消斑通脉合剂中药血清对大鼠血管平滑肌细胞增殖与凋亡的影响

【作者】 武跃华；

【导师】 杨积武；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 经皮腔内冠脉成形术(percutaneous transluminal coronary angioplasty,PTCA)后再狭窄(restenosis,RS)是影响冠状动脉粥样硬化性心脏病介入治疗预后的关键问题,严重制约了经皮冠状动脉介入治疗(percutaneous coronary intervention,PCI)的远期疗效,成为心血管领域的研究热点和难点。其病理基础是新生内膜的形成,而血管平滑肌细胞(vascular smooth muscle cell,VSMC)异常增殖和凋亡不足则是其主要成因。因此,VSMC是RS的主要作用细胞,也是防治RS研究最重要的靶细胞。随着对RS分子水平机理研究的不断深入,发现许多因素如细胞因子、生长因子等可通过活化细胞外信号调节蛋白激酶(ERK)、磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)信号通路影响细胞周期蛋白及凋亡相关基因,在VSMC增殖、分化、凋亡以及转移中发挥重要的调控作用。所以,针对VSMC增殖与凋亡调控因素的干预是RS防治的切入点。中医学虽无再狭窄病名,但从中医整体观念及辨证论治的角度出发,其仍归属“胸痹”、“心痛”、“厥心痛”的范畴。认为此病为本虚标实之证,以脏气亏虚为本,瘀血、痰浊阻滞为标。我们根据临床再狭窄患者的年龄特点,结合其证候转归,并运用中医学阴阳气血的基本理论,从中医角度治疗PTCA术后RS应在治重阳气的基础上,根据,临床应以益气助阳与活血化瘀并用,才能充分发挥中医药调节整体改善局部的治疗特色。本研究拟通过观察中药复方消斑通脉合剂中药血清对体外培养的大鼠VSMC增殖及凋亡的影响,初步探讨具有益气助阳,活血通脉作用的消斑通脉合剂对VSMC增殖及凋亡的可能作用机理,旨在为消斑通脉合剂对临床RS的防治提供切实的理论依据。第一部分理论研究中医防治PTCA术后再狭窄的辨证论治探讨研究目的:中医理论角度阐述具有益气助阳、活血通脉功效的中药复方消斑通脉合剂对PTCA术后再狭窄防治的可行性。研究方法:根据PTCA术后再狭窄患者发病的年龄特点、临床症状、体征,以中医学的核心理论阴阳学说为出发点,结合《黄帝内经》、《金匮要略》等历代中医古典文献的相关记载,运用中医学阴阳气血基本理论和辨证论治的理论思想,并结合现代药理学的研究成果,探寻具有益气助阳、活血通脉功效的中药复方消斑通脉合剂防治PTCA术后再狭窄的理论依据。研究结果:消斑通脉合剂可以应用于PTCA术后再狭窄的防治。第二部分实验研究实验一消斑通脉合剂对大鼠VSMC增殖的影响研究目的:通过观察不同体积分数的消斑通脉合剂中药血清对体外培养的大鼠VSMC增殖的影响,了解消斑通脉合剂中药血清是否具有抑制体外培养的大鼠VSMC增殖的作用,并确定最佳给药浓度。研究方法1.中药血清的制备:SD大鼠30只,雄性,体重180～220g,6～8周龄。随机分为正常血清组、中药血清组,中药血清组给予大鼠消斑通脉合剂中药复方常规煎剂(含生药2g/ml),按35g/kg/d灌胃,每天灌胃两次,每次间隔12h;正常血清组灌服等体积生理盐水。连续灌胃7d,末次给药2次,中间间隔lh,最后给药后lh,水合氯醛麻醉,无菌操作下取血。血样常温放置4h后4℃过夜, 2000rpm离心15min,0.22um微孔滤膜过滤,分装于EP管中,-20℃保存备用,用之前56℃水浴灭活30min。2.大鼠血管平滑肌细胞体外培养:大鼠A10主动脉VSMC细胞株采用高糖DMEM培养基培养,其中含有10%的FBS,青霉素(100U/ml)和链霉素(100ug/ml),细胞在37℃、5%C02、饱和湿度条件下培养。每2～3天更换培养基一次。待细胞生长至80～90%融合时,进行传代。并经血管平滑肌肌动蛋白(α-actin)免疫组织化学鉴定。3.MTT比色法测定消斑通脉合剂中药血清对VSMC增殖的影响将细胞分为4组,即正常血清组、5%中药血清组、10%中药血清组、20%中药血清组,每组10个复孔,5%中药血清与10%中药血清为20%中药血清用正常血清稀释而成,每孔保持20%血清浓度。细胞于37℃、5%C02、饱和湿度条件下分别培养为24h、48h、72h后,在不同的终点时间均换成无血清DMEM100ul,并加入MTT(5mg/ml) 20uL,继续孵育4h后,吸弃培养液,然后加入二甲基亚砜(DMSO) 150uL/孔,放于水平摇床轻微震荡10 min;用酶标仪在490nm波长处测定24h、48h、72h每个时间点各组细胞的吸光度(OD值),结果取均值(空白孔调零);并计算细胞增殖抑制率。对不同时间点各组细胞的OD值结果进行统计分析,得出最佳给药浓度。研究结果:MTT比色法结果显示,5%的中药血清组在24h、48h、72h时间点的与正常血清组比较,无统计学差异。而10%、20%的中药血清组在24h、48h、72h时间点的与相同体积分数正常血清组比较,其OD值在24h、48h、72h均有明显统计学差异,证明10%、20%的中药血清组具有抑制VSMC增殖的作用,在24h～48h之间表现最为明显。并且20%的中药血清组在24h、48h、72h时间点的抑制率均明显大于10%中药血清组,提示消斑通脉合剂中药血清呈时间和剂量依赖性的抑制VSMC增殖。实验二消斑通脉合剂对大鼠VSMC细胞周期及相关调控蛋白的影响研究目的:通过观察消斑通脉合剂中药血清对大鼠VSMC细胞周期分布及细胞周期相关调控蛋白的影响,了解消斑通脉合剂中药血清抑制VSMC增殖的作用机理。研究方法:根据MTT的结果,选择最佳中药血清浓度,分为正常血清组、中药血清组分别对VSMC作用12h、24h,用流式细胞仪碘化丙啶(PI)染色法分析正常血清组、中药血清组VSMC在12h、24h时间点细胞周期分布情况,对12h、24h时间点的流式细胞仪分析结果进行统计分析;选择最佳中药血清浓度,分为正常血清组、中药血清组,分别对VSMC作用6h、12h、18h、24h后收集细胞,应用Western blot的方法检测VSMC中Cyclin Dl、Cyclin E、p27蛋白在不同时间点的表达变化,并对结果进行统计分析。研究结果:流式细胞仪细胞周期分析结果表明,消斑通脉合剂中药血清在一定的范围内抑制VSMC由G0/G1期向S期转化,与正常血清组在12、24h两个时间点比较,中药血清组明显延缓了细胞周期进程,其G0/G1期比例较对正常血清组显著增多,而S期所占比例明显下降,使细胞周期阻滞于G1期。Western blot检测结果显示消斑通脉合剂20%中药血清呈时间依赖性的明显延缓Cyclin Dl、Cyclin E蛋白表达,与正常血清组比较有显著性差异。中药血清组对p27蛋白在不同时间点的表达没有影响,但与正常血清组比较,在12h、18h、24h时间点有显著差异。实验三消斑通脉合剂对大鼠VSMC增殖信号通路ERK的影响研究目的:通过观察消斑通脉合剂中药血清对大鼠VSMC磷酸化ERK1/2蛋白的表达变化,探讨消斑通脉合剂中药血清抑制VSMC增殖作用的信号转导途径。研究方法:选择最佳中药血清浓度,分为正常血清组、中药血清组分别对VSMC作用4h,分别在1h、2h、3h、4h收集细胞,应用Western blot的方法检测VSMC磷酸化ERK1/2蛋白表达变化。研究结果:Western blot法检测结果显示:消斑通脉合剂中药血清可以降低VSMC中磷酸化ERK1/2蛋白的表达水平,与正常血清组比较有显著性差异。实验四消斑通脉合剂对大鼠VSMC凋亡及Akt信号通路的影响研究目的:通过观察消斑通脉合剂中药血清对大鼠VSMC凋亡及凋亡相关信号通路Akt蛋白表达变化的影响,探讨消斑通脉合剂中药血清诱导VSMC凋亡作用的信号转导途径。研究方法:选择最佳中药血清浓度,细胞分为正常血清组、中药血清组分别对VSMC作用48h后,用透射电镜观察VSMC的超微结构改变;选择最佳中药血清浓度,细胞分为正常血清组、中药血清组分别对VSMC作用24h、48h后,应用流式细胞仪通过AnnexinV-FITC/PI双染方法分析VSMC在24h、48h凋亡率;选择最佳中药血清浓度,细胞分为正常血清组、中药血清组继续孵育4h,分别于1h、2h、3h、4h收集细胞,应用Western blot的方法检测磷酸化Akt蛋白表达变化。研究结果:透射电镜观察结果显示:正常血清组细胞形态正常,细胞核仁形态规则,核仁清晰;中药血清组对VSMC作用48h后,出现了典型的细胞凋亡表现,如细胞核形态不规则,胞质浓缩,核仁消失,核染色质固缩,边集于核膜下,内质网肿胀等。流式细胞仪Annexin V-FITC/PI双染方法凋亡率检测结果示:中药血清组对VSMC作用24h、48h后,随着作用时间的延长VSMC凋亡率明显增加;在24h、48h两个时间点与正常血清组比较有显著性差异;中药血清组在24h、48h两个时间点比较亦有显著性差异。Western blot法检测结果:中药血清组可以降低VSMC磷酸化Akt蛋白的表达水平,与正常血清组比较有显著性差异。结论1.消斑通脉合剂中药血清通过阻滞VSMC由G0/G1期向S期转化,延缓细胞周期进程而达到抑制VSMC增殖的作用。2.消斑通脉合剂中药血清延缓细胞周期进程与其降低ERK1/2蛋白与Akt蛋白磷酸化的表达水平密切相关,通过降低ERK1/2及Akt蛋白磷酸化的表达水平,延缓细胞周期蛋白Cyclin Dl的表达,并减少了p27蛋白的降解,p27蛋白的降解减少又可进一步抑制Cyclin/CDK复合物激活,从而使G0/G1期细胞向S期的转化减少。3.消斑通脉合剂中药血清抑制VSMC增殖的作用与其具有诱导VSMC凋亡作用直接相关。通过降低Akt蛋白磷酸化的表达水平,影响其下游底物的抗凋亡作用,VSMC凋亡增加,进而起到抑制VSMC增殖。4.消斑通脉合剂中药血清可通过多角度、多靶点抑制VSMC增殖,其作用机理符合现代分子生物学关于再狭窄机理的研究结果,可以作为临床再狭窄的防治药物。5.再狭窄应用益气助阳、活血化瘀并用的治疗方法是切实可行的,符合中医辨证论治的理论思想。更多还原

【Abstract】 Background: Restenosis after percutaneous transluminal Coronary Angiography (PTCA)is the main problem to influence the prognosis of intervention treatment to Coronary Heart Disease,and restricts Long-term effect of Percutaneous coro nary intervention seriously(PCI),it proves to be the hot point and difficulty in the field of cardiovascular. Abnormal proliferation of vascular smooth musc -le cells (VSMCs) is a critical component of atherosclerosis and arterial rest -enosis after angioplasty. Corresponding to the further study to the mechanism of RS molecule,finds that many factors has been related to a response to injury in which the growth factors such as basic fibroblast growth factor (FGF-2) and plateletderived growth factor (PDGF) are released, stimulating proliferation and migration of VSMCs, leading to the formation of a neointima. Binding of PDGF to its receptor leads to the activation of several cell-signaling pathways associated with both VSMC proliferation and VSMC migration which plays a important role in the proliferation and migration of VSMC, such as those related to mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase(ERK)1/2, Phospha-Tidy linositol 3-kinase (PI3-kinase).The pathological base of restenosis is the formation of neointima, while the abnormal proliferation and insufficient apoptosis of vascular smooth muscle cells (VSMCs)are the main courses. Apoptosis of vascular smooth muscle cells (VSMCs) is a well established component of the remodeling that occurs during normal development of the circulatory system as well as during the course of neointimal formation after intervention for atherosclerosis. Because increased total cellularity is a prominent feature of an occluding neointima, the balance between proliferation and apoptosis during vessel healing appears central to this pathologic process. Indeed, accumulating evidence suggests that abnormal VSMC apoptosis leads toneointimal hyperplasia.However, despite the importance of VSMC apoptosis, the precise molecular mech anism underlying the regulation of apoptotic pathways in VSMC remains largely undetermined. Apoptosis is a multistage, genetically controlled process of selective cell deletion. Protein kinases regulate the early stages of apopt osis by phosphorylating key proteins, whereas akt, a family of signaling passway, are the main effectors whose activation results in the characteristic morpho- logical changes associated with programmed cell death such as membrane blebbing, chroma tin condensation, and DNA fragmentation.So vascular smooth muscle cell plays important role in restenosis, it is also the target cell in the research of restenosis prevention. The proliferation of vascular smooth muscle cells is a crucial pathophysiological process in the development of restenosis after coronary angioplasty. Corresponding to the further study to the mechanism of restenosis in the stage of molecular level, people find that there are many factors participate in the process of prolifer ation and apoptosis of vascular smooth muscle cell such as cell factor, growth factor et al, which affect cyclin and relating apoptosis gene of cell by active ting ERK, PI3K/Akt the passageways of signal transmission. So aiming at inter -vening the regulating proliferation and apoptosis of vascular smooth muscle cell is the point of penetration.The pathogenesis of restenosis asthenia in origin and asthenia in superf icia lity in the eyes of Traditional Chinese Medicine (TCM), the deficiency of viscera qi is root, blood stasis and Phlegm are the sign. The main therapies to prevent restenosis are invigorate the circulation of blood and remove stagnate, dispel Phlegm, regulating liver and spleen and so on. We consider that the main therapy to cure restenosis after PTCA operation is to nourish qi and assist yang, invigo -rate the circulation of blood and remove stagnate as complement in the angle of TCM. To play the role of regulating entirety and ameliorate local as the characteristic of Traditional Chinese Medicine fully. The aim of this study is to observe the influence of the blood serum of compound Xiao ban tong mai mixture to the proliferation and apoptosis of rat aortic vascular smooth muscle cell by cultivating cell in vitro, investigating the possible mechanism of Xiao ban tong mai mixture with the effect of nourishing qi and assisting yang, Clearing stasis to unimpeded vein, aiming at supplying feasible theory evidence to preven -tion of clinical restenosis by using Xiao ban tong mai mixture.PartⅠTo investigate the treatment to RS after PTCA operation to pattern differentiationObjective: To elaborate on the feasibility of Xiaobantongmai mixture which had nourishing qi and assisting yang, invigorating the circulation of blood and removing stagnate to prevent artery from restenosis after PTCA operation in the eyes of Traditional Chinese Medicine.Methods: According to the features of age, symptom, sign that restenosis patients had, taking the theory of yin-yang which the core of TCM as starting point, combining related classical literature that record in Hang Di Nei Jing and Jin Kui Yao Lue,applying the basic theory of yin- yang, qi and blood in TCM and ideology of treatment according to pattern differentiation, and associating research findings of modern pharmacology seek theoretical evidence of Xiao ban tong mai mixture which had nourishing qi and assisting yang, invigorating the circulation of blood and removing stagnate to prevent artery from restenosis after PTCA operation.Results: Xiao ban tong mai mixture could be applied to prevent restenosis after PTCA operation.PartⅡTo observe the influence of Xiaobantongmai mixture to the proliferation of the rat aortic vascular smooth muscle cellObjective: According different proportion serum of Xiaobantongmai mixture to affect proliferation of the rat aortic vascular smooth muscle cell cultivated in vitro, understanded whether had the effect of Xiaobantongmai mixture to affect proliferation of the rat aortic vascular smooth muscle cell cultivated in vitro or not, and made sure the best giving concentration.Methods: The preparation of herbal serum. Choosing thirty male SD rats, weight between 180 and 220 gram, age between 6 and 8 weeks, rats were randomly assigned into two groups with 15 in each group (the normal serum group, the herbal serum group),the herbal serum group:Taking in Xiao ban tongmai mixture apozemwhose dose of intragastric administration was forty gram every kilogram everyday, Xiaobantongmai mixture was boiled in routine, the frequency of Xiaobantongmai mixture was twice a day, 12 hours interval, while giving the same dose normal saline to the normal serum group, continuing 7 days. At last time, rats were gave the herb two times as original dose,the interval of the two times was one hour ,after 1 hour at the last time, then took blood in a sterile operation manner 1 hour before gave herb. Rats were anesthesed by chloral hydrate.The sample of blood was laid up in a four centigrade degree refrigerator till the next day after placed in normal temperature 4 hours. 2000 rpm in the centrifuge for 15 minutes, then filtrated though 0.22um filter membrane, and packed the blood serum respectively in EP pipe, conserved in negative 80 centigrade degree in refrigerator, inactivated it in 56 centigrade degree water for 30 minutes before herb serum being used.Cells culture. The A10 rat aortic vascular smooth muscle cell line was obtained from the American Type Culture Collection (ATCC). The A10 rat VSMC derived from thoracic aorta of fetal rats were prepared as previously described. The cells were maintained in a humidified incubator with 5% CO2 and grown on 100-mm culture dishes in Dulbecco’s modified Eagle’s medium(DMEM) with high glucose, that was supplemented with 10% fetal bovine serum 100 units/ml penicillin, and 100 ug/ml streptomycin. The cells were split 1:10 when approaching confluence. Aliquots in DMEM/fetal bovine serum/DMSO (4:5:1) were prepared for storage in liquid nitrogen. The medium was replaced every 2 days until the time of all experiments.the medium was changed to serum-free DMEM 24h before experiments. The influence of serum of Xiaobantongmai mixture to proliferation of rat aortic vascular smooth muscle cell were detected using MTT color metric assay. There are four groups in all according to different proportion serum of Xiao ban tong mai mixture,the concentration of herbal blood serum was five percentage, ten percentage,twenty percentage, and the normal serum.There were ten repeated holes in each group, the five percentage serum and ten percentage serum were consisited of twenty percentage serum and the normal serum,every hole kept concentration as twenty percentage serum .cells were cultivated in 37 centigrade, a humidified incubator with 5%CO2 and grown on 100-mm culture dishes in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, that was supplemented with 10% fetal bovine serum 100 units/ml penicillin, and 100 ug/ml streptomycin for 24 hours,48 hours,72 hours respectively, then exchanged Dulbecco’s modified Eagle’s medium with no fetal bovine serum at different time, then added MTT (5mg/ml )20ul,continued cultured for 4 hours,throwed the nutrient solution away ,detected the each time point value of OD by Microplate reader device, Data are presented as mean±S.E.M. of the number of independent experiments shown in each Figure,then obtain the optimal experimental concentration.Results: The result of MTT color metric assay revealed that there was no significant difference between the five percentage herbal serum at different time with 24 hours, 48 hours, 72 hours and the normal serum. There was no significant difference between normal serum and 5 percentage herbal serum, while on the percentage of 10 and 20 herbal serum, there was significant difference about the value of OD comparing to normal serum, illuminated that 10 and 20 herbal serum had effect on the proliferation of rat vascular smooth muscle cell, and the most evident effect was between 24 hours and 48 hours, in addition, the inhibition ratio of 20 percentage was obviously higher than 10 percentage. The herbal blood serum inhibited the proliferation of rat aortic vascular smooth muscle cell in a concentration-dependent and time-dependent manner.PartⅢObjective: According to observe the influence of Xiaobantongmai mixture to the cell cycle of the rats’aortic vascular smooth muscle cell and regulated the protein that related, understanded the active mechanism of blood serum of Xiaobantongmai mixture to inhibite proliferation of the rat aortic vascular smooth muscle cell.Methods: The rat vascular smooth muscle cells were plated at culture dish. According to the result of MTT, choosed the optimal concentration of herbal serum, cells was divided into normal serum and herbal serum. The media were then removed and fresh growth media with drugs were added, acted on the cell for 12 hours and 24 hours respectively. The medium was carefully removed and combined with a 1ml phosphate-buffered saline (PBS) rinse to constitute the“detachedsample”. The adherent cells were trypsinized and suspended in growth medium. Samples of the adherent and detached suspensions were counted and assessed for viability by exclusion of trypan blue. The remaining cell suspensions were washed 1×with PBS and then suspended in PBS before being fixed and processed for flow cytometric analyses of DNA content as described previously. Percentages of proliferation cells and cells in the G1, S, and G2/M stages of the cell cycle were determined with a DNA histogram-fitting program.Attempts were made to collect a minimum of 104events/sample for subse quent analyses.Choosed the optimal concentration of herbal serum, cell was divided to normal serum and herbal serum, collected cell after serum acted on the cell for 6 hours,12 hours,18 hours,24 hours respectively .Then VSMC were starved as described above for 24 hours and then treated with the various agents indicated in the text. They were then washed twice with phosphate-buffered saline (PBS) and lysed in ice-cold lysis buffer. Cells were detected the expression of cyclin Dl、cyclin E、p27 protein of cells were detected by Western blot. Then carried out statistical analysis on the result. Data are presented as mean±S.E.M. of the number of independent experiments shown in each Figure. Differences from control were assessed by analysis of variance with ANOVA tests when multiple comparisons were required.Results: The analytical result of cell cycle detected by Flow cytometry show that serum of Xiaobantongmai mixture inhibited the transformation from G0/G1 phase to S phase in a definite range. comparing to the control group , herbal serum delayed the process of cell cycle obviously,the proportion that on G0/G1 phase was more than normal serum, while on the S phase the proportion went down sharply, cell blocked on the G1 phase. The analytical result of Western blot revealed that 20 percentage of Xiaobantongmai mixture serum was in a time- dependent manner, postponed the expression of cyclin Dl、cyclin E obviously, there was significant difference between the control group and the herb group, p27 was not affected by herbal serum, but comparing to normal serum, there was no distinction on the 6 hours , on the 12 hours, 18 hours, 24 hours, there was significant difference.PartⅣObjective: According to observe the influence of Xiaobantongmai mixture to the expression of phosphorylatal ERK1/2, discussed the signal pathway of Xiao bantongmai mixture serum to inhabited the proliferation of rat vascular smooth muscle cell.Methods: Choosing the optimal experimental concentration, divided into the normal serum and the herbal serum, acted on the rat aortic vascular smooth muscle cell for 24 hours repectively. VSMC were starved as described above for 24 hours and then treated with the various agents indicated in the text.then went on cultured for 4 hours, while collected cell. They were then washed twice with phosphate-buffered saline(PBS) and lysed in ice-cold lysis buffer.To detected the expression of phosphorylatal ERK1/2 protein of rat vascular smooth muscle cell in the way of Western blot.Results: The result of Western blot revealed that Xiaobantongmai mixture serum could down-regulate the expression of phosphorylatal ERK1/2 of rat vascular smooth muscle cell, there was significant difference comparing to normal serum.PartⅤObjective: According to observe the influence of Xiaobantongmai mixture serum to the apoptosis of rat vascular smooth muscle cell, the expression of Akt protein which related the passageways of apoptosis, discussed signal transductive channel of apoptosis that induced by Xiaobantongmai mixture serum.Methods: Choosing the optimal experimental concentration, divided into the normal serum and the herbal serum, acted on the rat vascular smooth muscle cell for 48 hours repectively, then observed the ultrastructure through transmission electron microscope; Choosing the optimal experimental concentration, divided into the control group and the herbal group, acted on the rat aortic vascular smooth muscle cell for 24 hours and 48 hours. Apoptosis was determined by flow cytometry using the“Annexin V-FITC Apoptosis Detection Kit I”, according to the manufacturers instructions. Choosing the optimal experimental concentration, divided into the control group and the herb group, acted on the rat aortic vascular smooth muscle cell for 24 hours repectively, VSMC were starved as described above for 24 hours and then treated with the various agents indicated in the text. Then went on cultured for 4 hours, while collected cell. They were then washed twice with phosphate buffered saline (PBS) and lysed in ice-cold lysis buffer. And analyzed the expression of phosphorylatal Akt of rat vascular smooth muscle cell in the way of Western blot.Results: The result of transmission electron microscope revealed that: the morphology of cell in normal serum was in a average state, the nucleolus was clear, while in the herbal serum the morphology of cell manifested typical apoptosis, for example, cell nucleus was irregular, concentratd cytoplasm, the nucleolus of the cell was disappear, the pyknosis of chromatin, gathered under the caryotheca aside, the swelling of endoplasmic reticulum etc. The result of Annexin V-FITC/PI double staining detected via Flow cytometry (FCM) showed that: Herbal blood serum acted on rat vascular smooth muscle cell for 24 hours and 48 hours, apoptotic rat vascular smooth muscle cell of rat could be increased obviously along with prolong acted time, there was significant difference comparing to normal blood serum on the time point of 24 hours and 48 hours,it also had significant difference between 24 hours and 48 hours. The result of Western blot revealed that Xiaobantongmai mixture serum could down-regulate the expression of phosphorylatal Akt of rat vascular smooth muscle cell in the herbal blood serum, and there was significant difference comparing to normal blood serum.Conclusions:1.The herbal serum of Xiaobantongmai mixture inhibited the proliferation of rat vascular smooth muscle cell in a concentration-dependent and time-dependent manner.2.The herbal serum of Xiaobantongmai mixture postponed the expression of Cyclin Dl、Cyclin E, promoted the expression of p27, as a result, the proliferation of rat vascular smooth muscle cell was inhabited.3.The herbal serum of Xiao ban tong mai mixture that influenced the trans for mation from G0/G1 phase to S phase and inhibited the proliferation of rat vascular smooth muscle cell was connected with the fall of the expression of phosphorylatal ERK1/2.4.The herbal serum of Xiaobantongmai mixture that influenced the transformation from G0/G1 phase to S phase and inhibited the proliferation of rat vascular smooth muscle cell was connected with the fall of the expression of phosphorylatal Akt, according to reduced the expression of phosphorylatal Akt to induce the apoptosis of rat vascular smooth muscle cell.5.According to influence the signal passageways of ERK1/2 and Akt, the proliferation of rat vascular smooth muscle cell was inhabited and the apoptosis of rat vascular smooth muscle cell was induced to some extent.6.The herbal serum of Xiaobantongmai mixture could influence the biological behavior of VSMC through angle and multi-point,could be used for the prevention from restenosis in clinical.7.To nourish qi and assist yang as the main therapy and invigorate the circulation of blood and remove stagnate as complement are feasible to cure restenosis, conform to the theory of Traditional Chinese Medicine that treatment according to pattern differentiation, and conform to research findings of modern molecular biology.更多还原