【作者】 高琳琳；

【导师】 王浩；

【作者基本信息】 山东中医药大学 ， 中西医结合基础， 2008， 博士

【摘要】 目的:对中药蚤休进行提取,应用其两种产物研究对H₂O₂诱导的脐静脉内皮细胞ECV304损伤的保护作用及其机制。方法:首先制备蚤休醇提物,并进行蚤休总皂苷的提取; MTT法检测蚤休醇提物和蚤休皂苷对H₂O₂诱导的ECV-304损伤的保护作用及确定两种药物保护作用的实验剂量;体外培养脐静脉内皮细胞ECV304,建立氧化损伤的细胞模型,然后分为八个实验处理组:1正常对照组2氧化损伤组3高浓度蚤休醇提物（EFB）（500pg/ml）组4中浓度蚤休醇提物（50pg/ml）组5低浓度（5pg/ml）组6高浓度蚤休皂苷（Pa）（1ug /ml）组7中浓度蚤休皂苷（100pg/ml）组8低浓度蚤休皂苷（10pg /ml）组;在第二部分实验中,应用流式细胞仪Annexin V/PI染色检测两种药物干预H₂O₂氧化损伤前后的细胞凋亡、PI染色检测细胞周期与凋亡的关系、激光共聚焦显微镜观察Annexin V/PI染色后各组细胞的形态学改变;激光共聚焦显微镜观测药物各组细胞内Ca2+的含量的表达,500pg/ml蚤休醇提物与1ug/ml蚤休皂苷预处理10分钟加入损伤浓度的H₂O₂,观察细胞内Ca2+的浓度的动态变化。RT-PCR检测各实验组caspase-3 mRNA表达的变化;免疫荧光染色检测各实验组caspase-3的定性表达。在第三部分实验中,以RT-PCR检测各实验组血管内皮细胞中细胞间粘附分子（intracelluar adhesion molecule,ICAM-1）、血管细胞粘附分子（vasculer cell adhesion molecule , VCAM-1 ）、细胞因子--单核细胞趋化蛋白（monocytechemoattractant protein,MCP-1）mRNA水平的变化的表达;应用流式细胞仪定量检测ICAM-1、VCAM-1的阳性细胞数;免疫荧光染色检测各实验组MCP-1的表达。结果:建立了细胞过氧化损伤的模型;经鉴定正确提取制备了蚤休醇提物和蚤休皂苷;MTT显示加入损伤因素后细胞吸光度OD值低于正常对照组（P<0.01）;两种药物预处理后OD值增加,高浓度蚤休醇提物组与高浓度蚤休皂苷组的OD值与正常组相比无显著性差异（P>0.05）;流式细胞结果显示:采用流式细胞仪PI染色检测结果对细胞周期进行分析,表明损伤作用于ECV-304细胞后,细胞在G0/G1期的数目增多,在S期的数目减少,同时流式细胞仪检测可见亚二倍体峰,而蚤休醇提物和蚤休皂苷预处理以后,S期细胞数明显增多,各药物干预组G0/G1期细胞数与正常组无差异。流式细胞仪Annexin V/PI检测方法显示,损伤组的早期与晚期凋亡率之和最高,,预处理后细胞的早期与晚期凋亡率之和降低,在蚤休皂苷,其降低凋亡的效应呈剂量依赖性。激光共聚焦显微镜观察Annexin V/PI染色后的各组细胞的形态学表现为,在正常对照组,镜下仅见较少的着色为绿色的细胞膜,未见染成红色的细胞核。在损伤组早期以及晚期凋亡均较明显,表现为密度高的红色的细胞核着色,细胞膜绿色或者细胞核将游移出细胞,即典型的凋亡小体表现。还有较多的细胞仅呈现红色的细胞核,而不见细胞膜,呈现晚期凋亡典型的形态学表现。而蚤休醇提物与蚤休皂苷干预后,均随药物浓度的增加,表现为红色细胞核的细胞量即晚期凋亡量逐渐减少,表现为深红色的胞核显著减少,多表现为细胞膜着色为绿色。各实验组细胞内静态Ca²⁺的含量测定结果显示,损伤组的Ca²⁺荧光强度最高,与正常对照组相比差异显著（P<0.01）;药物预处理后细胞内Ca²⁺浓度荧光强度显著下降,此效应在两种药物均呈现剂量依赖性增大。Ca²⁺的动态含量测定结果显示,H₂O₂作用于细胞后,细胞内Ca²⁺的浓度呈现升高趋势,荧光强度持续上升达一个高峰,然后缓慢下降至平台值;蚤休醇提物与蚤休皂苷预处理后,再加入损伤因素,则Ca²⁺的浓度上升峰值下降,峰值与平台期之间的差值减小,与损伤组相比差异显著（P<0.01）,细胞内的Ca²⁺的浓度呈现较为稳定的状态。caspase-3 mRNA水平在各实验组的表达显示;损伤组与内参照相比的灰度值最大,经过预处理之后其值明显下降（P<0.01）;免疫荧光染色也证实了这个结果,损伤组的caspase-3荧光强度最高,而预处理后明显下降（P<0.01）。在实验的第三部分中,RT-PCR实验结果显示,两种药物预处理氧化损伤后,ICAM-1、VCAM-1和MCP-1 mRNA的表达水平与损伤组相比明显减弱（P<0.01）;损伤组中与内参照的灰度值之比最大,与正常组相比差异显著（P<0.01）;免疫荧光染色显示损伤组MCP-1的荧光强度最大,与正常组相比差异显著（P<0.01）;而经过预处理之后表达减弱,效应均呈剂量依赖性;且均与损伤组相比有显著性差异（P<0.01）;流式细胞仪定量检测ICAM-1、VCAM-1的结果显示,损伤组中表达ICAM-1、VCAM-1的阳性细胞数均最多,与正常组相比差异显著（P<0.01）;两种药物预处理氧化损伤后,阳性细胞数明显减少。结论:所提取的两种药物蚤休醇提物以及皂苷可以保护H₂O₂造成的氧化损伤,推测蚤休的保护作用由其有效成分蚤休皂苷发挥。是通过阻抑正常的细胞周期诱导凋亡、抑制凋亡蛋白caspase-3介导的凋亡信号转导通路实现,而这个通路的激活与钙离子激活后介导的德信号转导有关;以及通过抑制内皮细胞粘附分子与细胞因子的表达从而抑制炎症性损伤,达到保护内皮细胞、抗动脉粥样硬化的目的。更多还原

【Abstract】 Objectives : To extract traditional Chinese medicine bistortae,investigate the protection of the injury on the umbiliar vein endothelial cell ECV 304 induced by hydrogen peroxide（H₂O₂） of ethanol from bistortae and pariphyllin, and to study the mechanism of anti一atherosclerosis （AS） of these medicines.Methods: Prepration ethanol from bistortae（EFB） ,then extracte pariphyllin（Pa） from this ; examine the protection of the two above medicine to the injury in ECV 304 induced by H₂O₂,decide the protection dose of the two medicine by MTT reduction assay. ECV 304 had been cultured in vitro, a model of endothelial cell oxidative damage induced by H₂O₂ was established.then cells were divided by eight experimental groups:1 normal;2oxidative damage group;3 high density ethanol from bistortae（500pg/ml）; 4 medium density ethanol from bistortae（50pg/ml）; 5 low density ethanol from bistortae（5pg/ml） ;6 high density pariphyllin（1ug /ml）; 7 medium density pariphyllin（100pg/ml）; 8 low density pariphyllin（10pg /ml）.In second part experiment, Flow cytometry （FCM） methods Annexin V/PI staining and PI staining were used to analysis the effection of two medicine to the apoptosis and cell cycle in all groups, Laser confocal microscopy（LCM） was used to observe the appearance of apoptosis and quiet state and the dynamic change of the density of calcium ion (Ca²⁺) , Reverse transcription PCR （RT-PCR） and immunofluorescene（IFM） staining were used to detect the protein express in mRNA level of apoptosis associated protein caspase-3 in all experiment groups. In third part experiment, we used RT-PCR to detect the protein expression in mRNA level of intracelluar adhesion molecule （ICAM-1）、vasculer cell adhesion molecule （VCAM-1）、monocyte chemoattractant protein（MCP-1）in all experiment groups,FCM were used to quantitate the ICAM-1 and VCAM-1 and in all group cells,then, and IFM was used to decide the qualitation expression of MCP-1 in all groups Results:The model of oxidative damage was established, ethanol from bistortae and pariphyllin were got out successfully ;MTT assay discovered that cell optical density in oxidation damaged group was lower than nomal control （P<0.01）;when pretreated by the two kind of medicine ,the OD value was increased,and was not significant difference in the high density ethanol from bistortae and the high density pariphyllin than normal control（P>0.05）.FCM Annexin V/PI staining result showed that early period and advanced stage apoptosis rate was lower when predisposed than that of damaged group,and the effection was dose dependent in pariphyllin groups. FCM PI staining showed that cells of G0/G1 stage were more in cell cycle in oxidation damage group,but less in S stage and hypodiploid peak could observe ,The LCM showed that there appeared apoptosis body in damage group, Furthermore we studied H₂O₂ mediated Ca²⁺ responses in ECV304 and found that the increase of Fluo-3 fluorescence intensity in the presence of external Ca²⁺: H₂O₂ elicited an transient peak of [Ca²⁺]i and then fell to a subsequent sustained higher plateau.When pretreatment by the ethanol from bistortae（500pg/ml）and pariphyllin（1ug/ml）for 10 min and which significantly prevented the transient increase and sustained increase of [Ca²⁺]i,RT-PCR showed that the expression of caspase-3 weakened when predisposed by the two kind of medicine than without this disposal（P<0.01）;IFM staining result showed that same effection;RT-PCR result showed that the expression of ICAM-1、VCAM-1 and MCP-1 mRNA was significantly descended（P<0.01） in medicine treatment groups than in H₂O₂ damage group, IFM staining showed that MCP-1 had the greatest fluorescence intensity in H₂O₂ damage group,but in all groups pretreatment with medicine it obviously weakened（P<0.01）; FCM result discovered that the numbers of masculine cells which express ICAM-1 or VCAM-1 was obviously more than other groups（P<0.01）,but pretreated groups the was lower.Conclusion: Ethanol from bistortae and Pariphyllin has the protective action on oxidative damage of ECV304 induced by H₂O₂, the mechanism of protective action may relate to decrease the cell signal transduction mediated of apoptosis,which may be activated by calcium ion mediated signal transduction ; and related the stabilize the cell wall, inhibit the inflammatory reaction induced by ICAM-1、VCAM-1 and MCP-1, and then protect endothelial cell,prevent AS come true.更多还原