PTA术后益气活血解毒方药物血清对兔VSMC在RS中作用的研究

【作者】 彭哲；

【导师】 王硕仁；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2004， 博士

【摘要】 PTCA 术后 RS 的病理生理机制研究表明,炎症反应和平滑肌细胞增殖在 PTCA 术后RS 的发生发展中起着关键作用。中医认为本病的病机为本虚标实,以脏气虚弱为本,血瘀、痰浊为标。目前中医防治本病主要通过活血化瘀、化痰去浊、益气养血、调补脾肾、理虚调肝等法的配合运用。我们认为 PTCA 术后 RS 从其病理过程与创伤修复相似,则辨治当类同于中医修复创伤的治法。因此,中药治疗的原则应为修复新伤,针对正气亏虚当扶助正气,同时根据病变局部瘀毒互结的病机特点治以活血解毒,而使心脉通畅,瘀毒两清。益气活血解毒方的组方用药体现了这一基本治法。目的 对比研究中医治疗创伤法及其益气活血解毒方与活血理气法经典方血府逐瘀胶囊在 PTA 后防治 RS 的血清药对培养的兔 VSMC 药效、药理作用的研究。方法 1.药物血清的制备:①整体动物模型及给药:用电刺激兔一侧颈总动脉,同时喂饲高脂饲料的方法造成局部动脉粥样硬化斑块和血栓形成后,运用球囊原位扩张和喂饲高脂饲料致 PTA 术后 RS 模型,对模型颈动脉进行 B 型超声和病理切片相结合研究评价,造模成功后给予含益气活血解毒方、血府逐瘀胶囊和西药(普伐他汀、巴米尔)的饲料进行治疗四周;②药物血清获取:获取模型动物和给药动物血清,正常组不施加干预因素,与其它动物同期采血。 2.兔血管平滑肌细胞(VSMC)原代培养:采用组织贴块法进行兔 VSMC 的原代培养,在倒置显微镜下观察细胞形态,并经血管平滑肌肌动蛋白(α-SM actin)免疫组织化学鉴定。 3.观察药物血清对离体定位靶细胞——VSMC 作用 12h、24h 的影响:①药物血清对VSMC 增殖和细胞周期的影响:采用四唑蓝(MTT)比色法测定 VSMC 存活率,采用流式细胞仪碘化丙啶(PI)染色法测定 VSMC 细胞周期、DNA 合成及凋亡;②药物血清对 MDA、SOD、NO、NOS 的影响:采用 MDA 测定试剂盒微量操作法、黄嘌呤氧化酶法分别测定培养上清液中 MDA 含量和 SOD 活力;采用硝酸还原法、一氧化氮合酶测定试剂盒分别测定培养上清液中 NO、NOS 含量;③药物血清对 IL-6、TNF-α、MIP-1、MCP-1 的影响:采用双抗体夹心 ABC-ELISA 法分别测定培养上清液中 IL-6、TNF-α、MIP-1、MCP-1 含量;④ 药物血清对 VSMC 分泌羟脯氨酸的影响:采用羟脯氨酸比色法,测定培养上清液中羟脯氨酸的含量。结果 1.益气活血解毒方药物血清通过 12h 调控 G2-M 转折点、24h 调控 G1-S 转折点,调控细胞周期进而显著抑制 VSMC 增殖,12h、24hMTT 结果(0.396±0.015、0.397±0.031)<WP=5>-2- PTA 术后益气活血解毒方药物血清对兔 VSMC 在 RS 中作用的研究与模型血清组(0.435±0.019、0.479±0.027)比较,p<0.05。血府血清组通过 24h 调控 G1-S 转折点抑制 VSMC 增殖,24hMTT 结果(0.422±0.043)与模型血清组比较,p<0.05。西药血清组有相同作用; 2.益气活血解毒方药物血清 24h 增加 SOD 活力(34.71±1.23)与模型血清组(32.44±0.78)比较,p<0.05;其 24h 降低 MDA 含量(1.79±0.17)与模型血清组(2.14±0.15)比较,p<0.05,其降低 MDA 的作用优于血府血清组(2.09±0.13),二者比较,p<0.05;益气活血解毒方药物血清 12h、24h 均具有显著促进 VSMC 分泌 NO(27.78±2.62、33.07±2.31)的作用,与模型血清组(20.63±4.56、18.54±4.06)比较,p<0.05。而血府血清组12h显著增加 SOD活力(35.56±0.53)与模型血清组(33.21±0.79)比较,p<0.05,但 24h(33.94±0.67)无显著作用;血府血清组 24h(28.64±4.99 )有显著促进 VSMC分泌 NO 的作用,与模型血清组比较,p<0.05。西药血清组有相同作用; 3. 西药血清组 12h、24h 均可抑制 VSMC 分泌 IL-6(278.90±56.16、365.24±28.16),与模型血清组(560.16±88.11、498.98±32.18)比较,p<0.05;西药血清组12h、24h 还可抑制 VSMC 分泌 TNF-α(208.58±25.15、199.54±21.99),与模型血清组(457.14±54.62、308.43±16.83)比较,p<0.05。益气活血解毒方药物血清 12h 能显著抑制 VSMC 分泌 IL-6(353.80±47.06),与模型组比较,p<0.05;该方 12h 亦可显著抑制 VSMC 分泌 TNF-α(294.58±26.01), 与模型血清组比较,p<0.05;该方与血府血清组 12h 抑制 VSMC 分泌 IL-6(370.84±58.37)和 TNF-α(291.28±27.89)的作用相似,24h 血府血清组抑制 VSMC 分泌 IL-6(336.84±80.22)的作用强于益气活血解毒血清组(485.04±99.43),p<0.05。三组药物血清以西药血清组的药效为佳; 4. 西药血清组在 12h、24h 均有显著抑制 VSMC 分泌趋化因子 MIP-1(355.56±31.41、350.08±18.92)的作用,与模型血清组(572.40±99.53、438.94±59.65)比较,p<0.05。血府血清组在 12h(364.18±32.82)有显著抑制作用,与模型血清组比较,p<0.05。益气活血解毒方药物血清未显示出作用;各药物血清组均未显示出抑制 VSMC 分泌 MCP-1 的作用; 5. 益气活血解毒方药物血清 12h、24h 均显示出抑制 VSMC 分泌胶原(0.2825±0.017、0.2870±0.027)的作用,与模型血清组(0.3807±0.037、0.3494±0.037)比较,p<0.05,12h 其抑制 VSMC 分泌胶原的作用较血府血清组(0.3501±0.047)和西药血更多还原

【Abstract】 The study of pathophysiological mechanism of RS after PTCA indicates inflammatoryreaction and smooth muscle cell proliferation play important roles in development of RS afterPTCA. It’s considered in Chinese medicine that the pathogenesis is deficiency in fundamentalaspect and excess in accidental aspect, the fundamental aspect is deficiency in Zhang Qi, theaccidental aspect is blood stasis and sputum turbid. The current preventing and treatingmethods in Chinese medicine are combination of activating blood and removing stasis,removing sputum and dispelling turbid, replenishing Qi and nourishing blood, regulating andnourishing spleen and kidney, treating deficiency and regulating liver. We consider that thepathological process of RS after PTCA is similar to repair in trauma, so the differentiation andtreatment are similar to the treatment of repair in trauma by Chinese medicine. So, theprinciple in Chinese medicine is repairing the new wound, helping the real Qi for Qideficiency, activating the blood and detoxicating according to the mechanism character oflocal combination of stasis and heat, so the heart channel flows fluently, the stasis and hearare removed. The composing of replenishing Qi, activating blood, and detoxicating formula(RADF) embodies this principle.PURPOSE To compare the effect and pharmacological effect of the serum medicine of replenishingQi, activating blood, and detoxicating formula(RADF) which treats trauma and Xuefuzhuyucapsule which is classical formula of activating blood and regulating Qi which can preventand treat RS after PTCA on cultured rabbit VSMC.METHODS 1.Preperation of the serum medicine: ①Replication of animal model and administration:one side common carotid of the rabbits were stimulated by the electricity, meanwhile, theywere fed with high fatty feedstuff, which resulted in local atherosclerosis plaque andthrombosis. The RS model was replicated by balloon in situ expanding and feeding high fattyfeedstuff and PTA, the common carotids of the model were assessed by B-mode ultrasonicsand part pathological section. The feedstuff with replenishing Qi and activating blood formula,Xuefuzhuyu capsule, and modern medicine(Pravastatin, Aspirin) had been given to theanimals for four weeks when the model were replicated; ②Taking of medicine serum: theserum of the model animals and the administrated animals was taken, the serum of the controlgroup animals which received no intervene factor was taken at same time. 2.Primary culture of Rabbit’s vascular smooth muscle cell: rabbit’s VSMC were primarycultured through tissue anchoring method, the cell form were observed under the invert<WP=8>英文摘要 -5-microscope, and the vascular smooth actin(α-SM actin) was assayed by immunohistoche-mistry method. 3.Observation of effects of serum containing medicine on localizing target cell—VSMCat 12h, 24h: ①Effects of serum containing medicine on VSMC proliferation and cell cycle:the survival rate of VSMC was determined through MTT, the cell cycle, DNA synthesis, andapoptosis were assayed by PI stain and flow cytometer methods;②Effects of serumcontaining medicine on MDA, SOD, NO, NOS concentration: MDA and SOD concentrationwere assayed by MDA test kit trace method and xanthine oxidase method respectively; NOand NOS concentration in the upper liquid were assayed by nitric reduction and NOS test kit;③Effects of serum containing medicine on IL-6, TNF-α, MIP-1, MCP-1 concentration: IL-6,TNF-α, MIP-1, MCP-1 concentration had been tested through double antibody sandwichABC-ELISA method;④Effects of serum containing medicine on hydroxyproline excretion ofVSMC: hydroxyproline concentration in upper liquid of the cultured cell was determinedthrough hydroxyproline chromatometry.RESULTS 1.The serum containing RADF could regulate cell cycle ,and then inhibit VSMCproliferation significantly through regulating G2-M turning point at 12h,G1-S turning point at24h, the MTT更多还原