly lower than control group (98.1610±2.0889) and BSA group (97.4307±3.5394)(P<0.05).5. Comparison ALP activities of cells after being incubated with AOPPs for different time showed that a significant negative correlation exists between the ALP activities of MSCs and time (0,24,48,72h)(P=0.000)6. After incubated with AOPPs, red calcified nodules were observed in control group and BSA group, but few in AOPPs group.7. After treatment with50,100,200,400μg/ml AOPPs for72h, the ALP mRNA relative levels were0.4557±0.0661,0.2945±0.0464,0.2182±0.0311,0.1632±0.0379, respectively, and significantly lower than BSA group (0.9058±0.0876)(P=0.000). The Collagen I mRNA relative levels were0.5575±0.0276,0.4755±0.0457,0.3827±0.0507,0.1893±0.0219, respectively, and significantly lower than BSA group (0.9123±0.0108)(P=0.000).ConclusionThe cells isolated were identified as MSCs. The inhibition of proliferation and ALP activities were induced by AOPPs in a dose, time-dependent manner. AOPPs could also down-regulate ALP and Collagen I mRNA levels, and inhibit the calcified nodules formation. All the results indicated that AOPPs could inhibit osteogenic differentiation of MSCs.Section Ⅱ The effects of AOPPs on oxidative stress and RAGE expression in MSCsObjectivesWe analyzed the level of oxidative stress by fluorescence microscop, and tested the mean fluorescence intensity (MFI) by flow cytometry. RAGE mRNA and protein levels were detected using RT-PCR and western blot respectively. All were to discuss the mechanism of AOPPs on MSCs.Methods1. Detection of reactive oxygen species (ROS) MSCs were treated by AOPPs at concentrations of50,100,200,400μg/ml for2h. The level of ROS was quantified by2’,7’-dichlorofluorescein diacetate assay using fluorescence microscop and flow cytometry.2. Detection of RAGE mRNA level by RT-PCRMSCs were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h. Then total RNA was extracted using Trizol, and was detected by RT-PCR.3. Detection of RAGE protein level by western blotCells with various treatments were collected and lysed in protein lysis buffer. The supernatant contained the cell extracts and the protein concentration was measured using the BCA protein assay. Equal amounts of protein (25μg) from each sample were separated on SDS-polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in blocking buffer.for4h and incubated with anti-RAGE (1:500) and anti-GAPDH (1:500) antibody for4h. Secondary specific horseradish peroxidase-linked antibodies (1:5000,1:4000) were added for50minh, and immune complexes were detected by ECL chemiluminescence, the band intensity was measured and quantified with BIO-RAD software.Statistical AnalysisAll analyses were carried out with SPSS18.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p <0.05.Results1. Compared with control group, DCF fluorescence in cells exposed to AOPPs was strikingly increased by fluorescence microscop. The ratio of MFI in different concentration groups and the control group were175.7478±18.0156,328.8604±15.8096,441.6609±17.5993,579.6729±26.2623, respectively, and significantly higher than the ratio of BSA and control (111.4910±4.3167)(P=0.000).2. After treatment with50,100,200,400μg/ml AOPPs for72h, the RAGE mRNA relative levels were1.6144±0.2747,3.0609±0.4656,5.2401±0.3257, 5.6851±1.0159, respectively, and significantly higher than BSA group (1.2209±0.1104)(P=0.000) except50μg/ml AOPPs.3. After treatment with50,100,200,400μg/ml AOPPs for72h, the RAGE protein relative levels were0.3980±0.0145,0.4566±0.0122,0.5398±0.0208,0.6249±0.0246, respectively, and significantly higher than control group (0.3799±0.0249) and BSA group (0.3898±0.0209)(P<0.01) except50μg/ml AOPPs.ConclusionExposure to AOPPs for2h caused a significant increase in ROS generation, and AOPPs also increased RAGE expression in both mRNA and protein levels. All these indicate that AOPPs may increase the RAGE expression after combined with RAGE, meanwhile increase the ROS level in MSCs, so that AOPPs could inhibit MSCs proliferation and osteogenic differention.Part Ⅱ The effects and mechanisms of AOPPs on proliferation and osteogenic differentiation of rat OBSection I The effects of AOPPs on proliferation and osteogenic differentiation of rat OBObjectivesWe aimed to establish the injury model by treating rat OB with AOPPs, and to analyze the effects of AOPPs on proliferation and osteogenic differentiation of OB.Methods1. Isolation, culture and identification of cellsOB were isolated from newborn SD rat’s skull and were cultured with osteogenic differentiation medium at37℃in a humidified atmosphere of5%CO2. These cells were identified using alkaline phosphatase assay.2. MTT assayOB were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h; cells were cultrured by200μg/ml AOPPs alone for0,24,48,72h respectively.The cells are planted in96-well plates. After each experimental treatment, add MTT solution to each hole, and add150μl DMSO to each hole. Absorption was measured at570nm with a microplate reader. The absorbance measured by MTT assay reflects cells injury.3. Assessment of ALP activity by P-nitrophenyl Phosphate (PNPP) assayOB were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h; cells were cultrured by200μg/ml AOPPs alone for0,24,48,72h respectively. ALP activity was measured with the instruction of ALP kit.4. Detection of ALP and collagen I mRNA levels by RT-PCR OB were treated by AOPPs at concentrations of50,100,200,400μg/ml for72h. Then total RNA was extracted using Trizol, and was detected by RT-PCR.Statistical AnalysisAll analyses were carried out with SPSS18.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p <0.05.Results1. OB were isolated from newborn SD rat’s skull and formed fusiform or polygon. The ALP staining was positive, and the red brown granules were observed in cytoplasm.2. After treatment with50,100,200,400μg/ml AOPPs for72h, the OD value were0.5207±0.0431,0.4947±0.0164,0.4087±0.0170,0.2673±0.0031, respectively, and significantly lower than control group (0.6290±0.0092) and BSA group (0.6170±0.0168)(P=0.000).3. Comparison OD value of cells after being incubated with AOPPs for different time showed that a significant negative correlation exists between the OD value of OB and time (0,24,48,72h)(P=0.000).4. After treatment with50,100,200,400μg/ml AOPPs for72h, the ALP activities (U/L) were40.1066±0.7870,37.0065±1.4205,31.2424±0.930520.5793±0.7350, respectively, and significantly lower than control group (53.2817±3.2130) and BSA group (52.3129±3.7520)(P=0.000). 5. Comparison ALP activities of cells after being incubated with AOPPs for different time showed that a significant negative correlation exists between the ALP activities of OB and time (0,24,48,72h)(P=0.000)6. After treatment with50,100,200,400μg/ml AOPPs for72h, the ALP mRNA relative levels were0.8960±0.0187,0.7017±0.0438,0.6044±0.0299,0.4765±0.0323, respectively, and significantly lower than BSA group (0.9648±0.0366)(P<0.05). The Collagen I mRNA relative levels were0.8811±0.0007,0.8151±0.0075,0.6378±0.0309,0.5730±0.0061, respectively, and significantly lower than BSA group (0.9664±0.0113)(P=0.000).ConclusionThe cells isolated were identified as OB. The inhibition of proliferation and ALP activities were induced by AOPPs in a dose, time-dependent manner. AOPPs could also down-regulate ALP and Collagen I mRNA levels wich indicated that AOPPs could inhibit osteogenic differentiation of OB.Section II The effects of AOPPs on oxidative stress and RAGE expression in OBObjectivesWe analyzed the level of oxidative stress by fluorescence microscop, and tested the mean fluorescence intensity (MFI) by flow cytometry. RAGE mRNA level was detected using RT-PCR. All were to discuss the mechanism of AOPPs on OB.Methods1. Detection of reactive oxygen species (ROS) Use the same method with that of Part Ⅰ-SectionⅡ.2. Detection of RAGE mRNA level by RT-PCR Use the same method with that of Part Ⅰ-Section Ⅱ.Statistical AnalysisAll analyses were carried out with SPSS18.0software. Data are expressed as mean±standard deviation (SD). Differences between groups were tested by one-way ANOVA followed by a LSD test. Statistical significance was defined as two-sided p <0.05.Results1. Compared with control group, DCF fluorescence in cells exposed to AOPPs was strikingly increased by fluorescence microscop. The ratio of MFI in different concentration groups and the control group were183.6783±14.1578,307.5650±13.8079,366.9337±20.0321,606.2912±46.2415, respectively, and significantly higher than the ratio of BSA and control (112.7752±6.7175)(P<0.01).2. After treatment with50,100,200,400μg/ml AOPPs for72h, the RAGE mRNA relative levels were1.3626±0.1090,2.9922±0.3198,5.1792±0.3936,5.8637±0.3561, respectively, and significantly higher than BSA group (1.0703±0.0557)(P=0.000) except50μg/ml AOPPs.ConclusionExposure to AOPPs for2h caused a significant increase in ROS generation, and AOPPs also increased RAGE expression in mRNA level. All these indicate that AOPPs may increase the RAGE expression after combined with RAGE, meanwhile increase the ROS level in OB, so that AOPPs could inhibit OB proliferation and osteogenic differention.更多还原