【作者】 王建礼；

【导师】 王家富； 商战平；

【作者基本信息】 泰山医学院 ， 病理学与病理生理学， 2007， 硕士

【摘要】 目的：研究蜂胶水提物(water extract propolis,WEP)对氧化型低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)诱导的人脐动脉平滑肌细胞(human umbilical artery smooth muscle cell,HUASMC)胆固醇酯(cholesteryl ester,CE)聚集及凋亡的影响,探讨WEP抗动脉粥样硬化作用可能的机制,为蜂胶的进一步开发应用提供依据。方法：1.制备WEP：采用超声波法萃取泰山蜂胶,其水溶性成分用于实验研究。2.培养HUASMC及鉴定：取新鲜脐带脐动脉的中膜平滑肌层,采用贴块法培养HUASMC;经免疫细胞化学方法进行鉴定后用于实验。3.制备ox-LDL,测定其氧化修饰程度：以Cu2+诱导法催化低密度脂蛋白氧化,采用硫代巴比妥酸反应物法测定其中MDA含量,以确定其氧化修饰程度。4.制备平滑肌源性泡沫细胞模型：分别以25mg/L、50mg/L、75mg/L三个浓度ox-LDL与平滑肌细胞共同培养,于12h、24h、36h、48h、60h五个时间点检测细胞内总胆固醇(total cholesterol,TC)和游离胆固醇(free cholesterol,FC)含量,以TC减FC得出CE的含量,CE/TC比值>50%作为泡沫细胞的标志,选择50mg/L ox-LDL作用48h作为制备平滑肌源性泡沫细胞模型的适宜条件。5.实验分组：培养的细胞随机分为五组对照组：正常培养基模型组：50 mg/L ox-LDL作用48hWEP干预组：分别以50mg/L、100mg/L、200mg/L三个浓度的WEP与50mg/Lox-LDL共同作用48h；6.通过油红O染色法对细胞内脂质成分进行染色,观察各组细胞荷脂情况；7.采用高效液相色谱法测定细胞内TC和FC的含量,从中得出CE的含量,根据细胞蛋白定量。8.采用流式细胞仪技术Annexin V/PI双染法检测细胞凋亡率。结果：1.平滑肌源性泡沫细胞模型的制备：采用50mg/Lox-LDL与平滑肌细胞共同培养48h,可使细胞内CE/TC比值>50%,而转变为泡沫细胞。2.WEP对细胞内CE聚集影响的形态学观察：经油红O染色后显示,模型组与对照组相比细胞内脂滴含量明显增多,50 mg/L、100mg/L、200mg/L三个浓度WEP干预组细胞内脂滴比模型组少,且随WEP浓度的增高,脂滴的数量有减少的趋势。3.WEP对细胞内CE含量的影响：模型组细胞内CE含量高于对照组(P<0.01),50 mg/L、100mg/L、200mg/L三个浓度WEP干预组细胞内CE的含量都低于模型组(P<0.01),且随WEP浓度的增高细胞内CE含量有减少的趋势;4.WEP对细胞凋亡的影响：模型组细胞凋亡率高于对照组(P<0.01),50 mg/L、100mg/L、200mg/L三个浓度WEP干预组细胞凋亡率都低于模型组(P<0.01),且随WEP浓度的增高细胞凋亡率有降低的趋势。5.细胞内CE含量与细胞凋亡率之间相关性分析：细胞内CE含量与细胞凋亡率之间存在正相相关关系,相关系数为0.964,P<0.01。结论：1.ox-LDL可引起体外培养的HUASMC细胞内CE聚集,而转变为泡沫细胞,同时也能导致HUASMC凋亡率增加；2.WEP能够降低ox-LDL所致的HUASMC细胞内CE聚集的程度,从而抑制泡沫细胞的形成。3.WEP能够降低ox-LDL诱导的HUASMC凋亡,可能与WEP降低细胞内CE含量有关。4.WEP抑制ox-LDL所致的HUASMC细胞内CE聚集并降低其凋亡率,可能是其抗动脉粥样硬化作用的机制之一。更多还原

【Abstract】 [Objective]The aim of this study to investigate the effect of water extract propolis on cholesteryl ester accumulation and apoptosis in human umbilical artery smooth muscle cell induced by oxidized low density lipoprotein, and explore the mechanism of water extract propolis on inhibition of atherosclerosis and it’s implications.[Mothods]1.Preparation of WEP:Extract propolis by ultrasonic waves,chose the composition which can dissolved in water for the experiment.2.Culture human umbilical artery smooth muscle cell and identificate them:get umbilical cord and dissecte umbilical artery,culture HUASMC by explant method. Identificate them by immunocytochemistry technique.3.Preparation of oxidized low density lipoprotein and determination of the extent of oxidized modification:ox-LDL was produced from LDL after being incubated with CuSO4. in vitro;The extent of lipid peroxidation was estimated as for thiobarbituric acid-reactive substances. Tetramethoxypropane was used as a standard, and results are expressed as nanomoles of malondialdehyde equivalents per mg protein.4.Preparation of the smooth muscel cell derived foam cell model:coculture HUASMC with ox-LDL in concentration of 25mg/L,50mg/L,75mg/L respectively, the intracellular total cholesterol (TC)and free cholesterol(FC) at 12h,24h,36h,48h,60h were measured by high performance liquid chromatogram,the content of cholesterol ester was obtained by subtracting the FC from TC.The ratio of CE/TC>50% is the mark of foam cell,so coculture HUASMC with ox-LDL in concentration of 50mg/L for 48h is the optimum condition for foam cell formation.5.Experiment groups: Cultured HUASMCs were randomly divided into 5 groups:Control:without any drugModel:with 50mg/L ox-LDL for 48hPropolis groups:with 50mg/L ox-LDL and 50mg/L,100mg/L,200mg/LWEP respectively for 48h6.0il red O staining was used to stain lipids in the cultured HUASMC of various groups.7.Intracellular total cholesterol and free chelosterol were measured by high performance liquid chromatogram, the content of cholesterol ester was obtained by subtracting the FC from TC.8.Cellular apoptosis index was tested by flow cytometry.[Result]l.Estabilishment of smooth muscle cell derived foam cell:HUASMC were Cocultured with 50mg/Lox-LDL for 48h,the ratio of intracellular CE/TC is more than 50%,and the cells were transformed into foam cell.2.The morphology observation of the effect of WEP on cholesteryl ester accumulation in HUASMC induced by ox-LDLThe amount of intracellular red lipid granules in the model group are more than that in the control group;The amount of intracellular red lipid granules in 50 mg/L,100mg/L,200mg/L WEP groups are less than that in the model group,and with the increase of the concentration of WEP the amount of intracellular red lipid granules has the tendency of decreasing.3.Effect of WEP on the content of intracellular CE:The content of intracelluar CE in model group is more than that in control group (P<0.01); The content of intracellular CE in 50 mg/L,100mg/L,200mg/L WEP groups are less than that in the model group(P<0.01), and with the increase of the concentration of WEP the content of intracellular CE has the tendency of decreasing.4.The effect of WEP on cell apoptosis index:The cell apoptosis index in model group is more than that in control group (P<0.01); The cell apoptosis index in 50 mg/L,100mg/L,200mg/L WEP groups are less than that in the model group(P<0.01),and with and with the increase of the concentration of WEP the cell apoptosis index has the tendency of decreasing.5.The relationship between the content of intracellular CE and cell apoptosis:The content of intracellular CE was correlated with the cell apoptosis (rs=0.964,P<0.01).【Conclusion】1.ox-LDL can induces the cholestreyl ester accumulation in cultured HUASMC in vitro,and transforms it into foam cell, simultaneously it can raise the apoptosis index of HUASMC.2.WEP can reduce the extent of intracellular CE accumulation in HUASMC induced by ox-LDL.3.WEP can reduce the apoptosis index of HUASMC induced by ox-LDL which maybe concerned with the effect of reducing the content of intracellular CE.4.WEP reduce the extent of intracellular CE accumulation and cell apoptosis maybe one of the mechanisms of it’s anti-atherosclerosis effect.更多还原