【作者】 陈晓瑜；

【导师】 邝枣园；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2008， 硕士

【摘要】 目的动脉粥样硬化（atherosclerosis,AS）是一种严重危害人类健康的常见血管疾病,是其他多种心脑血管疾病的病理基础。巨噬细胞凋亡贯穿于动脉粥样硬化的整个过程,凋亡的巨噬细胞形成动脉硬化斑块的坏死核心,该坏死区域与动脉硬化斑块的不稳定,心肌梗塞,中风和外周血管病的发生有密切关系。进一步探讨巨噬细胞的凋亡机制,有利于形成处理病变进展的新策略。中医药具有治疗心脑血管疾病的丰富经验,痰热内阻临床冠心病患者的常见证型。黄连温胆汤是具有清痰热、畅气机、宁心神的作用。已有的研究显示,该方在临床上对冠心病疗效显著。本研究采取体外培养巨噬细胞的方法,人工诱导其内质网压力途径的凋亡过程,并观察黄连温胆汤含药血清对其凋亡过程的影响。方法1、小鼠巨噬细胞（RAW264.7）的培养RAW264.7细胞在25cm²的培养瓶内,以含10%胎牛血清、4.5g/L葡萄糖、IOOU/ml双抗的RPIM-1640培养液,在含有5%体积分数的CO₂培养箱内,保持恒温37℃培养3-5天。细胞长成单层,铺满瓶底的70%左右时即可以0.25%胰酶-EDTA消化传代。2、小鼠巨噬细胞（RAW264.7）凋亡模型的诱导取对数生长期的RAW264.7以1×10⁵/ml浓度接种于24孔板,每孔1000μl,待长成单层后,加入Tg与Fuc作为凋亡诱导剂的为模型组,不做处理的为空白对照组,每组设3个复孔。放置37℃、5%CO₂的培养箱内培养24h;结束时24孔板内的细胞用胰蛋白酶-EDTA消化,PBS洗后,按Annexin/PI试剂盒的说明书处理细胞后,流式细胞仪检测凋亡率,结果以凋亡百分率表示。3、黄连温胆汤含药血清与对照血清的制备黄连温胆汤药物按中药常规煎煮,在恒温箱内浓缩至所需体积。新西兰兔标号、称重、随机分为空白对照与药物组。分别以黄连温胆汤和生理盐水灌胃。每天灌胃1次,连续4天,第4天上午灌胃后1小时取血。采取无麻醉下心脏取血。所取全血分离血清,56℃,30分钟灭活;过滤灭菌并分装;-30℃冰箱保存备用。4、黄连温胆汤含药血清对正常小鼠巨噬细胞增殖的影响取对数生长期的RAW264.7按2.5×10⁵/mL浓度接种100ul到96孔板中;24h后,细胞贴壁,加入黄连温胆汤含药血清为血清组,加入空白血清为血清对照组;两组分别设立高中低剂量组,另设一组正常普通培养,不加刺激物为空白细胞对照。24h后,加入20ul MTT,继续培养4h;每孔加入150ul的DMSO液置室温下37℃培养箱中15min;待孔底沉淀完全溶解后,用酶标仪在波长570测OD值。5、黄连温胆汤含药血清对小鼠巨噬细胞凋亡模型的影响培养后的RAW264.7以1×10⁵/ml的浓度接种于24孔板,待长成单层后,加入黄连温胆汤预处理24h后,加入凋亡诱导剂;设立不加任何刺激物者为正常组;只加凋亡诱导剂为模型组,每组设3个复孔。放置37℃、5%CO₂的培养箱内培养。上述24孔板内的细胞用胰蛋白酶-EDTA消化,PBS洗后,按Annexin V/PI试剂盒的说明书处理细胞后,流式细胞仪检测凋亡率,结果以凋亡百分率表示。6、黄连温胆汤对凋亡模型细胞清道夫受体表达的影响培养后的RAW264.7以1×10⁵/ml的浓度接种于24孔板,待长成单层后,加入黄连温胆汤预处理24h后,加入凋亡诱导剂;设立不加任何刺激物者为正常组;只加凋亡诱导剂为模型组,每组设3个复孔。放置37℃、5%CO₂的培养箱内培养。上述24孔板内的细胞用胰蛋白酶-EDTA消化,PBS洗后,按SR-A荧光抗体试剂盒的说明书处理细胞后,流式细胞仪检测凋亡率,结果以荧光强度与荧光百分比表示。7.统计方法采用SPSS11.0软件包的方差分析（ANOVA）模块进行数据分析,多组间两两比较采用LSD法。结果1.倒置显微镜下观察RAW264.7的生长在倒置显微镜下观察到RAW264.7为贴壁细胞,呈三角形、椭圆形、多角形等不规则形状,或有突触。2.以0.5uM Ta+50ug/ml Fuc以及2uM Tg+50ug/ml Fuc刺激RAW264.7细胞48h,均能够稳定诱导出凋亡模型,用SPSS分析,与正常组（空白细胞）数据有显著差异,p＜0.05。3.不同浓度黄连温胆汤含药血清（1:10;1:20;1:40,1:80;1:160）刺激RAW264.7细胞,以MTT法检测细胞增殖情况,1:10浓度的含药血清可以促进细胞增殖,以SPSS分析,与空白细胞及同浓度空白血清组比较,OD值具有显著差别,p＜0.05。4.黄连温胆汤含药血清（1:10终浓度）预刺激RAW264.7细胞,再以凋亡诱导剂诱导凋亡,经检测,预刺激组的凋亡率下降,与凋亡模型及空白血清对照组均有显著差异,p＜0.05。5.与模型组比较时,黄连温胆汤血清不能影响SR-A表达。结论以0.5μM Tg联合50μg/ml Puc可诱导小鼠巨噬细胞凋亡,与空白对照组相比,具有统计学意义。黄连温胆汤含药血清能够明显抑制凋亡模型细胞的凋亡,与模型对照组以及空白血清对照组相比,均有统计学意义。实验结果表明,抑制巨噬细胞的凋亡可能是黄连温胆汤具有治疗动脉粥样硬化作用的机制之一。黄连温胆汤含药血清并不具有下调SR-A表达的作用,这一结论说明了黄连温胆汤抑制凋亡的机理可能是作用于p38或JNK这两条途径之一,或两者兼有。另外,黄连温胆汤含药血清中抑制凋亡的有效成分,可能是对温度变化敏感、易于在反复冻融情况下失效的成分。更多还原

【Abstract】 OjectiveAtherosclerosis(AS)is one of the main diseases that danger human health.It is also the pathology substructure of cardiovascular disease.The apoptosis of macrophage is a persistent factor in the whole process of atherosclerosis.Macrophages that apoptosised form the necrotic core of the atherosclerotic plaque,which take an important part in the instability of necrosis,myocardial infarction,stroke and peripheral vascular disease.So the investigation in the mechanisms of macrophages apoptosis could be an important potential way for anti-AS.The experiences of TCM for treating cardiovascular and cerebrovascular diseases are abundant.And according to researches and reports,the phlegm-heat is an important and common cause of atherosclerosis(AS)in clinic.Huanglianwendan Medicinal Broth is good at clearing phlegm-heat, regulating the Qi and stabilizing the mind.Researches have revealed that the clinical effects of Huanglianwendan Medicinal Broth to cure coronary heart disease are significantly.In this study,macrophages arecultured in vitro and are inducted by artificial method to apoptosis in ER stress pathway,and then the effect of Huanglianwendan Medicinal Broth is observed.Method1.The culture of RAW264.7.2.The apoptosis of RAW264.7 conducted by Tg and Fuc. 3.Preparation of the serum contains Huanglianwendan and the control serum.4.The effect of serum contains Huanglianwendan on the proliferation of RAW264.7.5.Huanglianwendan serum’s effect on the macrophage apoptosis.6.Huanglianwendan serum’s effect on the expression of scavenger receptor in macrophage.7.Statistical methodUses the variance analysis(ANOVA)of SPSS11.0 software package to carry on the data analysis,during the multi-groups comparisons uses the LSD.Inhibition ratio=(1-experimental group/comparison group)×100%Results1.The normal RAW264.7 under the inverted microscope are adherent cells, to assume the triangle;oval-shaped,the polygon,or synapses.2.Stimulate RAW264.7 cells 48 h with 0.5 uM Tg +50 ug/ml Fuc and 2 uM Tg +50 ug / ml Fuc are able to induce stable apoptosis.When analysis, the difference between normal group(control cells)and apoptosis group is significant,P＜0.05.3.After detected the effect of different concentrations of Huanglianwendan -containing serum(1:10,1:20;1:40,1:80;1:160)to stimulate RAW264.7’ proliferation by MTT method,We found1:10 concentrations can promote cell proliferation.Under analysis,the data of experimental group and control group are significant different,p＜0.05.4.Huanglianwendan-containing serum(1:10 final concentration) pre-stimulation RAW264.7 cells can decline the rate of apoptosis,and the data among the serum group,the apoptosis group and the blank Serum control group were significantly different,p＜0.05.5.The Huanglianwendan-containing serum group can not decline SR-A expression.Conclusion0.5μM Tg joint 50μg/ml Fuc can induce the apoptosis of macrophage.Huanglianwendan-containing serum can inhibit apoptosis of RAW264.7. and the data is significant compared both with apoptosis group and the blank serum group.The results showed that the inhibition of macrophage apoptosis may be one of the Huanglianwendan’s mechanisms of treating atherosclerosis.Huanglianwendan soup does not have the effect to decline the SR-A.The effect about SR-A declinate the apoptosis would be happened in p38 may or JNK way or both.In addition,the active ingredients of inhibiting apoptosis contains in Huanglianwendan serum may be sensitive to temperatures,and were easy to failure under repeated freeze-thaw.更多还原