【作者】 沈瑾秋；

【导师】 龚婕宁；

【作者基本信息】 南京中医药大学 ， 中医临床基础， 2009， 博士

【摘要】 研究目的本研究理论部分目的在于探讨内生热毒与慢性炎症的相关性,提出慢性炎症相关病变从热毒论治的思路;总结运用清热解毒法治疗慢性炎症相关病变的治疗规律,为临床辨证治疗此类疾病提供理论基础。实验部分以慢性炎症相关病变之一血管生成为靶点,研究清热解毒代表中药黄芩主要成分黄芩苷、金银花主要成分绿原酸对这一事件的影响作用,从而为临床用药提供实验依据,为清热解毒法实质的阐释提供科学的支持。研究方法理论部分以文献研究为主,全面总结前人研究成果,深度挖掘中西医不同领域理论内在联系,归纳出病理共性和治疗规律。实验方案分为体内和体外两部分,体内部分以鸡胚绒毛尿囊膜(CAM)为模型,观察药物黄芩苷、绿原酸对血管新生的影响作用;体外实验主要通过观察黄芩苷、绿原酸对血管生成不同环节(血管内皮基底膜的降解、内皮细胞的增殖、迁移以及管腔形成过程)的影响作用,来评价药物是否具有抗血管生成的活性。具体方法为:对血管内皮细胞Ea.hy926进行常规培养,采用MTT法检测药物对细胞增殖的影响;划痕实验、Transwell实验观察药物对内皮细胞迁移的影响;人工基质胶法观察药物对内皮细胞管腔形成的影响:明胶酶谱法检测药物对内皮细胞分泌MMP9、MMP2活性的影响。实验数据采用SPSS统计软件进行处理。研究结果本研究理论上对慢性炎症的内涵进行了全面总结,追踪国内外最新研究情况,重点阐述了慢性炎症相关病变尤其是血管生成的相关背景;从内生热毒与慢性炎症相关病变的各自特点,展开对二者相关性的探讨,总结了内在联系和规律:并由此进一步论述了运用清热解毒法治疗慢性炎症相关病变的用药规律。实验部分的总结如下:1、体内实验即CAM法发现,无论黄芩苷还是绿原酸,各个浓度对血管生成有一定的抑制作用,且黄芩苷(浓5度0.01、0.1、1mM的抑制率分别为53.33%、66.67%、73.33%)呈现浓度依赖性。2、MTT实验表明,黄芩苷可以抑制Ea.hy926的增殖,OD值与空白组比较有显著性差异(p＜0.01),各个浓度(1、0.1、0.01mM)下细胞存活率分别为49.976%、70.78%、71.01%;绿原酸高浓度(1 mM)能够抑制细胞增殖,OD值与空白组比较有显著性差异(p＜0.05),但是无浓度依赖性。3、对划痕实验中各组细胞迁移率的统计数据来看,以姜黄素作对比,黄芩苷各个浓度(1、0.1、0.01mM)迁移率分别为20.67%、8.67%、97.33%,呈现较为明显的抑制作用;绿原酸各个浓度(1、0.1、0.01mM)迁移率分别为60.67%、1.0067%、1.2467%,有一定的浓度依赖性。4、Transwell实验结果显示,黄芩苷组的迁移细胞分别为29±4、41.8±17.12、36.7342.8±6.98、76.6±7.74,与对照组相比,具有显著性差异(p＜0.01),绿原酸组的迁移细胞分别为11.8±3.03、26.8±5.68、85.6±13.24、99±7.88,与对照组相比,具有显著性差异(p＜0.01)。5、在内皮小管形成实验中,高浓度黄芩苷、绿原酸(1mM)对Ea.hy926形成的内皮小管具有抑制和断裂作用。6、明胶酶谱实验中,黄芩苷有效地抑制了Ea.hy926分泌的胶原酶MMP-9、MMP-2,从而抑制内皮细胞的迁移,其中以MMP-2的抑制作用最为显著,且呈现明显的浓度依赖性。绿原酸的高浓度(1mM)能明显减弱MMP9、2的负染条带,低浓度差异不明显。更多还原

【Abstract】 Research purposesThe purpose of the theory part in this study is to investigate the relevance of chronic inflammation and the toxicity and heat.the chronic inflammatory disease-related drugs from the heat of the idea of governance;summary of the use of heat-clearing and detoxifying treatment of chronic inflammatory diseases related to the treatment of the law,for clinical differentiation therapy in this provide a theoretical basis for diseases.Experimental part of one of chronic inflammatory disease-related angiogenesis as a target to study the heat-clearing and detoxifying the representative major components of Chinese medicine baicalin baicalin,honeysuckle main component of chlorogenic acid on the impact of the incident,so as to provide clinical experimental basis for the clearing away heat and toxic method to provide a scientific explanation of real support.Research MethodsTheoretical part of research-oriented literature,a comprehensive summary of the results of previous studies,the depth of excavation in the Western theory of internal relations in different fields,into a pathology and treatment of common law.Experimental program is divided into two parts,in vivo and in vitro,in vivo portion of chicken chorioallantoic membrane(CAM) as a model to observe the drug of baicalin,chlorogenic acid on the impact of the role of angiogenesis;vitro mainly through the observation of baicalin,chlorogenic acid on the different aspects of angiogenesis(vascular endothelial basement membrane degradation,endothelial cell proliferation,migration and lumen formation) effect,to evaluate whether a drug of anti-angiogenesis activity.Specific methods for:Ea.hy926 of vascular endothelial cells to conventional training,the use of MTT assay of drugs on cell proliferation;scratch experiments, Transwell experiments to observe the drug on endothelial cell migration;artificial plastic matrix observed drugs on endothelial cells lumen formation;gelatin zymography detection of drugs on endothelial cell secretion of MMP9,MMP2 activity.Experimental data using statistical software SPSS for processing.The resultsIn this study,in theory,chronic inflammation of the connotation of a comprehensive summary of the latest follow-up study at home and abroad,focusing on the chronic inflammatory disease,especially related to angiogenesis in the background;from the inside heat-related toxicity and chronic inflammatory disease of the respective characteristics carried out on the relevance of the two,summed up the internal links and the law;and thus further discusses the use of heat-clearing and detoxifying treatment of chronic inflammatory disease of drug-related law.Part of the experiment are summarized as follows:1,in vivo experiments found that the CAM method,regardless of baicalin or chlorogenic acid,various concentrations of a certain degree of angiogenesis inhibition,and baicalin(0.01,0.1,1 mM concentration,respectively,the inhibitory rate for 53.33%,66.67%,73.33%) showed dose-dependent manner.2,MTT experiment showed that baicalin Ea.hy926 can inhibit the proliferation,OD values compared with the blank control group were significantly different(p＜0.01),various concentrations(1, 0.1,0.01 mM) under the cell survival rate was 49.976%,70.78%,71.01%;high concentration of chlorogenic acid(1 mM) could inhibit cell proliferation,OD values compared with the blank control group were significantly different(p＜0.05),but no dose-dependent manner.3, experiments on the scratch cell migration rate in each group statistical data,compared to curcumin,various concentrations of baicalin(1,0.1,0.01 mM) migration rates were 20.67%, 58.67%,97.33%,showed more obvious inhibition;various concentrations of chlorogenic acid (1,0.1,0.01 mM) migration rates were 60.67%,1.0067%,1.2467%,a certain degree of concentration-dependent.4,Transwell experiments showed that baicalin group migrated cells were 29±4,41.8±17.12,36.7342.8±6.98,76.6±7.74,compared with the control group with significant difference(p＜0.01),chlorogenic acid in cell migration group were 11.8±3.03,26.8±5.68,85.6±13.24,99±7.88,compared with the control group with significant difference (p＜0.01).5,in the endothelial tubule formation assay,the high concentration of baicalin, chlorogenic acid(1mM) on endothelial Ea.hy926 tubule formation and fracture with the role of inhibition.6,gelatin zymography experiments,Baicalin Ea.hy926 effectively inhibited the secretion of collagenase MMP-9,MMP-2,thereby inhibiting migration of endothelial cells, with inhibition of MMP-2 most significant,and a clear dose-dependent manner.Chlorogenic acid in high concentrations(1mM) significantly reduced MMP9,2 bands of negative staining, no obvious difference in low concentration.更多还原