【作者】 张晓；

【导师】 徐又佳；

【作者基本信息】 苏州大学 ， 骨外科（专业学位）， 2017， 博士

【摘要】 第一部分丹参对小鼠前成骨细胞的增殖分化的影响及其与MAPK信号通路关系研究目的:研究丹参对于小鼠前成骨细胞(MC3T3-E1)的增殖、分化及矿化的影响,并研究其与细胞外信号调节蛋白激酶(ERK)和p38细胞信号通路的关系。方法:分别用生药浓度为75mg/L、150mg/L、300mg/L的丹参注射液稀释液干预MC3T3-E1小鼠前成骨细胞,并设对照组;用CCK8法检测细胞增殖活性的改变,培养21d后茜素红染色法检测各组钙结节生成情况,实时荧光定量PCR(qPCR)法检测各组骨钙素(BGP)、Runt相关基因2(Runx2)mRNA表达情况。设300mg/L浓度的丹参干预组和对照组,蛋白免疫印迹法检测p-ERK1/2、p-p38蛋白的表达情况;用ERK1/2信号通路抑制剂PD98059和p38信号通路抑制剂SB203580分别抑制ERK1/2、p38通路后,qPCR检测成骨相关基因BGP、Runx2基因mRNA表达改变。结果:与对照组相比,24h、48h、72h三个时间点,经不同浓度的丹参干预后MC3T3-E1细胞的OD值均较对照组升高,且呈浓度依赖性。培养21d后茜素红染色结果不同浓度丹参干预组钙结节生成数量比对照组明显增多(p<0.01)。在4d和7d两个时间点,各个浓度丹参干预组的BGPmRNA、Runx2 mRNA相对表达量较对照组均增加,并呈浓度、时间依赖性。经过丹参干预后(300mg/L),磷酸化ERK1/2蛋白表达相对表达量是对照组的1.4倍(P<0.05),磷酸化p38蛋白的表达与对照组相比差异无统计学意义。300mg/L丹参组比300mg/L丹参+PD98059组的BGPmRNA、Runx2mRNA相对表达量均明显增多,分别是1.35倍和1.08倍(P<0.01、P<0.05);但与300mg/L丹参+SB203580组相比,BGPmRNA、Runx2mRNA相对表达量差异无统计学意义。结论:丹参能促进成骨细胞增殖、分化,增进矿化作用,作用机制可能与激活MAPK信号通路中的ERK1/2通路有关,而与p38信号通路无明显相关。第二部分丹参通过P38MAPK和JAK2/STAT3信号通路对高铁环境下MC3T3-E1小鼠前成骨细胞起保护作用目的:研究丹参对高浓度铁离子环境下的MC3T3-E1小鼠前成骨细胞的增殖、成骨分化、矿化能力的影响,并探索其中的作用机制。方法:在培养基中加入高浓度铁离子模拟成骨细胞铁蓄积,设F1组:50μmol/L的枸橼酸铁铵(FAC);F2组:200μmol/L的FAC;D1+F2组:75mg/L的丹参+200μmol/L的FAC;D2+F2组:150mg/L的丹参+200μmol/L的FAC组;D3+F2组:300mg/L的丹参+200μmol/L的FAC组,及对照组;用CCK8法检测细胞增殖活性的改变;茜素红染色法检测各组钙结节生成情况;流式细胞术检测细胞凋亡率改变;实时荧光定量PCR法(qPCR)检测各组BGP、Runx2、Col1a基因mRNA表达情况。对F2组、D2+F2组及CON组用western blot检测磷酸化p38(p-p38)、p-JAK2、p-STAT3蛋白的表达情况。结果:经过CCK8法检测,用一定浓度丹参处理的MC3T3-E1细胞(D1+F2组、D2+F2组)的存活率高于高铁组(F2组)的存活率(p<0.01);经茜素红染色法检测,D2+F2组的钙结节生成率明显大于F2组;经流式细胞术检测结果提示在高铁培养环境中加了不同浓度的丹参各组比单纯高铁组(F1、F2组)的MC3T3-E1凋亡率明显下降(p<0.01);经qPCR检测,相比高铁组(F2组),加了不同浓度的丹参各组,成骨相关基因(BGP、Runx2、Col1a)的mRNA的表达均明显增加(P<0.05);Western blot检测发现在高铁培养环境下p-p38、p-JAK2和p-STAT3蛋白的表达量均明显增加(p<0.01)。而加入一定浓度丹参后p-p38和p-JAK2蛋白的表达又明显减少(p<0.01)。结论:丹参能拮抗高浓度铁离子环境对MC3T3-E1小鼠前成骨细胞的增殖、成骨分化、矿化的抑制作用,并且减少细胞凋亡,故对铁蓄积环境下的成骨细胞起保护作用,其中的机制可能与丹参抑制了P38MAPK信号通路和JAK2/STAT3信号通路的激活有关。第三部分丹参对小鼠巨噬细胞系RAW264.7的增殖、破骨分化和破骨功能的影响及其机制研究目的:研究丹参对小鼠前破骨细胞RAW264.7的增殖、分化和破骨相关功能的影响和机制。方法:采用RANKL诱导小鼠巨噬细胞系RAW264.7细胞在体外向破骨细胞分化的模型,用不同浓度的丹参的水溶性成分混合物(丹参注射液稀释液)处理细胞,浓度分别为75mg/L、150mg/L、300mg/L,用CCK8法检测细胞的增殖活性,通过抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色法检测各组破骨细胞数量;用荧光定量聚合酶反应(qPCR)检测破骨相关基因组织蛋白激酶(CTK)、基质金属蛋白酶9(MMP-9)、TRAP的mRNA转录情况;western blot检测破骨相关蛋白TRAP、CTK、NFATc1的表达情况;利用荧光探针DCFH-DA显示各组细胞内的活性氧(ROS),并用荧光显微镜观测和多功能酶标仪上检测吸光度值两种方法检测ROS水平。结果:CCK8检测结果提示丹参能抑制RAW264.7细胞的增殖活性,且呈浓度依赖性;TRAP染色提示丹参能抑制破骨细胞的生成和成熟;qPCR检测提示丹参能抑制破骨相关基因CTK、MMP-9、TRAP的mRNA的转录,且呈浓度依赖性;western blot检测结果示丹参能抑制破骨相关蛋白TRAP、CTK、NFATc1的表达;荧光显微镜和酶标仪检测均显示,丹参能减轻RAW264.7细胞内ROS水平且呈浓度依赖性。结论:丹参能抑制RAW264.7细胞向破骨细胞方向分化,并能抑制RAW264.7细胞的增殖、破骨分化以及破骨相关功能,其中的机制与减少细胞内ROS生成和抑制NFATc1通路有关。更多还原

【Abstract】 PARTⅠSalvia miltiorrhiza promotes osteogenic differentiation and proliferation of MC3T3-E1 preosteoblast by the ERK pathwayObjective:To study whether or not Salvia miltiorrhiza(SM)can promote osteogenic differentiation and proliferation of MC3T3-E1 preosteoblast,and examine the relationship between the mechanism and extracellular signal-regulated kinase(ERK)signal pathway.Method :Different groups with different Salvia miltiorrhiza concentration(75mg/L、150mg/L、300mg/L),the control group without Salvia miltiorrhiza,the proliferation of MC3T3-E1 cell was analyzed by cell counting kit-8(CCK8)assy,After 21 days culture,the mineralization of MC3T3-E1 was examined by alizarin red staining,and the calcium nodes were counted.The mRNA expressions of bone gla protein(BGP)and runt related transcription factor 2(Runx2)were examined by realtime fluorescence quantitative PCR(qPCR).MC3T3-E1 cells were treated with SM in high concentration(300mg/L),the protein expression of phosphorylated extracellular signal-regulated kinase(p-ERK1/2)and p-p38 were detected by Western bloting.After added inhibitor of ERK1/2 and p38 pathway,the mRNA expressions of BGP and Runx2 were examined by qPCR again.Results: Compared to control group,MC3T3-E1 preosteoblasts treated with SM had higher level of proliferation,and SM in 300mg/L showed extremely significant difference(p=0.006).MC3T3-E1 cells treated with SM in different concentration had more calcium nodes than the control group(p=0.000),the quantity was(11.40±2.30),(21.00±2.24),(35.60±1.52),(6.40±1.14)(control group)respectively.In general,the groups treated with SM had higher level of the mRNA expressions of osteoblasts-related gene BGP and Runx2.For BGPm RNA,the raletive expression were(1.02±0.04),(1.11±0.07),(1.26±0.05)respectively,the two groups(treated with SM in150mg/L and 300mg/L)haded significant statistical differences(p=0.019,p=0.000).ForRunx2 mRNA,the raletive expression were(1.38±0.25),(2.28±0.13,(2.48±0.06)respectively,the group treated with SM in 75mg/L showed significant differences(p=0.012),while the two groups treated with SM in 150mg/L and 300mg/L respectively,showed very significant statistical differences(p=0.000).The expression level of protein p-ERK1/2 was also remarkably elevated in SM group compared to the control group(p=0.014),but The expression level of protein p-p38 of the two groups was not significant difference.The two groups were compared(300mg/LSM vs 300mg/LSM+PD98059),the mRNA expressions of BGP and Runx2 obviously decreased(P=0.000).but between the two groups(300mg/L SM vs 300mg/L SM+ SB203580),there was no significant difference about the m RNA expressions of BGP and Runx2.Conclusion: Salvia miltiorrhiza can promote osteogenic differentiation,proliferation and mineralization of MC3T3-E1 preosteoblast by the ERK1/2 signal pathway and not by the p38 signal pathwayPARTⅡ Salvia miltiorrhiza prevents iron overload-induced apoptosis and decline of osteogenic capacity of MC3T3-E1 by blocking p38 MAPK and JAK2/STAT3 signalObjective:To study Salvia miltiorrhiza(SM)whether or not can prevent iron accumulation-induced decline of osteogenic differentiation,proliferation and mineralization of MC3T3-E1 preosteoblast,and examine the mechanism.Method: Different groups were set,group F1: 50μmol/L ammonium ferric citrate(FAC),group F2: 200μmol/L FAC,group D1+F2 : 75mg/L SM+200μmol/L FAC,group D2+F2:150mg/L SM+200μmol/L FAC,group D3+F2:300mg/L SM+200μmol/L FAC,CON as the control without any treatment.The proliferation of MC3T3-E1 cell was analyzed by cell counting kit-8(CCK8)assy,the mineralization of MC3T3-E1 was examined by alizarin red staining,the apoptosis rate of the cells was examined by flow cytometry.The mRNA expressions of BGP,Runx2 and Col1 a were examined by realtime fluorescence quantitative PCR(qPCR).The protein expression of p-p38、p-JAK2、p-STAT3 were detected by Western blotting in group F2,D2+F2 and CON.Result: Compared to group F2,the proliferation of group D1+F2 and group D2+F2had obviously rise[(87.38±1.03)% vs(82.35±1.79)%,(86.91±1.00)% vs(82.35±1.79)%,p<0.01].The generation rate of calcium nodes of group D2+F2 was higher than group F2 observably.Compered to the groups treated with iron of high concentration only(group F1,F2),the groups treated with iron and SM together(group D1+F2,D2+F2,D3+F2)had lower apoptosis rate obviously(p<0.01).The relative expression of osteogenesis-related genes(BGP,Runx2,Col1a)of different groups(groupD1+F2,D2+F2,D3+F2),that were added SM of different concentration and FAC,had higher level than group F2(p <0.05)[(for BGP,(1.95±0.04,2.08±0.17,2.78±0.10)vs 0.78±0.08;for RunX2,(0.64±0.02,0.75±0.02,0.83±0.03)vs 0.58±0.02;for Col1 a,(0.39±0.02,0.74±0.02,0.94±0.02)vs 0.35±0.03].Western blotting measure result : Compared to the control group,the protein expression level of p-p38,p-JAK2 and p-STAT3 in group F2 was significantly rised(p < 0.01),however,when added 150mg/L SM to culture medium(group D2+F2),expression level of p-p38、p-JAK2 declined obviously(p<0.01).Conclusion: Salvia miltiorrhiza can prevent iron overload-induced apoptosis of MC3T3-E1 preosteoblast,meanwhile reduce the decline of proliferation,differentiation and mineralization which induced by excess iron.the mechanism is related to suppressing the p38 MAPK and JAK2/STAT3 signal pathway.PART Ⅲ Salvia miltiorrhiza suppress RAW264.7 proliferation osteoclastic differentiation and function by reducing the production of ROS in the cells and suppressing NFATc1 signalObjective:To explore the mixture of water-soluble effective component of Salvia miltiorrhiza,whether or not it can affect proliferation,differentiation and osteoclastic function of osteoclast,and probe the mechanism.Method: using RANKL(receptor activator of NF-κB ligand)induce RAW264.7 to osteoclast as the model of osteoclast in vitro.Used Salvia miltiorrhiza in differentconcentration(75mg/L、150mg/L、300mg/L respectively)treat cells of different groups,and compare to control group.The proliferation of RAW264.7 cell was analyzed by cell counting kit-8(CCK8)assy,Osteoclast differentiation and number counting was assessed by tartrate resistant acid phosphatase(TRAP)staining and electron microscope,used fluorescence probe DCFH-DA to detect Reactive oxygen specie(ROS)in cells,and used fluorescence microscope and multiscan spectrum to measure the ROS level.The mRNA expression of cathepsin k(CTK),matrix metalloprotease-9(MMP9)and TRAP was detected by fluorescent quantitative polymerase chain reaction(qPCR).The expression of CTK、TRAP and NFATc1 protein was detected by western blot.Result: Salvia miltiorrhiza can suppress the proliferation of RAW264.7 cells concentration-dependently.The result of TRAP staining indicated that Salvia miltiorrhiza in different concentration could suppress the generation and maturity of osteoclast in dose-dependent manner,the average number of osteoclast in one well,which was the treated with different concentration of Salvia miltiorrhiza,was few than the control group obviously.According to the result of quantitative polymerase chain reaction(qPCR),Salvia miltiorrhiza could suppress the mRNA transcription of cathepsin K(CTK),matrix metallopeptidase-9(MMP9)and tartrate resistant acid phosphatase(TRAP),the three important gene of osteoclast.According the result of observation by fluorescence microscope and assessement by multiscan spectrum,Salvia miltiorrhiza significantly reduced intracellular ROS level,especially in high concentration(300mg/L).Conclusion:Salvia miltiorrhiza can suppress RAW264.7 cell to differentiate to osteoclast,and suppress the proliferation and differentiation of RAW264.7 cell,and restrain the osteoclastic function.one of the mechanism was relating to reduce the production of ROS in the cells and suppress NFATc1 signal.更多还原