【作者】 凌家杰；

【导师】 李赛美；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2006， 博士

【摘要】 1 研究目的 通过观察加味桃核承气汤及其配伍组方对体外培养糖尿病损伤血管内皮细胞的干预作用,寻找该方的有效组分,并探讨其组方配伍规律及抗糖尿病血管病变的作用机理,为研制、开发抗糖尿病血管病变有效方剂,阐明《伤寒论》经典方剂桃核承气汤“下瘀血”理论提供实验依据。 2 研究内容、方法与结果 2.1 理论与文献研究 对伤寒方实验研究思路与方法进行探讨,认为作为中药复方中较为特殊的一类方剂——《伤寒论》经方,其临床疗效确切、配伍合理、药物精少,是进行中药复方实验研究的首选对象,其成果可作为研究其他方剂的基础。探讨的内容主要包括:伤寒方实验研究的总体要求;伤寒方化学、药理研究特点及其思路和方法。这是本课题研究的指导思想,自始至终贯穿于整个实验研究过程。 探讨体外培养细胞中药血清药理学研究的若干问题,为整个课题的实验设计、具体方法奠定理论基础。 对中西医糖尿病血管内皮功能损伤研究以及经方治疗糖尿病进行文献综述,由此寻求本课题的突破口与创新点。内皮素-1(ET-1)、一氧化氮(NO)是目前研究血管内皮损伤具有较好代表性的指标。对于糖尿病血管内皮损伤的中医研究方面,有人证实了血瘀证与血管内皮损伤有密切的关系。目前罗格列酮对血管内皮功能影响的研究备受学术界关注,并取得了丰富的成果。 2.2 实验研究 目的是观察加味桃核承气汤不同剂量组(加味高剂量组、加味中剂量组、加味低剂量组)及其不同配伍组方(活血组、泻热通下组、益气养阴组、《伤寒论》原方组)大鼠含药血清对模拟糖尿病环境(高糖和高胰岛素)下血管内皮细胞功能的影响:基更多还原

【Abstract】 1. ObjectiveIn order to study and research the effectual ingredient of Modified Taohe Chengqi Decoction, discussed its composition regulation of prescription and mechanism of treating diabetes blood vessel pathological changes, we Observed the effect of Modified Taohe Chengqi Decoction and its composition of prescription on the diabetes mellitus endothelial cell. We hoped to achieve a effectual decoction for diabetes blood vessel pathological changes and illuminated the reason why Taohe Chengqi Decoction could activate blood flow and remove blood stasis.2. Content, method and result2.1. Theory and literature studyDiscussed the thinking and method of the Treatise on Febrile’ s classical prescription experimental research. Classical prescription were effectual. Their composition of prescription were reasonable. They were fit for experimental research. The following was content we discussed: The main require of classical prescription experimental research;The trait, thinking, method of classical prescription experimental chemistry and pharmacody research. The project carried out follow the direction by studying on treatment of diabetic vascular complication by classical prescription.Disscused some problems about cell culture and serum pharmacology.Investigated literatures about diabetic endothelium dysfunction and found the hinge and creative point of this project. Endothelin-1 (ET-1) and nitric oxide (NO) were representative chemical targets of endothelium dysfunction. It was reported that blood stasis was well connected with endothelium dysfunction.2.2. Experimental studyThe objective of experimental study was to observe Sprague Dawley (SD) rats serum containing different dosage Modified Taohe Chengqi Decoction and its composition of prescription, to find out their effect on diabetes vascular endothelial cell. Three different dosage Modified Taohe Chengqi Decoction (MTCD) were high dosage (hMTCD), middle dosage (mMTCD) and low dosage (1MTCD). Its composition of prescription contained Huoxue (HX) group, Xieretongxia (XT) group, Yiqiyangyin (YY) group, Yuanfang (YF) group. First, obtained SD rats serum containing different decoction, then observed the serum and found out its effect on diabetes human umbilical vein endothelial cell (HUVEC). The comparison was rosiglitazone (RSG). The whole experiment contained three part: Part one, culture HUVEC and research the method to gain diabetes HUVEC;Part two, obtain SD rats serum containing drugs;Part three, research the effect of Modified Taohe Chengqi Decoction and its composition of prescription on diabetes HUVEC (mensurate some targets).2.2.1. Culture HUVEC and gain diabetes HUVECAt present, HUVEC was used in most of researching vascular endothelial cell internationally. The VEC304 was confirmed by authoritative ATCC that its aberrance made it have no characteristic of vascular endothelial cell. In our experiment, we adopted HUVEC via primary culture. There were lots of research about culture HUVEC, but it was absolutely necessarily that we should research its culture condition because of the culture difficulty and no authoritative culture project. The research contents were following: Observed the different growth of HUVEC between normal culture condition (without high consistency glucose and insulin) and diabetes culture condition (high consistency glucose and insulin). The conclusion was that high consistency glucose (20mmol/L) and high consistency insulin (300mU/L) made the diabetes HUVEC.2.2.2. Obtain SD rats serum containing drugsWe obtained SD rats serum (normal serum and serum contain drugs), researched how much serum and how many drugs were fit for HUVEC. The research was based on the limitation of serum pharmacology. Firstly, culture cells were prone to stop growing, be polluted or die if the culture circumstance was bad for them. Both SD rats surm and drugs impacted the growth of culture HUVEC. Serum come from different species hurt the cells. Too many drugs did harm to the culture cells and few drugs make no effect on the morbid cells. Secondly, the mode that culture cells contact serum containing drugs was not as sameas it in body. It was not difficult to understand that the cells in body were not all dip in blood and the intensity of drugs was different. Thirdly, the method make diabetes HUVEC was not unified. So we did some advance experiments before the formal ones. The advance experiments told us that the proportion of SD rats serum adding into cell culture medium was 20 percent and the drugs dose of SD rats was as same as the equivalent dosage.2.2.3. Effect of modified taohe chengqi decoction and its composition of prescription on the diabetes HUVECIt was reported that blood stasis was well connected with endothelium dysfunction and activating blood circulation to dissipate blood stasis was main function of Modified Taohe Chengqi Decoction, so we selected three targets for researching as following: tissue plasminogen activator (tPA), plasminogen activator inhibitor l(PAI-l)and intercellular adhesion molecule(ICAM-1). The research content contained effect of modified taohe chengqi decoction and its composition of prescription on the change of ET-1, NO, tPA, PAI-1 and ICAM-1 in HUVEC culture medium.Effect of Modified Taohe Chengqi Decoction and its composition of prescription on the change of ET-1 and NO in HUVEC culture medium: All decoction groups could restrain the secretion of ET-1 (P<0. 05) . mMTCD group and 1MTCD group restrained the secretion of ET-1 are more effectual than RSG group (P<0. 05) o mMTCD group restrained the secretion of ET-1 were less than 1MTCD group, but it was no signification difference compare with 1MTCD (P >0. 05) . YY group restrained the secretion of ET-1 were less than model group, but it was no signification difference compare with mMTCD (P>0. 05) . HX group’ s effect to restrain ET-1 was the best, there had signification difference compare with RSG group (P<0. 01) .Compared with model group, all groups could promote the secretion of NO (P<0. 01) except for YY group (P >0. 05) .The level of NO in mMTCD group, hMTCD group, HX group, XT group, YF group and RSG group were all higher than normal group (P<0. 01) . The level of NO in hMTCD group was highest, there had signification difference compare with RSG group (P<0.01) .Effect of Modified Taohe Chengqi Decoction and its composition ofprescription on the change of tPA and PAI-1 in HUVEC culture medium: The levelof tPA in RSG group was higher than model group (P < 0.05) and there hadsignification difference compare with hMTCD group, mMTCD group and 1MTCD group(P<0. 05) .The level of tPA had signification difference among hMTCD group.mMTCD group and 1MTCD group(P<0.01). The level of tPA had signification difference between composition group and model grouop (P<0. 01) .The effect of HX group was the best among all decoction groups and approached RSG group (P>0. 05) . The effect of YF group was in the second place among all composition groups, it was good for promoting the level of tPA. The level of PAI-1 in model group was higher than that in normal group (P<0. 01) , hMTCD group, mMTCD group, 1MTCD group and RSG group could restrain the rise trend (P<0. 01) and RSG group was the best one to restrain the rise trend. The level of PAI-1 had signification difference among hMTCD group, mMTCD group and 1MTCD group(P <0. 01). The noteworthiness were two points: the effect of HX group was better than RSG group;the level of PAI-1 in YY group was higher than model group (P<0.05).Effect of Modified Taohe Chengqi Decoction and its composition of prescription on the change of ICAM-1 in HUVEC culture medium: The level of ICAM-1 in model group was higher than other groups (P<0. 01) .hMTCD group> 1MTCD group could decrease ICAM-1 lightly and mMTCD group could promote ICAM-1 lightly, but none of them had signification difference(P>0. 05). HX group, XT group and YF group could decrease ICAM-1 (P<0. 05). YY group could promote ICAM-1.3. ConclusionConclusion about ET-1 and NO: All groups serum containing drugs could decrease ET-1 in diabetes HUVEC culture medium, but YY group did not have signification difference. HX group was the best one to decrease ET-1. The turns of effect was following: HX group > mMTCD group > YF group > 1MTCD group > XT group > RSG group > hMTCD group. All groups serum containing drugs could promote NO in diabetes HUVEC culture medium, but YY group did not have signification difference. hMTCD group could promote NO greatly. The turns of effect was following: hMTCD group > HX group > YF group > RSG group > XT group > mMTCD group> 1MTCD group.Conclusion about tPA and PAI-1: Most of serum containing drugs could promote tPA in diabetes HUVEC culture medium, but YY group did not have signification difference. The effect of HX group was the best among all decoction groups and approached RSG group. The effect of YF group was in the second place among all composition groups, it was good for promoting the level of tPA. The turns of effect was following: RSG group > HX group > hMTCD > YF group > mMTCD group > XT group. Most of serum containing drugs coulddecrease PAI-1 in diabetes HUVEC culture medium. The effect of HX group was better than RSG group. The turns of effect was following: HX group > RSG group > YF group > XT group > hMTCD group > mMTCD group > 1MTCD group.Conclusion about ICAM-1: hMTCD group, mMTCD group and 1MTCD group could not decrease ICAM-1 in diabetes HUVEC culture medium. Only RSG group, HX group, XT group, YF group could decrease it. The turns of effect was following: RSG group > HX group > YF group > XT group.In a word, RSG was good for diabetes endothelium dysfunction, in especial for tPA, PAI-K ICAM-1. The effect of RSG was not so good for ET-1 and NO. HX group could improve diabetes endothelium dysfunction well, but the effect for tPA, PAI-K ICAM-1 was not so good as RSG. It was unexpected that RSG and HX group seem can complement each other in the effect of diabetes endothelium dysfunction. This conclusion maybe good for studying ETDF in the future.更多还原