防风有效成份抗动脉粥样硬化炎性反应的实验研究
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摘要
动脉粥样硬化(AS)是许多心血管病与脑卒中的诱发因素，其病因复杂；从19世纪中叶到20世纪70年代，Virehow、Dugwid等分别提出了动脉炎性损伤学说、脂源性假说，从不同角度解释了AS的形成机制。但进入20世纪90年代，对于动脉粥样硬化病理生理机制的理解发生了重大改变，超越了动脉粥样硬化是血管进行性阻塞这一传统观念的局限，Ross以新的论据在他损伤反应学说的基础上，明确提出：动脉粥样硬化是一种炎症性疾病而不是单纯的由于脂质沉积所致。随着对动脉粥样硬化发生机制的深入研究，炎症在AS形成中的作用已被越来越多的资料所证实，可以认为AS是以慢性炎症过程为基础，炎性细胞因子、血管内皮细胞、血管平滑肌细胞及免疫因素等环节共同参与的综合发生发展过程，为AS治疗学的进步及未来关于AS的理论研究提出了新思路。
在中医学中，有关AS的临床辩证分别归属于心悸、胸痹心痛，治疗多采用活血化瘀等具有综合调节功效的方剂，治疗过程中体现了中药多靶点、多环节、整体调节的作用特点，与AS发生机制的理论变革有密切相关性。防风是具有疏风解表、辛温通阳作用的风药，新近研究表明，风药具有活血化瘀的功效，开展防风抗AS的研究，有助于丰富中医风药活血的理论内涵。现代药理研究表明，防风具有显著的抗炎活性，进一步研究发现，升麻苷和5-O甲基维斯阿米醇苷是防风主要的抗炎成分，现已作为防风药材质量标准的定性、定量标准。因此，本研究项目从中西医结合的理论出发，根据AS的发生发展规律，针对炎性因子、血管内皮细胞、平滑肌细胞等综合因素在AS发生中的相互作用，研究升麻苷、5-O-甲基维斯阿米醇苷对AS炎性反应的影响，为拓展中医风药的临床应用提供科学依据。
根据Ross的AS炎症假说，ox-LDL等诱发因素引起血管内皮损伤或功能障碍，刺激内皮细胞、巨噬细胞等释放IL-6、TNF-α等炎性因子，这些炎症因子继而刺激平滑肌细胞的增殖，最终导致AS斑块的形成与发展。本研究采用体外ox-LDL损伤血管内皮细胞模型、促黏附模型及TNF-α刺激平滑肌细胞增殖模型，探讨升麻苷和5-O-甲基维斯阿米醇苷抗AS炎性反应，研究工作主要包括以下三个方向：
1升麻苷、5-O-甲基维斯阿米醇苷对于ox-LDL所致心肌微血管内皮细胞生存率和分泌炎性细胞因子的影响
参照Nishida等的方法，建立了大鼠乳鼠心肌微血管内皮细胞体外培养方法。采用免疫荧光法对心肌微血管内皮细胞进行了鉴定，并用细胞计数法绘制了细胞的生长曲线。在掌握乳鼠心肌微血管内皮细胞的生长特性后，选用第三代内皮细胞，进行下一步实验。通过测定升麻苷与5-O甲基维斯阿米醇苷对内皮细胞活性的影响，确定升麻苷和5-O甲基维斯阿米醇苷的实验浓度；通过测定ox-LDL对内皮细胞存活率的影响，确定ox-LDL造成内皮细胞损伤的造模浓度；然后通过MTT法测定了升麻苷与5-O甲基维斯阿米醇苷预处理后，对ox-LDL诱导血管内皮细胞损伤的影响，并分别采用ELISA与放免法的方法，测定了升麻苷与5-O甲基维斯阿米醇苷对ox-LDL致心肌微血管内皮细胞分泌炎性细胞因子的影响。
实验结果如下：
①免疫荧光法鉴定所培养的乳鼠心肌微血管内皮细胞因子Ⅷ相关抗原阳性，结合细胞呈单层铺路石样生长的形态学特征，可确定培养的细胞为心肌微血管内皮细胞；
②根据绘制细胞生长曲线，以一定细胞密度铺板(1×10~5·mL~(-1))，在铺板后24h-72h之间细胞处于指数增长期，是进行实验的最佳时期；
③采用MTT法测定升麻苷与5-O甲基维斯阿米醇苷对内皮细胞活性的影响，结果发现升麻苷与5-O甲基维斯阿米醇苷毒性较小，测不出对内皮细胞的最大无毒浓度，因此选择50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1)三个浓度作为升麻苷和5-O甲基维斯阿米醇苷的实验浓度；
④采用MTT法测定ox-LDL对内皮细胞存活率的影响，确定在MDA含量为3ng·ml~(-1)的ox-LDL作用下，内皮细胞损伤较明显，可建立稳定的内皮细胞损伤模型，故选用此浓度作为ox-LDL的造模浓度。
⑤采用MTT法测定升麻苷与5-O甲基维斯阿米醇苷预处理后，对ox-LDL导致的血管内皮细胞生存率的影响，结果表明MDA含量为3nmol·ml~(-1)的ox-LDL能够明显降低内皮细胞存活率，使模型组细胞吸光值明显下降，与空白组相比差异极显著(P＜0.01)。升麻苷200μg·ml~(-1)，5-O甲基维斯阿米醇苷50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1)均能明显抑制ox-LDL引起的内皮细胞损伤，与ox-LDL组相比差异极显著(P＜0.01)。升麻苷50μg·ml~(-1)、100μg·ml~(-1)具有减轻ox-LDL损伤作用的趋势，但与ox-LDL组相比，差异不显著。
⑥采用ox-LDL(MDA含量为3nmol·ml~(-1)造成血管内皮细胞损伤，升麻苷与5-O甲基维斯阿米醇苷(浓度均分别为50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1)预处理，观察这两种药物对ox-LDL诱导血管内皮细胞炎性因子释放的影响，结果表明：ox-LDL能够明显增加内皮细胞IL-6、TNF-α的分泌量，与空白组相比差异极显著(P＜0.01)。升麻苷和5-O甲基维斯阿米醇苷100μg·ml~(-1)、200μg·ml~(-1)组均能明显抑制ox-LDL引起的内皮细胞分泌IL-6，与模型组相比差异显著(P＜0.05)：升麻苷三个剂量组和5-O甲基维斯阿米醇苷200μg·ml~(-1)组能明显抑制Ox-LDL引起的内皮细胞分泌TNF-α，与模型组相比差异极显著(P＜0.01)。
2升麻苷、5-O-甲基维斯阿米醇苷对于心肌微血管内皮细胞与单核细胞黏附的影响
参照Nishida等的方法，建立了大鼠乳鼠心肌微血管内皮细胞体外培养方法，采用第三代内皮细胞，观察升麻苷与5-O甲基维斯阿米醇苷对ox-LDL诱导血管内皮细胞与单核细胞黏附性的影响。
实验结果如下：
采用MDA含量为3nmol·ml~(-1)的ox-LDL增加血管内皮细胞对单核细胞的黏附性，并用升麻苷与5-O甲基维斯阿米醇苷(浓度均分别为50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1))预处理，观察这两种药物对ox-LDL诱导血管内皮细胞黏附性增加的影响，结果表明：ox-LDL能明显增加单核细胞与内皮细胞的黏附，与空白组相比，差异极显著(P＜0.01)。升麻苷50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1)，5-O甲基维斯阿米醇苷200μg·ml~(-1)均能明显抑制ox-LDL引起的单核细胞与内皮细胞的黏附，与模型组相比差异极显著(P＜0.01)。5-O甲基维斯阿米醇苷50μg·ml~(-1)、100μg·ml~(-1)能明显抑制ox-LDL引起的单核细胞与内皮细胞的黏附，与ox-LDL组相比差异显著(P＜0.05)。
3升麻苷、5-O-甲基维斯阿米醇苷对于TNF-α所致大鼠胸主动脉平滑肌细胞增殖的影响
采用组织贴块法，建立了大鼠胸主动脉平滑肌细胞原代培养方法。采用免疫化学方法对该细胞进行鉴定，并用细胞计数法绘制细胞生长曲线。根据细胞的生长特性，选用第三代平滑肌细胞，进行下一步实验。首先确定这两种药物的最大无毒浓度及实验浓度：然后确定TNF-α能够刺激平滑肌细胞的促增殖的造模浓度；通过MTT法测定升麻苷、5-O-甲基维斯阿米醇苷对TNF-α刺激平滑肌细胞增殖的影响；用流式细胞仪测定这两种药物对平滑肌细胞增殖周期的影响。
实验结果如下：
①采用SABC法对平滑肌细胞进行肌动蛋白免疫组化染色，99％的细胞染色阳性，再结合细胞呈典型的“峰谷”状排列的形态学特征，可确定培养的细胞为胸主动脉平滑肌细胞；
②通过绘制细胞生长曲线，确定以一定密度(1x10~5·mL~(-1))铺板后，24h-96h为该细胞的指数增长期，选择这段时间进行实验；
③通过观察不同浓度升麻苷、5-O-甲基维斯阿米醇苷对平滑肌细胞活性的影响，发现这两种药物毒性较小，测不出对平滑肌细胞的最大无毒浓度，因此选择50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1)三个浓度作为升麻苷和5-O甲基维斯阿米醇苷的实验浓度；
④通过观察TNF-α对平滑肌细胞增殖的影响，发现5ng·ml~(-1)的TNF-α促增殖作用最明显，因此选择5ng·ml~(-1)作为TNF-α的造模浓度；
⑤采用5ng·ml~(-1)的TNF-α诱导血管平滑肌细胞增殖，用升麻苷与5-O甲基维斯阿米醇苷(浓度均分别为50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1))进行处理，观察这两种药物对TNF-α诱导的平滑肌细胞增殖的作用，结果表明：5ng·ml~(-1)TNF-α能明显刺激平滑肌细胞增殖，与空白组相比差异极显著(P＜0.01)。升麻苷和5-O甲基维斯阿米醇苷三个剂量组均能明显抑制TNF-α引起的平滑肌细胞增殖，与TNF-O组相比差异极显著(P＜0.01)；
⑥采用5ng·ml~(-1)的TNF-α诱导血管平滑肌细胞增殖，用升麻苷与5-O甲基维斯阿米醇苷(浓度均分别为50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1))进行处理，观察这两种药物对平滑肌细胞周期的影响。结果表明5ng·ml~(-1)TNF-α能明显增加平滑肌细胞G2期与S期比例，与空白组相比差异极显著(P＜0.01)。升麻苷和5-O甲基维斯阿米醇苷均使G1／G1期细胞比例增加，S期、G2／M期细胞比例减少，与TNF-α组相比差异极显著(P＜0.01)。
从以上结果可以看出，升麻苷与5-O甲基维斯阿米醇苷具有抑制ox-LDL刺激血管内皮细胞引起的损伤及炎性因子释放、抑制ox-LDL引起的单核细胞与内皮细胞黏附性增高、抑制TNF-α引起的平滑肌细胞增殖的作用，总的来说，升麻苷与5-O甲基维斯阿米醇苷具有抗AS炎性反应的作用，为升麻苷与5-O甲基维斯阿米醇苷发展成为抗AS的药物，提供了实验依据。
Atherosclerosis (AS) is characterized by complicate mechanisms. Since 19 century, theories concerning pathological mechanisms were raised. The most important one is inflammatory hypothesis by Ross that AS is a synthetic process caused by common actions of cytokine, vascular endothelial cell, vascular smooth muscle cell and immunity factors.
Based on the theory of TCM, Fangfeng is a wind-dispersing medicine, which has the effect of activating blood circulation to dissipate blood stasis. Modern Pharmacology research discovered that Fangfeng has significant activityof anti-inflammatory, and the primary anti-inflammatory component is prim-O-glucosyl-cimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con. In this paper we investigate the effect of prim-O-glucosylcimifugin and 4'-O-β-D-gluco syl-5-O-methylvisamminol con on the inflammatory reaction in AS in order to provide the scientific evidence for wide clinical application.
According to the inflammatory hypothesis of Ross, the factor like oxidizedlow density lipoprotein (ox-LDL) can cause injury or dysfunction of vascular endothelium, stimulate the release of inflammatory factor like IL-6, TNF-αin endothelial cell and macrophage,which can stimulate the proliferation of smoothmuscle cell subsequently, and result in the formation and development of AS plaque finally. We employed in vitro vascular endothelial cell injured model,adhesion model and smooth muscle cell proliferation model to investigate the anti-inflammatory effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-met-hylvisamminol con. Our research work includes three parts as follows: 1 The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisa-mminol con on the survival rate and secretion of cytokines of cardiac microvas-cular endothelial cell.
Referred to Nishida's method, the primary culture method of rat cardiac microvascular endothelial cell (rCMEC) in vitro was established. The rCMEC was identified by immumofluorescence method, and its growth curve was drawed by cytometry. The third generation of cells was adopted in our experiment. By determining the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the activity of EC, the concentration of two medicines was decided. By determining the effect of ox-LDL on the survival rate of EC, it's concentration to make the EC injured was decided. And then the effect ofprim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamm-inol con onthe survival rate and secretion of cytokines of EC was investigated by MTT,ELISA and radioimmunoassay respectively.
The results were as follows:
1．The cultured cell was identified to be rCMEC by immumofluorescence method and morphology observation, because of its positive dyeing of factorⅧrelated antigen and its appearance like paving stones.
2．Based on the growth curve, the best time of experiment was limited in the period between 24h to 72h after inoculating cell into culture plates in the density of 1×10~5mL~(-1) .
3．The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methy-lvisamminol con on the activity of EC was determined by MTT. It was foundthat the maximum of no toxicity concentration can not be determined because of the low toxicity of these two medicines on EC. Thus, 50μg·ml~(-1) ,100μg·ml~1~(-1) , 200μgml~(-1) were chosen as the concentration of prim-O-glucosylcimifugin and 4 '-O-β-D-glucosyl-5-O-methylvisamminol con in experiment.
4．The effect of ox-LDL on the survival rate of EC was determined by MTT. The ox-LDL of 3ng·ml~(-1) DA content was chosen to injure EC in experiment.
5．After pretreatment with prim-O-glucosylcimifugin(50,100,200μg·ml~(-1) 4'-O-β-D-glucosyl-5-O-methylvisamminol con(50,100,200μgml~(-1) 24 hours, the rCMEC was incubated with ox-LDL for another 24 hours, then the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the survival rate of EC injured by ox-LDL was determined by MTT. The res-ult indicated that the survival rate of EC was reduced by ox-LDL of 3ng·ml~(-1) as MDA content significantly, the model group had great significant difference (P<0．01) compared with control. 200μg·ml~(-1) group of prim-O-glucosyl-cimifugin, 50μg·ml~(-1) , 100μg·ml~(-1) , 200μg·ml~(-1) groups of 4'-O-β-D-glucosyl-5-O-methylvisam-minol con can inhibit EC injury made by ox-LDL, had great significant differ-ence(P<0．01) compared with model group. 50μg·ml,100μg·ml~(-1) groups of prim-O-glucosylcimifugin had the tendency to lighten ox-LDL injury, but had no significant difference compared with model group.
6．After pretreatment with prim-O-glucosylcimifugin(50, 100, 200μg·ml~(-1) ) and 4'-O-β-D-glucosyl-5-O-methylvisamminol con(50, 100, 200μg·ml) for 24 hours, the rCMEC was incubated with ox-LDL for another 24 hours, then the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the secretion of cytokines of EC was determined. The result indicated that ox-LDL can increase the secretion of IL-6 and TNF-αof EC, model group had great significant difference (P<0．01) compared with control. 100μg·ml~(-1) ,200μg·ml~(-1) groups of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the secretion of IL-6, and had significant difference (P<0．05) compared with model group. The three dose groups of prim-O-glucosylcimifugin and 200μg·ml~(-1) group of 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the secretion of TNF-α, and had great significant difference (P<0．01) compared with model group. 2 The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisa-mminol con on the adhesion of monocyte to rCMEC
Referred to Nishida's method, the primary culture method of rCMEC in vitro was established. The third generation EC was adopted to investigate the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the adhesion of monocyte to rCMEC induced by ox-LDL.
The result was as follows:
ox-LDL of 3ng·ml~(-1) as MDA content can increase the adhesion of monocyte to EC, which has great significant difference (P<0．01) compared with control. 50μg·ml~(-1) ,100μg·ml~(-1) , 200μg·ml~(-1) groups of prim-O-glucosylcimifugin, 200μg·ml~(-1) group of 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the adhesion of monocyte to EC, which has great significant difference(P<0．01) compared with model group. 50μg·ml~(-1) , 100μg·ml~(-1) groups of 4'-O-β-D-glucosyl-5-O-methylvisamminol con inhibit the adhesion, which has significant difference (P<0．05) compared with model group. 3 The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisa-mminol con on the proliferation of smooth muscle cell stimulated by TNF-α
The primary culture method of smooth muscle cell (SMC) was established by attachment-block. The SMC was identiflcated by immunochemistry method, and the growth curve was drawed by cytometry. The third generation of SMC was adopted in our experiment. By determining the effect of prim-O-glucosylc-imifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the activity of SMC , the concentration of the two medicine was decided. By determining the effect of TNF-αon the proliferation of SMC, it's concentration to stimulate the SMC proliferate was dicided. And then the effect of prim-O-glucosylcimif-ugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the proliferation and cell cycle of SMC was investigated by MTT and flow cytometry respectively.
The result was as follows:
1 The cultured cell was identificated to be SMC by immunochemistry method and morphology observation, because of its positive dyeing ofα-smooth actin and its appearance like peak valley.
2．Based on the growth curve, the best time of experiment was limited in the period between 24h to 96h after inoculating cell into culture plates in the density of 1×10~5mL~(-1) .
3．The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methy-lvisamminol con on the activity of SMC was determined by MTT. It was found that the maximum of no toxicity concentration can not be determined because of the low toxicity of these two medicines on SMC. Thus, 50μg·ml~(-1) , 100μ·gml~(-1) , 200μg·ml~(-1) were chosen as the concentration of prim-O-glucosylcimi-fugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con in experiment.
4．The effect of TNF-αon the proliferation of SMC was determined by MTT. The concentration of 5ng·ml~(-1) was chosen to stimulate SMC in experiment.
5 The SMC was stimulated to proliferate by TNF-αof 5ng·ml~(-1) , and incubated with prim-O-glucosylcimifugin (50μg·ml~(-1) , 100μg·ml~(-1) , 200μg·ml~(-1) ) and 4'-O-β-D-glucosyl-5-O-methylvisamminol con (50μg·ml~(-1) , 100μg·ml~(-1) , 200μg·ml~(-1) ), the effect of these two medicines was investigated. The result indicate-d that TNF-αof 5ng·ml~(-1) can stimulate the proliferation of SMC, which has great significant difference (P<0．01) compared with control. The three dose groups of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisammin-ol con can inhibit the proliferation of SMC, which has great significantdifference(P<0．01) compared with model group.
6 The SMC was stimulated to proliferate by TNF-αof 5ng·ml~(-1) , and incubated with prim-O-glucosylcimifugin(50μg·ml~(-1) ,100μg·ml~(-1) , 200μg·ml~(-1) ) and 4'-O-β-D-glucosyl-5-O-methylvisamminol con(50μg·ml~(-1) ,100μg·ml~(-1) ,200μg·ml~(-1) ),the effect of these two medicines on the cell cycle was investigated. The result indicated that TNF-αof 5ng·ml~(-1) can increase the proportion of G2 phase and S phase in cell cycle, which has great significant difference (P<0．01) compared with control. The three dose groups of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con can increase the proportion of G0/G1 phase, decrease the proportion of S phase and G2/M phase, which havegreat significant difference(P<0．01) compared with model group.
It can be concluded that prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the injury and secretion of inflammatorycytokines of EC, the adhesion of monocytes to EC and the proliferation of SMC stimulated by TNF-α. In conclusion, prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con has the effect of anti-inflammatory reaction in AS, and provides the scientific evidence for wide clinical application.
在中医学中，有关AS的临床辩证分别归属于心悸、胸痹心痛，治疗多采用活血化瘀等具有综合调节功效的方剂，治疗过程中体现了中药多靶点、多环节、整体调节的作用特点，与AS发生机制的理论变革有密切相关性。防风是具有疏风解表、辛温通阳作用的风药，新近研究表明，风药具有活血化瘀的功效，开展防风抗AS的研究，有助于丰富中医风药活血的理论内涵。现代药理研究表明，防风具有显著的抗炎活性，进一步研究发现，升麻苷和5-O甲基维斯阿米醇苷是防风主要的抗炎成分，现已作为防风药材质量标准的定性、定量标准。因此，本研究项目从中西医结合的理论出发，根据AS的发生发展规律，针对炎性因子、血管内皮细胞、平滑肌细胞等综合因素在AS发生中的相互作用，研究升麻苷、5-O-甲基维斯阿米醇苷对AS炎性反应的影响，为拓展中医风药的临床应用提供科学依据。
根据Ross的AS炎症假说，ox-LDL等诱发因素引起血管内皮损伤或功能障碍，刺激内皮细胞、巨噬细胞等释放IL-6、TNF-α等炎性因子，这些炎症因子继而刺激平滑肌细胞的增殖，最终导致AS斑块的形成与发展。本研究采用体外ox-LDL损伤血管内皮细胞模型、促黏附模型及TNF-α刺激平滑肌细胞增殖模型，探讨升麻苷和5-O-甲基维斯阿米醇苷抗AS炎性反应，研究工作主要包括以下三个方向：
1升麻苷、5-O-甲基维斯阿米醇苷对于ox-LDL所致心肌微血管内皮细胞生存率和分泌炎性细胞因子的影响
参照Nishida等的方法，建立了大鼠乳鼠心肌微血管内皮细胞体外培养方法。采用免疫荧光法对心肌微血管内皮细胞进行了鉴定，并用细胞计数法绘制了细胞的生长曲线。在掌握乳鼠心肌微血管内皮细胞的生长特性后，选用第三代内皮细胞，进行下一步实验。通过测定升麻苷与5-O甲基维斯阿米醇苷对内皮细胞活性的影响，确定升麻苷和5-O甲基维斯阿米醇苷的实验浓度；通过测定ox-LDL对内皮细胞存活率的影响，确定ox-LDL造成内皮细胞损伤的造模浓度；然后通过MTT法测定了升麻苷与5-O甲基维斯阿米醇苷预处理后，对ox-LDL诱导血管内皮细胞损伤的影响，并分别采用ELISA与放免法的方法，测定了升麻苷与5-O甲基维斯阿米醇苷对ox-LDL致心肌微血管内皮细胞分泌炎性细胞因子的影响。
实验结果如下：
①免疫荧光法鉴定所培养的乳鼠心肌微血管内皮细胞因子Ⅷ相关抗原阳性，结合细胞呈单层铺路石样生长的形态学特征，可确定培养的细胞为心肌微血管内皮细胞；
②根据绘制细胞生长曲线，以一定细胞密度铺板(1×10~5·mL~(-1))，在铺板后24h-72h之间细胞处于指数增长期，是进行实验的最佳时期；
③采用MTT法测定升麻苷与5-O甲基维斯阿米醇苷对内皮细胞活性的影响，结果发现升麻苷与5-O甲基维斯阿米醇苷毒性较小，测不出对内皮细胞的最大无毒浓度，因此选择50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1)三个浓度作为升麻苷和5-O甲基维斯阿米醇苷的实验浓度；
④采用MTT法测定ox-LDL对内皮细胞存活率的影响，确定在MDA含量为3ng·ml~(-1)的ox-LDL作用下，内皮细胞损伤较明显，可建立稳定的内皮细胞损伤模型，故选用此浓度作为ox-LDL的造模浓度。
⑤采用MTT法测定升麻苷与5-O甲基维斯阿米醇苷预处理后，对ox-LDL导致的血管内皮细胞生存率的影响，结果表明MDA含量为3nmol·ml~(-1)的ox-LDL能够明显降低内皮细胞存活率，使模型组细胞吸光值明显下降，与空白组相比差异极显著(P＜0.01)。升麻苷200μg·ml~(-1)，5-O甲基维斯阿米醇苷50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1)均能明显抑制ox-LDL引起的内皮细胞损伤，与ox-LDL组相比差异极显著(P＜0.01)。升麻苷50μg·ml~(-1)、100μg·ml~(-1)具有减轻ox-LDL损伤作用的趋势，但与ox-LDL组相比，差异不显著。
⑥采用ox-LDL(MDA含量为3nmol·ml~(-1)造成血管内皮细胞损伤，升麻苷与5-O甲基维斯阿米醇苷(浓度均分别为50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1)预处理，观察这两种药物对ox-LDL诱导血管内皮细胞炎性因子释放的影响，结果表明：ox-LDL能够明显增加内皮细胞IL-6、TNF-α的分泌量，与空白组相比差异极显著(P＜0.01)。升麻苷和5-O甲基维斯阿米醇苷100μg·ml~(-1)、200μg·ml~(-1)组均能明显抑制ox-LDL引起的内皮细胞分泌IL-6，与模型组相比差异显著(P＜0.05)：升麻苷三个剂量组和5-O甲基维斯阿米醇苷200μg·ml~(-1)组能明显抑制Ox-LDL引起的内皮细胞分泌TNF-α，与模型组相比差异极显著(P＜0.01)。
2升麻苷、5-O-甲基维斯阿米醇苷对于心肌微血管内皮细胞与单核细胞黏附的影响
参照Nishida等的方法，建立了大鼠乳鼠心肌微血管内皮细胞体外培养方法，采用第三代内皮细胞，观察升麻苷与5-O甲基维斯阿米醇苷对ox-LDL诱导血管内皮细胞与单核细胞黏附性的影响。
实验结果如下：
采用MDA含量为3nmol·ml~(-1)的ox-LDL增加血管内皮细胞对单核细胞的黏附性，并用升麻苷与5-O甲基维斯阿米醇苷(浓度均分别为50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1))预处理，观察这两种药物对ox-LDL诱导血管内皮细胞黏附性增加的影响，结果表明：ox-LDL能明显增加单核细胞与内皮细胞的黏附，与空白组相比，差异极显著(P＜0.01)。升麻苷50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1)，5-O甲基维斯阿米醇苷200μg·ml~(-1)均能明显抑制ox-LDL引起的单核细胞与内皮细胞的黏附，与模型组相比差异极显著(P＜0.01)。5-O甲基维斯阿米醇苷50μg·ml~(-1)、100μg·ml~(-1)能明显抑制ox-LDL引起的单核细胞与内皮细胞的黏附，与ox-LDL组相比差异显著(P＜0.05)。
3升麻苷、5-O-甲基维斯阿米醇苷对于TNF-α所致大鼠胸主动脉平滑肌细胞增殖的影响
采用组织贴块法，建立了大鼠胸主动脉平滑肌细胞原代培养方法。采用免疫化学方法对该细胞进行鉴定，并用细胞计数法绘制细胞生长曲线。根据细胞的生长特性，选用第三代平滑肌细胞，进行下一步实验。首先确定这两种药物的最大无毒浓度及实验浓度：然后确定TNF-α能够刺激平滑肌细胞的促增殖的造模浓度；通过MTT法测定升麻苷、5-O-甲基维斯阿米醇苷对TNF-α刺激平滑肌细胞增殖的影响；用流式细胞仪测定这两种药物对平滑肌细胞增殖周期的影响。
实验结果如下：
①采用SABC法对平滑肌细胞进行肌动蛋白免疫组化染色，99％的细胞染色阳性，再结合细胞呈典型的“峰谷”状排列的形态学特征，可确定培养的细胞为胸主动脉平滑肌细胞；
②通过绘制细胞生长曲线，确定以一定密度(1x10~5·mL~(-1))铺板后，24h-96h为该细胞的指数增长期，选择这段时间进行实验；
③通过观察不同浓度升麻苷、5-O-甲基维斯阿米醇苷对平滑肌细胞活性的影响，发现这两种药物毒性较小，测不出对平滑肌细胞的最大无毒浓度，因此选择50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1)三个浓度作为升麻苷和5-O甲基维斯阿米醇苷的实验浓度；
④通过观察TNF-α对平滑肌细胞增殖的影响，发现5ng·ml~(-1)的TNF-α促增殖作用最明显，因此选择5ng·ml~(-1)作为TNF-α的造模浓度；
⑤采用5ng·ml~(-1)的TNF-α诱导血管平滑肌细胞增殖，用升麻苷与5-O甲基维斯阿米醇苷(浓度均分别为50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1))进行处理，观察这两种药物对TNF-α诱导的平滑肌细胞增殖的作用，结果表明：5ng·ml~(-1)TNF-α能明显刺激平滑肌细胞增殖，与空白组相比差异极显著(P＜0.01)。升麻苷和5-O甲基维斯阿米醇苷三个剂量组均能明显抑制TNF-α引起的平滑肌细胞增殖，与TNF-O组相比差异极显著(P＜0.01)；
⑥采用5ng·ml~(-1)的TNF-α诱导血管平滑肌细胞增殖，用升麻苷与5-O甲基维斯阿米醇苷(浓度均分别为50μg·ml~(-1)、100μg·ml~(-1)、200μg·ml~(-1))进行处理，观察这两种药物对平滑肌细胞周期的影响。结果表明5ng·ml~(-1)TNF-α能明显增加平滑肌细胞G2期与S期比例，与空白组相比差异极显著(P＜0.01)。升麻苷和5-O甲基维斯阿米醇苷均使G1／G1期细胞比例增加，S期、G2／M期细胞比例减少，与TNF-α组相比差异极显著(P＜0.01)。
从以上结果可以看出，升麻苷与5-O甲基维斯阿米醇苷具有抑制ox-LDL刺激血管内皮细胞引起的损伤及炎性因子释放、抑制ox-LDL引起的单核细胞与内皮细胞黏附性增高、抑制TNF-α引起的平滑肌细胞增殖的作用，总的来说，升麻苷与5-O甲基维斯阿米醇苷具有抗AS炎性反应的作用，为升麻苷与5-O甲基维斯阿米醇苷发展成为抗AS的药物，提供了实验依据。
Atherosclerosis (AS) is characterized by complicate mechanisms. Since 19 century, theories concerning pathological mechanisms were raised. The most important one is inflammatory hypothesis by Ross that AS is a synthetic process caused by common actions of cytokine, vascular endothelial cell, vascular smooth muscle cell and immunity factors.
Based on the theory of TCM, Fangfeng is a wind-dispersing medicine, which has the effect of activating blood circulation to dissipate blood stasis. Modern Pharmacology research discovered that Fangfeng has significant activityof anti-inflammatory, and the primary anti-inflammatory component is prim-O-glucosyl-cimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con. In this paper we investigate the effect of prim-O-glucosylcimifugin and 4'-O-β-D-gluco syl-5-O-methylvisamminol con on the inflammatory reaction in AS in order to provide the scientific evidence for wide clinical application.
According to the inflammatory hypothesis of Ross, the factor like oxidizedlow density lipoprotein (ox-LDL) can cause injury or dysfunction of vascular endothelium, stimulate the release of inflammatory factor like IL-6, TNF-αin endothelial cell and macrophage,which can stimulate the proliferation of smoothmuscle cell subsequently, and result in the formation and development of AS plaque finally. We employed in vitro vascular endothelial cell injured model,adhesion model and smooth muscle cell proliferation model to investigate the anti-inflammatory effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-met-hylvisamminol con. Our research work includes three parts as follows: 1 The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisa-mminol con on the survival rate and secretion of cytokines of cardiac microvas-cular endothelial cell.
Referred to Nishida's method, the primary culture method of rat cardiac microvascular endothelial cell (rCMEC) in vitro was established. The rCMEC was identified by immumofluorescence method, and its growth curve was drawed by cytometry. The third generation of cells was adopted in our experiment. By determining the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the activity of EC, the concentration of two medicines was decided. By determining the effect of ox-LDL on the survival rate of EC, it's concentration to make the EC injured was decided. And then the effect ofprim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamm-inol con onthe survival rate and secretion of cytokines of EC was investigated by MTT,ELISA and radioimmunoassay respectively.
The results were as follows:
1．The cultured cell was identified to be rCMEC by immumofluorescence method and morphology observation, because of its positive dyeing of factorⅧrelated antigen and its appearance like paving stones.
2．Based on the growth curve, the best time of experiment was limited in the period between 24h to 72h after inoculating cell into culture plates in the density of 1×10~5mL~(-1) .
3．The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methy-lvisamminol con on the activity of EC was determined by MTT. It was foundthat the maximum of no toxicity concentration can not be determined because of the low toxicity of these two medicines on EC. Thus, 50μg·ml~(-1) ,100μg·ml~1~(-1) , 200μgml~(-1) were chosen as the concentration of prim-O-glucosylcimifugin and 4 '-O-β-D-glucosyl-5-O-methylvisamminol con in experiment.
4．The effect of ox-LDL on the survival rate of EC was determined by MTT. The ox-LDL of 3ng·ml~(-1) DA content was chosen to injure EC in experiment.
5．After pretreatment with prim-O-glucosylcimifugin(50,100,200μg·ml~(-1) 4'-O-β-D-glucosyl-5-O-methylvisamminol con(50,100,200μgml~(-1) 24 hours, the rCMEC was incubated with ox-LDL for another 24 hours, then the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the survival rate of EC injured by ox-LDL was determined by MTT. The res-ult indicated that the survival rate of EC was reduced by ox-LDL of 3ng·ml~(-1) as MDA content significantly, the model group had great significant difference (P<0．01) compared with control. 200μg·ml~(-1) group of prim-O-glucosyl-cimifugin, 50μg·ml~(-1) , 100μg·ml~(-1) , 200μg·ml~(-1) groups of 4'-O-β-D-glucosyl-5-O-methylvisam-minol con can inhibit EC injury made by ox-LDL, had great significant differ-ence(P<0．01) compared with model group. 50μg·ml,100μg·ml~(-1) groups of prim-O-glucosylcimifugin had the tendency to lighten ox-LDL injury, but had no significant difference compared with model group.
6．After pretreatment with prim-O-glucosylcimifugin(50, 100, 200μg·ml~(-1) ) and 4'-O-β-D-glucosyl-5-O-methylvisamminol con(50, 100, 200μg·ml) for 24 hours, the rCMEC was incubated with ox-LDL for another 24 hours, then the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the secretion of cytokines of EC was determined. The result indicated that ox-LDL can increase the secretion of IL-6 and TNF-αof EC, model group had great significant difference (P<0．01) compared with control. 100μg·ml~(-1) ,200μg·ml~(-1) groups of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the secretion of IL-6, and had significant difference (P<0．05) compared with model group. The three dose groups of prim-O-glucosylcimifugin and 200μg·ml~(-1) group of 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the secretion of TNF-α, and had great significant difference (P<0．01) compared with model group. 2 The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisa-mminol con on the adhesion of monocyte to rCMEC
Referred to Nishida's method, the primary culture method of rCMEC in vitro was established. The third generation EC was adopted to investigate the effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the adhesion of monocyte to rCMEC induced by ox-LDL.
The result was as follows:
ox-LDL of 3ng·ml~(-1) as MDA content can increase the adhesion of monocyte to EC, which has great significant difference (P<0．01) compared with control. 50μg·ml~(-1) ,100μg·ml~(-1) , 200μg·ml~(-1) groups of prim-O-glucosylcimifugin, 200μg·ml~(-1) group of 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the adhesion of monocyte to EC, which has great significant difference(P<0．01) compared with model group. 50μg·ml~(-1) , 100μg·ml~(-1) groups of 4'-O-β-D-glucosyl-5-O-methylvisamminol con inhibit the adhesion, which has significant difference (P<0．05) compared with model group. 3 The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisa-mminol con on the proliferation of smooth muscle cell stimulated by TNF-α
The primary culture method of smooth muscle cell (SMC) was established by attachment-block. The SMC was identiflcated by immunochemistry method, and the growth curve was drawed by cytometry. The third generation of SMC was adopted in our experiment. By determining the effect of prim-O-glucosylc-imifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the activity of SMC , the concentration of the two medicine was decided. By determining the effect of TNF-αon the proliferation of SMC, it's concentration to stimulate the SMC proliferate was dicided. And then the effect of prim-O-glucosylcimif-ugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con on the proliferation and cell cycle of SMC was investigated by MTT and flow cytometry respectively.
The result was as follows:
1 The cultured cell was identificated to be SMC by immunochemistry method and morphology observation, because of its positive dyeing ofα-smooth actin and its appearance like peak valley.
2．Based on the growth curve, the best time of experiment was limited in the period between 24h to 96h after inoculating cell into culture plates in the density of 1×10~5mL~(-1) .
3．The effect of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methy-lvisamminol con on the activity of SMC was determined by MTT. It was found that the maximum of no toxicity concentration can not be determined because of the low toxicity of these two medicines on SMC. Thus, 50μg·ml~(-1) , 100μ·gml~(-1) , 200μg·ml~(-1) were chosen as the concentration of prim-O-glucosylcimi-fugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con in experiment.
4．The effect of TNF-αon the proliferation of SMC was determined by MTT. The concentration of 5ng·ml~(-1) was chosen to stimulate SMC in experiment.
5 The SMC was stimulated to proliferate by TNF-αof 5ng·ml~(-1) , and incubated with prim-O-glucosylcimifugin (50μg·ml~(-1) , 100μg·ml~(-1) , 200μg·ml~(-1) ) and 4'-O-β-D-glucosyl-5-O-methylvisamminol con (50μg·ml~(-1) , 100μg·ml~(-1) , 200μg·ml~(-1) ), the effect of these two medicines was investigated. The result indicate-d that TNF-αof 5ng·ml~(-1) can stimulate the proliferation of SMC, which has great significant difference (P<0．01) compared with control. The three dose groups of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisammin-ol con can inhibit the proliferation of SMC, which has great significantdifference(P<0．01) compared with model group.
6 The SMC was stimulated to proliferate by TNF-αof 5ng·ml~(-1) , and incubated with prim-O-glucosylcimifugin(50μg·ml~(-1) ,100μg·ml~(-1) , 200μg·ml~(-1) ) and 4'-O-β-D-glucosyl-5-O-methylvisamminol con(50μg·ml~(-1) ,100μg·ml~(-1) ,200μg·ml~(-1) ),the effect of these two medicines on the cell cycle was investigated. The result indicated that TNF-αof 5ng·ml~(-1) can increase the proportion of G2 phase and S phase in cell cycle, which has great significant difference (P<0．01) compared with control. The three dose groups of prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con can increase the proportion of G0/G1 phase, decrease the proportion of S phase and G2/M phase, which havegreat significant difference(P<0．01) compared with model group.
It can be concluded that prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con can inhibit the injury and secretion of inflammatorycytokines of EC, the adhesion of monocytes to EC and the proliferation of SMC stimulated by TNF-α. In conclusion, prim-O-glucosylcimifugin and 4'-O-β-D-glucosyl-5-O-methylvisamminol con has the effect of anti-inflammatory reaction in AS, and provides the scientific evidence for wide clinical application.
引文
[1] 绍耕主编．现代冠心病．北京：北京医科大学，中国协和医科大学联合出版社，1994．43-44
[2] 1, ROSS R. Atherosclerosis-an inflammatory disease[J] . N Engl J Med, 1999, 340 (2): 115-126)
[3] 黄薇、蔡运昌．幽门螺旋杆菌感染与冠心病关系的研究现状及进展．贵州医药，2006，30(2)：174-176
[4] 王守东，郭新贵，林宪如．幽门螺旋杆菌与冠心病发病的关系[J] ．心脏杂志，2004，16(5)：454-456
[5] 杨久亮．动脉粥样硬化发病机制的研究进展．人民军医，2003，46(8)：484-486
[6] 张轶轩．同型半胱氨酸粗炎症反应与动脉粥样硬化．中国医药导报，2006，3(27)：18-19
[7] 董艳玲．冠心病新危险因素的研究进展．医学综述，2006，12(15)：917-919
[8] Fanzo JC, Reaves SK, Cui L, et al. Am J Physiol, 2002;283(2):C631-C638
[9] Fukamachi Y, Karasaki Y, Sugiura T, et al. Biochem Biophys Res Commun, 1998;246(2):364-369
[10] Connell P, Young VM, Toborek M, et al. Am J Coll Nutr, 1999;18(2):152-158
[11] Perry DK, Smyth MJ, Stennicke HR, et al. J Biol Chem, 1992;272(30):18 530-18 533
[12] Truony-Tran AQ, Carter J, Ruffin RE, et al. Biometals, 2001;14(3-4):315-330
[13] 颜利求．缺锌—动脉粥样硬化形成的危险因素．国外医学·心血管疾病分册，2005，32(1)：9-11
[14] Li D, Saldeen T, Romeo F, et al. Oxidized LDL upregulates angiotensin Ⅱ type 1 receptor expression in cultured human coronary artery endothelial cells:the potential role of transcription factor NF-kappa B[J] . Circulation, 2000, 102(16): 1970-1976
[15] Neve BP, Fruchart JC, Staels B. Role of the peroxisome proliferator-activated receptors(PPAR) in atherosclerosis. Biochem Pharmacol. 2000, 60(8):1245-1250
[16] Lglarz M, Touyz RM, Amin F, Lavoie MF, Diep QN, Schiffrin El. Effect of peroxisome proliferators-activated receptor-alpha and -gamma activators on vascular remodeling in endothelin-dependent hypertension. Arterioscler Thromb Vasc BioI, 2003, 23(1):45-51
[17] Vosper H, Patel L, Graham TL, Khoudoli GA, Hill A, Macphee CH, et al. The peroxisome proliferator-activated receptor delta promotes lipid accumulation in human macrophages. J Biol CHem, 2001, 276(47):44 258-265
[18] Welch JS, Ricote M, Akiyama TE, Gonzalez FJ, Glass CK. PPAR gamma and PPAR delta negatively regulate specific subsets of lipopolysaccharide and IFN-gamma target genes in macrophages. Proc Natl Acad Sci USA, 2003, 100(11):6712-717
[19] 高凌云，李福平等．PPARγ与动脉粥样硬化，中国微循环，2005，9(5)：369-372
[20] Verrier E 等 Verrier E, Wang L, Wadham C, et al. PPARγ agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway. Role of Diacylglycerol Kinase [J] . Cire Res. 2004, 94(11): 1515-1522
[21] Cominacini L, Garbin U, Pasini AF, et al. The expression of adhesion molecules on endothelial cells in inhibited by troglitazone through its antioxidant activity[J] . Cell Adhesion Commun. 1999, 7:223-231
[22] Nicholson AC, Han J, Febbraio M, et al. Role of CD36, the macrophage class B scavenger receptor in atherosclerosis[J] . Ann NY Acad Sci, 2001, 947:224-228
[23] Argmann CA, Sawyez CG, McNeil CJ, et al. Activation of peroxisome proliferator-activated receptor gamma and retinoid X receptor results in net depletion of cellular cholesteryl esters in macrophages exposed to oxidized lipoproteins[J] . Arterioscler Thromb Vasc Biol, 2003, 23(3):457-482
[24] Oram JF. Atp-binding cassette transporter AI and cholesterol trafficking[J] . Curr Opin Lipidol, 2002, 13:373-381
[25] Wakino S, Kintscher U, Kim S, et al. Peroxisome proliferators-activated receptor gamma ligands inhibits retinoblastoma phosphorylation and G1-S transition in vascular smooth muscle cells[J] . J Biol Chem. 2000, 36:1529-1535
[26] 袁展群，尚可，刘勇．肝X受体与动脉粥样硬化．中国分子心脏病学杂志，2006，6(2)：115-118
[27] Joseph SB, Castrillo A, Laffitte BA, et al. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med, 2003, 9(2):213-219
[28] Sanguigni V, Ferro D, Pignatelli P, Del Ben M, Nadia T, Saliola M, et al. CD40 ligand enhances monocyte tissue factor expression and thrombin generation via oxidative stress in patients with hypercholesterolemia[J] . J Am Coll Cardiol, 2005, 45(1):35-42
[29] Li DY, Liu L, Chen HJ, Sawamura T, Mehta J L. LOX-1, an oxidized LDL endothelial receptor, induces CD40-CD40L signaling in human coronary artery endothelial cells[J] . Arter Thromb Vas Biol, 2003, 23(5):816-821
[30] Pratico D Tillmann C, Zhang ZB, et al. Cyclooxygenase-2 promotes early atherosclerotic lesion formation in ApoE-deficient and C57BL/6 mice. J Mol Cell Cardiol, 2005, 39:443-452
[31] Sasu S, La Verda D, Qureshi N et al. Chlamydia pneumoniae and heat shock protein 60 simulate proliferation of human vascular smooth muscle cells via Toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation. Circ Res, 2001, 89:244-250
[32] Ouchi N, Kihara S, Arita Y, et al. Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-kappaB signaling through a cAMP-dependent pathway[J] . Circulation, 2000, 102(11): 1296-1301
[33] Yokota Y, Oritani K, Takahashi L, et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages[J] . Blood, 2000, 96(5): 1723-1732
[34] Ouchi N, Kihara S, Arita Y, et al. Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages[J] . Circulation, 2001, 103(8):1057-1063
[35] 王铁成，李晓燕．脂联素—动脉粥样硬化的保护性因子．泰山医学院学报，2005，5(26)：500-502
[36] Huang Y, Jaffa A, Koskinen S, et al. Arterioscler Thromb Vasc Biol, 1999; 19(7):1600-1607
[37] Brown M. A vaccine against atherosclerosis. DDT, 2002, 7(11):588
[38] Fricker J. A vaccine approach to healthy lipoprotein levels. Mol Med Today, 1999, 5(7):28
[39] Li J, Hirose N, Kawamura M, Arai Y. Antiatherogenic effect of angiotensin converting enzyme enhibitor and angiotensin Ⅱ receptor antagonist in the cholesterol fed rabbits. Atherosclerosis, 1999, 143(2):315-326
[40] 王允，丁华．血管紧张素Ⅱ 1型受体拮抗剂抗动脉粥样硬化机制研究进展．中国动脉硬化杂志，2005，13(6)：810-812
[41] 谈红，潘其兴，梁春香，魏敏，克丙申，尹格平等．氯沙坦、卡托普利及联合用药对肾动脉粥样硬化的干预．中国动脉硬化杂志，2003，11(4)：299-303
[42] Hayek T, Aviram M, Heinrich R, Saknini E, Keidar S. Losartan inhibits cellular uptake of oxidized LDL by monocyte-macrophages from hypercholesterolemic patients. Biochem Biophys Res Commun, 2000, 273(2):417-420
[43] Strawn WB, Chappell MC, Dean RH, Kivlighn S, Ferrario CM. Inhibition of early atherogenesis by losartan in monkeys with diet-induced hypercholesterolemia. Circulation, 2000, 101(13): 1586-1593
[44] Hussein O, Shneider J, Rosenblat M, Aviram M. Valsartan therapy has additive anti-oxidative effect to that of fluvastatin therapy against low density lipoprotein oxidation:studies in hypercholesterolemic and hypertensive patients. J Cardiovasc Pharmacol, 2002, 40(1):28-34
[45] Kohno M, Ohmori K, Nozaki S, Mizushige K, Yasunari K, Kano H, et al. Effects of valsartan on angiotensin Ⅱ-induced migration of human coronary artery smooth muscle cells. Hypertens Res, 2000, 23(6):677-681
[46] 董晓燕，张贵清，方向明，郑民安，胡继军，林桂珍．卡托普利对动脉粥样硬化家兔内皮素和血管紧张素Ⅱ的影响和原癌基因c-myc和c-fos的相互关系．中国动脉硬化杂志，2002，10(3)：217-220
[47] HomeBD, Muhlestein, JB, Carlquist JF, et al. lntermountain Heart Collaborative (HC) Study Group statin therapy interacts with cytomegalovirus seropositivity and high C-reactive protein in reducing mortality among patients with angiographically significant coronary disease Circulation, 2003, 107:258-263
[48] Grip O, Janciauskiene S, Lindgren S. A torvastatin activates PPAR- and attenuates the inflammatory response in human monocytes[J] . Infamm Res, 2002, 51:58-62
[49] Dichtl W, Dulak J, Frick M, et al. HMG-CoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells [J] . Arterioscler Thromb Vase Biol, 2003, 23(1):58.
[50] 何燕，秦明照．噻唑烷二酮类药物与动脉粥样硬化[J] ．中国医药导刊，2005，7(4)：293-294
[51] Matsuda M, Shimoura I, Sara M, et al. Role of adiponectin in reventing vascular stenosis the missing link of adipo vascular axis. J Biol Chem, 2002;277:37487-37491
[52] Combs TP, Wangner JA, Berger J, et al. Induction of adpocyte complement-releated protein of 30 kilo daltons by PPAR agonists: Apotential mechanism of insulin sensitization. Endocrinology, 2002; 143:998-1007
[53] Li AC, Brown KK, Silvestre MJ, et al. Peroxisome proliferators activated receptor gamma ligands inhibit development of atherosclerosis in LDL, receptor-deficient mice. J Clin Invest, 2000;106:523-531
[54] 沈洪，尹明，黎檀实，刘刚，王艳辉，冯丽洁，等．环氧化酶2在动脉粥样硬化中的表达及血清炎性细胞因子的变化．中国危重病急救医学，2002，14(4)：226-229
[55] Harari OA, Wickham TJ, Stocker CJ, Kovesdi I, Segal DM, Huehns TY, et al. Targeting an adenoviral gene vector to cytokine-activated vascular endothelium via E-selectin. Gene Ther, 1999, 6(5):801-807
[56] 张子新，赵卫华，宋丽新，曾定尹．阿奇霉素对动脉粥样硬化家兔炎症反应的影响[J] ．中国动脉硬化杂志，2006，14(7)：601-603
[57] Grayston JT, Kronmal RA, Juckson LA, Jackson LA, Parisi AF, Muhlestein JB, et al. Azithromycin for the secondary prevention of coronary events[J] . N Engl J Med, 2005, 352(16):1637-1645
[58] 贺圣文，范志涛，李佩贤，吕士军，韩连堂，洪舜华．野生马齿苋抗动脉粥样硬化形成的实验研究．营养学报，1997，19(1)：59-63
[59] 罗琰，调脂胶囊对大鼠动脉粥样硬化的影响．河南中医学院学报，2006，21(125)：6
[60] 于辉，李春香，宫凌涛，甘庆贤．甘草的药理作用概述[J] ．现代生物医学 进展，2006，6(4)：77-79
[61] 罗瑞芝，贾伟，赵利斌，邱明丰，孙霞．何首乌研究进展[J] ．中草药，2005，36(7)：1097-1100
[62] 金伟军．虎杖晶4号对人血PMNS呼吸爆发和氧自由基的作用[J] ．中国药理学通报，1993，9(5)：355
[63] 王绿娅，王大全，秦彦文等．余甘子抗脂质过氧化和保护血管内皮的实验研究[J] ．中国药学杂志，2003，38(10)：505
[64] 何书英，钱之玉．西红花苷对氧化性低密度脂蛋白致内皮细胞损伤的保护作用．中国新药杂志，2005，14(2)：169-171
[65] 孙秀才，冯建章，斐晓阳等．姜黄素对食高脂饲料家兔血管内皮功能的保护作用[J] ．岭南医学杂志，2004，10(1)：60-63
[66] 罗心平，李勇，范维琥等．麝香保心丸减少高脂血症对动脉壁损害作用的实验研究[J] ．中国中西医结合杂志，1998，18(8)：486-489
[67] 刘连噗．虎杖晶4号对兔血小板超微结构的影响[J] ．第一军医大学学报，1998，7(5)：17
[68] 王跃忠，骆苏芳，张佩文等．3，4，5-三羟基芪3-β-D-葡萄糖苷减轻动脉内皮损伤性血栓形成的作用[J] ．中国药理学报，1995 16(2)：169
[69] 戴敏，李后开．丹皮酚对动脉粥样硬化家兔血管平滑肌细胞增殖的影响[J] ．中国药理学通报，2006，22(5)：587-591
[70] Chen W S, Liu W Y, Yang G J, et al. Structural elucidation of a new tetrahydroxystilbene of Radix Polygoni Multiflori Prepatrata and a study on its cardiovascular activity[J] . Acta Pharm Sin, 2000, 35(12):906-908.
[71] Wu Y, Zhu B D. Effect of Danggui Buxue Decoction on proliferation and expression of intercellular adhesion molecule-1 in human umbilical vein endothelial cells[J] . J West China Univ Med Sci, 2001, 32(4):593-595.
[72] Zhang Y J, Zhao L G, Wu X Z. Effect of Huoxuehuayu Decoction on expression of adhesive molecular of intrapulmonic ecdothelial cell of rats suffered from SAP[J] . Chin Tradit Herb Drugs, 2001, 32(11): 1010-1012.
[73] Hao Y, Qiu Q Y, W J. Effect of astragalus polysaccharin(APS) on lymphocyte-endothelium adhesion and the molecular mechanism[J] . Immunnol J, 2000, 16(3):206-209.
[74] 王利民，蔡生业，姚成芳，王丽，周宪宾，王华亭，王恒孝．花刺参粘多糖对大鼠血管平滑肌细胞粘附分子表达的影响[J] ．中国动脉硬化杂志，2006，14(7)：565-568
[75] 陈利国，屈援，葛红颖，胡小勤，聂优爱，何孟栖．补阳还五汤对血瘀证大鼠血管内皮细胞粘附分子表达的影响，中草药．2005，36(5)：706-709．
[76] 徐茂锦，邹大进，叶江洪，张军，冯正康．斛皮酮对糖化终产物诱导的小鼠微血管内皮细胞血管细胞黏附分子-1表达的抑制作用．中华糖尿病杂志，2004，12(1)：60-61．
[77] 张艳军，赵连根，吴咸中．活血化瘀注射液(HHI-Ⅰ)对TNF-a诱发大鼠肺微血管内皮细胞黏附分子mRNA表达的影响．天津中医药，2005，22(3)：240-242。
[78] 王宗仁，李军昌，马爱玲，郑瑾，李晶华，杜永平，岳乔红．芪丹通脉片对动脉粥样硬化大鼠单个核细胞中细胞间黏附分子1及血管细胞黏附分子1 mRNA表达的影响．中国临床康复，2005，27(9)：77-79
[79] Orbe J, Femandez I, Rodriguez JA. Different expression of MMPs/TIMP-1 in human atherosclerotic lesion. Relation to Plaque features and vascular bed[J] . Atherosclerosis, 2003, 170(2):269-276.
[80] 鞠镐，程文立，董唏．丹参粉针剂对氧化低密度脂蛋白诱导的内皮细胞表达MMP9及ICAM-1的影响．中日友好医院学报，2005，19(4)：230-232．
[81] 刘锦，鲁映青．金雀异黄素对低密度脂蛋白氧化修饰及氧化型低密度脂蛋白诱导的血管内皮细胞c-myc mRNA表达的抑制作用．中国动脉硬化杂志，2002，10(6)：509-513．
[82] 薛宝云，李文，李丽等．防风色原酮甙类成分的药理活性研究．中国中药杂志．2000，25(5)：97
[83] NishidaM, CarleyWW, GerritsenME, et al. Isolation and characterization of human and rat cardiac microvascular endothelial cells. Am J Physiol, 1993, 264:H639-H652
[84] 司徒镇强，吴军正主编．细胞培养．西安：世界图书出版公司，1996．186
[85] Anderson JW, Diwadkar VA, Bridges SR. Selective effects of different antioxidants on oxidation of lipoproteins from rats. Proc Soc Exp Biol Med, 1998:218(4):376-381
[86] ChinDusting JP, Fisher LJ, Lewis TV, et al. The vascular activity of some isoflavone metabolites: implications for a cardioprotective role. Br J Pharmacol, 2001:133(4):595-604
[87] . Boyum A. (1968): Separation of leucocytes from blood and bone marrow. Scand J. Clin. Lab. Invest. 21, Suppl. 97.
[88] . Harris R. & Ukayiofo EV. (1969): Rapid preparation for lymphocytes for tissue typing. Lancet 327, 7615.
[89] . Boyum, A. , Isolation and removal of lymphocytes from bone marrow of rats and guinea-pigs. , Scand J. Clin. Lab. Invest. , 21, Suppl. 97, 91-109 (1968).
[90] Fischer DK, et al. Ultrastructural and immunohistochemical studies of primary cultures of aortic medial cells. Exp Mol Pathol, 1973, 18:162-176
[91] Diglio C, et al. Rat heart-derived endothelial and smooth muscle cell cultures isolation, cloning and characterization. Tissue and Cell, 1988, 20(4):477-492
[2] 1, ROSS R. Atherosclerosis-an inflammatory disease[J] . N Engl J Med, 1999, 340 (2): 115-126)
[3] 黄薇、蔡运昌．幽门螺旋杆菌感染与冠心病关系的研究现状及进展．贵州医药，2006，30(2)：174-176
[4] 王守东，郭新贵，林宪如．幽门螺旋杆菌与冠心病发病的关系[J] ．心脏杂志，2004，16(5)：454-456
[5] 杨久亮．动脉粥样硬化发病机制的研究进展．人民军医，2003，46(8)：484-486
[6] 张轶轩．同型半胱氨酸粗炎症反应与动脉粥样硬化．中国医药导报，2006，3(27)：18-19
[7] 董艳玲．冠心病新危险因素的研究进展．医学综述，2006，12(15)：917-919
[8] Fanzo JC, Reaves SK, Cui L, et al. Am J Physiol, 2002;283(2):C631-C638
[9] Fukamachi Y, Karasaki Y, Sugiura T, et al. Biochem Biophys Res Commun, 1998;246(2):364-369
[10] Connell P, Young VM, Toborek M, et al. Am J Coll Nutr, 1999;18(2):152-158
[11] Perry DK, Smyth MJ, Stennicke HR, et al. J Biol Chem, 1992;272(30):18 530-18 533
[12] Truony-Tran AQ, Carter J, Ruffin RE, et al. Biometals, 2001;14(3-4):315-330
[13] 颜利求．缺锌—动脉粥样硬化形成的危险因素．国外医学·心血管疾病分册，2005，32(1)：9-11
[14] Li D, Saldeen T, Romeo F, et al. Oxidized LDL upregulates angiotensin Ⅱ type 1 receptor expression in cultured human coronary artery endothelial cells:the potential role of transcription factor NF-kappa B[J] . Circulation, 2000, 102(16): 1970-1976
[15] Neve BP, Fruchart JC, Staels B. Role of the peroxisome proliferator-activated receptors(PPAR) in atherosclerosis. Biochem Pharmacol. 2000, 60(8):1245-1250
[16] Lglarz M, Touyz RM, Amin F, Lavoie MF, Diep QN, Schiffrin El. Effect of peroxisome proliferators-activated receptor-alpha and -gamma activators on vascular remodeling in endothelin-dependent hypertension. Arterioscler Thromb Vasc BioI, 2003, 23(1):45-51
[17] Vosper H, Patel L, Graham TL, Khoudoli GA, Hill A, Macphee CH, et al. The peroxisome proliferator-activated receptor delta promotes lipid accumulation in human macrophages. J Biol CHem, 2001, 276(47):44 258-265
[18] Welch JS, Ricote M, Akiyama TE, Gonzalez FJ, Glass CK. PPAR gamma and PPAR delta negatively regulate specific subsets of lipopolysaccharide and IFN-gamma target genes in macrophages. Proc Natl Acad Sci USA, 2003, 100(11):6712-717
[19] 高凌云，李福平等．PPARγ与动脉粥样硬化，中国微循环，2005，9(5)：369-372
[20] Verrier E 等 Verrier E, Wang L, Wadham C, et al. PPARγ agonists ameliorate endothelial cell activation via inhibition of diacylglycerol-protein kinase C signaling pathway. Role of Diacylglycerol Kinase [J] . Cire Res. 2004, 94(11): 1515-1522
[21] Cominacini L, Garbin U, Pasini AF, et al. The expression of adhesion molecules on endothelial cells in inhibited by troglitazone through its antioxidant activity[J] . Cell Adhesion Commun. 1999, 7:223-231
[22] Nicholson AC, Han J, Febbraio M, et al. Role of CD36, the macrophage class B scavenger receptor in atherosclerosis[J] . Ann NY Acad Sci, 2001, 947:224-228
[23] Argmann CA, Sawyez CG, McNeil CJ, et al. Activation of peroxisome proliferator-activated receptor gamma and retinoid X receptor results in net depletion of cellular cholesteryl esters in macrophages exposed to oxidized lipoproteins[J] . Arterioscler Thromb Vasc Biol, 2003, 23(3):457-482
[24] Oram JF. Atp-binding cassette transporter AI and cholesterol trafficking[J] . Curr Opin Lipidol, 2002, 13:373-381
[25] Wakino S, Kintscher U, Kim S, et al. Peroxisome proliferators-activated receptor gamma ligands inhibits retinoblastoma phosphorylation and G1-S transition in vascular smooth muscle cells[J] . J Biol Chem. 2000, 36:1529-1535
[26] 袁展群，尚可，刘勇．肝X受体与动脉粥样硬化．中国分子心脏病学杂志，2006，6(2)：115-118
[27] Joseph SB, Castrillo A, Laffitte BA, et al. Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat Med, 2003, 9(2):213-219
[28] Sanguigni V, Ferro D, Pignatelli P, Del Ben M, Nadia T, Saliola M, et al. CD40 ligand enhances monocyte tissue factor expression and thrombin generation via oxidative stress in patients with hypercholesterolemia[J] . J Am Coll Cardiol, 2005, 45(1):35-42
[29] Li DY, Liu L, Chen HJ, Sawamura T, Mehta J L. LOX-1, an oxidized LDL endothelial receptor, induces CD40-CD40L signaling in human coronary artery endothelial cells[J] . Arter Thromb Vas Biol, 2003, 23(5):816-821
[30] Pratico D Tillmann C, Zhang ZB, et al. Cyclooxygenase-2 promotes early atherosclerotic lesion formation in ApoE-deficient and C57BL/6 mice. J Mol Cell Cardiol, 2005, 39:443-452
[31] Sasu S, La Verda D, Qureshi N et al. Chlamydia pneumoniae and heat shock protein 60 simulate proliferation of human vascular smooth muscle cells via Toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation. Circ Res, 2001, 89:244-250
[32] Ouchi N, Kihara S, Arita Y, et al. Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-kappaB signaling through a cAMP-dependent pathway[J] . Circulation, 2000, 102(11): 1296-1301
[33] Yokota Y, Oritani K, Takahashi L, et al. Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages[J] . Blood, 2000, 96(5): 1723-1732
[34] Ouchi N, Kihara S, Arita Y, et al. Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages[J] . Circulation, 2001, 103(8):1057-1063
[35] 王铁成，李晓燕．脂联素—动脉粥样硬化的保护性因子．泰山医学院学报，2005，5(26)：500-502
[36] Huang Y, Jaffa A, Koskinen S, et al. Arterioscler Thromb Vasc Biol, 1999; 19(7):1600-1607
[37] Brown M. A vaccine against atherosclerosis. DDT, 2002, 7(11):588
[38] Fricker J. A vaccine approach to healthy lipoprotein levels. Mol Med Today, 1999, 5(7):28
[39] Li J, Hirose N, Kawamura M, Arai Y. Antiatherogenic effect of angiotensin converting enzyme enhibitor and angiotensin Ⅱ receptor antagonist in the cholesterol fed rabbits. Atherosclerosis, 1999, 143(2):315-326
[40] 王允，丁华．血管紧张素Ⅱ 1型受体拮抗剂抗动脉粥样硬化机制研究进展．中国动脉硬化杂志，2005，13(6)：810-812
[41] 谈红，潘其兴，梁春香，魏敏，克丙申，尹格平等．氯沙坦、卡托普利及联合用药对肾动脉粥样硬化的干预．中国动脉硬化杂志，2003，11(4)：299-303
[42] Hayek T, Aviram M, Heinrich R, Saknini E, Keidar S. Losartan inhibits cellular uptake of oxidized LDL by monocyte-macrophages from hypercholesterolemic patients. Biochem Biophys Res Commun, 2000, 273(2):417-420
[43] Strawn WB, Chappell MC, Dean RH, Kivlighn S, Ferrario CM. Inhibition of early atherogenesis by losartan in monkeys with diet-induced hypercholesterolemia. Circulation, 2000, 101(13): 1586-1593
[44] Hussein O, Shneider J, Rosenblat M, Aviram M. Valsartan therapy has additive anti-oxidative effect to that of fluvastatin therapy against low density lipoprotein oxidation:studies in hypercholesterolemic and hypertensive patients. J Cardiovasc Pharmacol, 2002, 40(1):28-34
[45] Kohno M, Ohmori K, Nozaki S, Mizushige K, Yasunari K, Kano H, et al. Effects of valsartan on angiotensin Ⅱ-induced migration of human coronary artery smooth muscle cells. Hypertens Res, 2000, 23(6):677-681
[46] 董晓燕，张贵清，方向明，郑民安，胡继军，林桂珍．卡托普利对动脉粥样硬化家兔内皮素和血管紧张素Ⅱ的影响和原癌基因c-myc和c-fos的相互关系．中国动脉硬化杂志，2002，10(3)：217-220
[47] HomeBD, Muhlestein, JB, Carlquist JF, et al. lntermountain Heart Collaborative (HC) Study Group statin therapy interacts with cytomegalovirus seropositivity and high C-reactive protein in reducing mortality among patients with angiographically significant coronary disease Circulation, 2003, 107:258-263
[48] Grip O, Janciauskiene S, Lindgren S. A torvastatin activates PPAR- and attenuates the inflammatory response in human monocytes[J] . Infamm Res, 2002, 51:58-62
[49] Dichtl W, Dulak J, Frick M, et al. HMG-CoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells [J] . Arterioscler Thromb Vase Biol, 2003, 23(1):58.
[50] 何燕，秦明照．噻唑烷二酮类药物与动脉粥样硬化[J] ．中国医药导刊，2005，7(4)：293-294
[51] Matsuda M, Shimoura I, Sara M, et al. Role of adiponectin in reventing vascular stenosis the missing link of adipo vascular axis. J Biol Chem, 2002;277:37487-37491
[52] Combs TP, Wangner JA, Berger J, et al. Induction of adpocyte complement-releated protein of 30 kilo daltons by PPAR agonists: Apotential mechanism of insulin sensitization. Endocrinology, 2002; 143:998-1007
[53] Li AC, Brown KK, Silvestre MJ, et al. Peroxisome proliferators activated receptor gamma ligands inhibit development of atherosclerosis in LDL, receptor-deficient mice. J Clin Invest, 2000;106:523-531
[54] 沈洪，尹明，黎檀实，刘刚，王艳辉，冯丽洁，等．环氧化酶2在动脉粥样硬化中的表达及血清炎性细胞因子的变化．中国危重病急救医学，2002，14(4)：226-229
[55] Harari OA, Wickham TJ, Stocker CJ, Kovesdi I, Segal DM, Huehns TY, et al. Targeting an adenoviral gene vector to cytokine-activated vascular endothelium via E-selectin. Gene Ther, 1999, 6(5):801-807
[56] 张子新，赵卫华，宋丽新，曾定尹．阿奇霉素对动脉粥样硬化家兔炎症反应的影响[J] ．中国动脉硬化杂志，2006，14(7)：601-603
[57] Grayston JT, Kronmal RA, Juckson LA, Jackson LA, Parisi AF, Muhlestein JB, et al. Azithromycin for the secondary prevention of coronary events[J] . N Engl J Med, 2005, 352(16):1637-1645
[58] 贺圣文，范志涛，李佩贤，吕士军，韩连堂，洪舜华．野生马齿苋抗动脉粥样硬化形成的实验研究．营养学报，1997，19(1)：59-63
[59] 罗琰，调脂胶囊对大鼠动脉粥样硬化的影响．河南中医学院学报，2006，21(125)：6
[60] 于辉，李春香，宫凌涛，甘庆贤．甘草的药理作用概述[J] ．现代生物医学 进展，2006，6(4)：77-79
[61] 罗瑞芝，贾伟，赵利斌，邱明丰，孙霞．何首乌研究进展[J] ．中草药，2005，36(7)：1097-1100
[62] 金伟军．虎杖晶4号对人血PMNS呼吸爆发和氧自由基的作用[J] ．中国药理学通报，1993，9(5)：355
[63] 王绿娅，王大全，秦彦文等．余甘子抗脂质过氧化和保护血管内皮的实验研究[J] ．中国药学杂志，2003，38(10)：505
[64] 何书英，钱之玉．西红花苷对氧化性低密度脂蛋白致内皮细胞损伤的保护作用．中国新药杂志，2005，14(2)：169-171
[65] 孙秀才，冯建章，斐晓阳等．姜黄素对食高脂饲料家兔血管内皮功能的保护作用[J] ．岭南医学杂志，2004，10(1)：60-63
[66] 罗心平，李勇，范维琥等．麝香保心丸减少高脂血症对动脉壁损害作用的实验研究[J] ．中国中西医结合杂志，1998，18(8)：486-489
[67] 刘连噗．虎杖晶4号对兔血小板超微结构的影响[J] ．第一军医大学学报，1998，7(5)：17
[68] 王跃忠，骆苏芳，张佩文等．3，4，5-三羟基芪3-β-D-葡萄糖苷减轻动脉内皮损伤性血栓形成的作用[J] ．中国药理学报，1995 16(2)：169
[69] 戴敏，李后开．丹皮酚对动脉粥样硬化家兔血管平滑肌细胞增殖的影响[J] ．中国药理学通报，2006，22(5)：587-591
[70] Chen W S, Liu W Y, Yang G J, et al. Structural elucidation of a new tetrahydroxystilbene of Radix Polygoni Multiflori Prepatrata and a study on its cardiovascular activity[J] . Acta Pharm Sin, 2000, 35(12):906-908.
[71] Wu Y, Zhu B D. Effect of Danggui Buxue Decoction on proliferation and expression of intercellular adhesion molecule-1 in human umbilical vein endothelial cells[J] . J West China Univ Med Sci, 2001, 32(4):593-595.
[72] Zhang Y J, Zhao L G, Wu X Z. Effect of Huoxuehuayu Decoction on expression of adhesive molecular of intrapulmonic ecdothelial cell of rats suffered from SAP[J] . Chin Tradit Herb Drugs, 2001, 32(11): 1010-1012.
[73] Hao Y, Qiu Q Y, W J. Effect of astragalus polysaccharin(APS) on lymphocyte-endothelium adhesion and the molecular mechanism[J] . Immunnol J, 2000, 16(3):206-209.
[74] 王利民，蔡生业，姚成芳，王丽，周宪宾，王华亭，王恒孝．花刺参粘多糖对大鼠血管平滑肌细胞粘附分子表达的影响[J] ．中国动脉硬化杂志，2006，14(7)：565-568
[75] 陈利国，屈援，葛红颖，胡小勤，聂优爱，何孟栖．补阳还五汤对血瘀证大鼠血管内皮细胞粘附分子表达的影响，中草药．2005，36(5)：706-709．
[76] 徐茂锦，邹大进，叶江洪，张军，冯正康．斛皮酮对糖化终产物诱导的小鼠微血管内皮细胞血管细胞黏附分子-1表达的抑制作用．中华糖尿病杂志，2004，12(1)：60-61．
[77] 张艳军，赵连根，吴咸中．活血化瘀注射液(HHI-Ⅰ)对TNF-a诱发大鼠肺微血管内皮细胞黏附分子mRNA表达的影响．天津中医药，2005，22(3)：240-242。
[78] 王宗仁，李军昌，马爱玲，郑瑾，李晶华，杜永平，岳乔红．芪丹通脉片对动脉粥样硬化大鼠单个核细胞中细胞间黏附分子1及血管细胞黏附分子1 mRNA表达的影响．中国临床康复，2005，27(9)：77-79
[79] Orbe J, Femandez I, Rodriguez JA. Different expression of MMPs/TIMP-1 in human atherosclerotic lesion. Relation to Plaque features and vascular bed[J] . Atherosclerosis, 2003, 170(2):269-276.
[80] 鞠镐，程文立，董唏．丹参粉针剂对氧化低密度脂蛋白诱导的内皮细胞表达MMP9及ICAM-1的影响．中日友好医院学报，2005，19(4)：230-232．
[81] 刘锦，鲁映青．金雀异黄素对低密度脂蛋白氧化修饰及氧化型低密度脂蛋白诱导的血管内皮细胞c-myc mRNA表达的抑制作用．中国动脉硬化杂志，2002，10(6)：509-513．
[82] 薛宝云，李文，李丽等．防风色原酮甙类成分的药理活性研究．中国中药杂志．2000，25(5)：97
[83] NishidaM, CarleyWW, GerritsenME, et al. Isolation and characterization of human and rat cardiac microvascular endothelial cells. Am J Physiol, 1993, 264:H639-H652
[84] 司徒镇强，吴军正主编．细胞培养．西安：世界图书出版公司，1996．186
[85] Anderson JW, Diwadkar VA, Bridges SR. Selective effects of different antioxidants on oxidation of lipoproteins from rats. Proc Soc Exp Biol Med, 1998:218(4):376-381
[86] ChinDusting JP, Fisher LJ, Lewis TV, et al. The vascular activity of some isoflavone metabolites: implications for a cardioprotective role. Br J Pharmacol, 2001:133(4):595-604
[87] . Boyum A. (1968): Separation of leucocytes from blood and bone marrow. Scand J. Clin. Lab. Invest. 21, Suppl. 97.
[88] . Harris R. & Ukayiofo EV. (1969): Rapid preparation for lymphocytes for tissue typing. Lancet 327, 7615.
[89] . Boyum, A. , Isolation and removal of lymphocytes from bone marrow of rats and guinea-pigs. , Scand J. Clin. Lab. Invest. , 21, Suppl. 97, 91-109 (1968).
[90] Fischer DK, et al. Ultrastructural and immunohistochemical studies of primary cultures of aortic medial cells. Exp Mol Pathol, 1973, 18:162-176
[91] Diglio C, et al. Rat heart-derived endothelial and smooth muscle cell cultures isolation, cloning and characterization. Tissue and Cell, 1988, 20(4):477-492