中药对动脉粥样硬化相关基因表达调控作用的研究

【作者】 林琦；

【导师】 李杰芬；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2000， 硕士

【摘要】 动脉粥样硬化（As）是影响人类健康常见的疾病之一。它以细胞增殖，尤其是血管平滑肌细胞（VSMC）的异常增生和迁移为特征。多年来医学界对SMC增殖的起因提出过各种学说，其中以Ross提出的损伤反应学说较有影响。自从癌基因发现以来，针对癌基因表达与肿瘤产生的关系进行了大量的研究。鉴于As也是一种以细胞增殖为主的疾病，所以人们应用分子生物学技术以癌基因为探针对As进行了一些探索性工作。结果发现As的发生与癌基因关系密切：动脉粥样硬化时，某些癌基因表达明显增强，一些诱发As的因素如内皮细胞（EC）损伤、胆固醇等亦可促进癌基因的表达。已经证明，癌基因sis、myc和fos在SMC增殖时表达增强，对As产生具有重要意义。内皮素（ET）作为一种促有丝分裂剂，可明显促进VSMC分裂增生。在人体As斑块中，增生的VSMC能产生丰富的ET，且VSMC增生的程度与ET含量在一定范围内呈正比。进一步研究发现，在氚标胸腺嘧啶核苷（~3H-TdR）掺入量和VSMC数增加之前先有c-fos和c-myc mRNA表达增加，说明ET可通过激活核内癌基因表达而促进细胞增生。此外，迄今发现与动脉粥样硬化较密切的基因还有血小板衍化生长因子-A链（PDGF-A）、过氧化物酶基因、一氧化氮合酶（NOS）基因、抑癌基因等。 从中医角度来看，As是一种全身性疾病，属本虚标实。心脾肾三脏为病之本，气滞血瘀痰积为病之标。由于心脾肾的亏损，导致气滞血瘀，痰积内生，脉络不通。治疗上以健脾益气、滋阴养血、补益肝肾以治其本，活血化瘀、软坚散结、消食化痰、通腑化浊以治其标。中药抗As的临床和实验研究均证实，依据上述理论所选的方药具有不同程度的抗As作用。而且，随着对As发病机制认识的不断深入，中药抗As的研究已经深入到基因水平。近年已有报道，某些中药或复方如血管通可通过抑制PDGF-A、c-myc mRNA 中药对动脉粥样硬化相夫基因表达调控作用的研究 毕业论文表达而抑制 SMC增生，阻止 AS形成。中药丹参和）；l穹均有活血化瘀之功，而灵芝则有补益肝肾作用，三药均是临床上常用于治疗AS的药物。本研究以上述的c。myc、ET为指标，分别观察三药对C－yC、ET m删A转录水平的影响，从而探讨此三药对AS相关基因表达的调控作用。 实验过程中，我们采用培养的新西兰兔主动脉平滑肌细胞第4～6代，给予同步处理后，分别加入灵芝提取物（三砧类）侣0ng／ml；160ug／mlL丹参酮＊ a磺酸钠u0 11 g／il人盐酸）I！穹嗓＊刀2％）及刺激剂血管紧张素＊ （ Allg 11，10“’mol／L），然后提取细胞总 RNA，利用 RT－PCR法，通过凝胶成像分析仪分析细胞内 C－iyC、ET 11：lltNA表达情况，从而观察药物的作用。结果发现：Aug 11能明显促进 SMC中 C－pC、ET tnRNA表达。盐酸川穹嗓、丹参酮 11a磺酸钠、灵芝提取物（5＠类）均能抑制 SMC中小m以c、ET mRNA的过量表达，提示川穹、丹参和灵芝可能具有抑制SMC增殖作用。但三药的作用强度却不同：其中灵芝作用最弱，丹参和j！I穹的作用较强。更多还原

【Abstract】 The proliferation of vascular smooth muscle cell (VSMC) is an important factor in forming atherosclerotic plaque. From the discovery of oncogene on, scientists find there are close relations between oncogenes and atherosclerosis (As). Many studies have indicated that oncogenes’expression increases obviously as As occurs; some factors such as cholesterol can also promote its’expression. For example: compared to the normal VSMCs, the expression of myc, fos in SMCs of atherosclerotic plaque reinforces. Endothelin (ET) can promote the fission and proliferation of VSMCs. Several studies showed that the expression of c-myc mRNA increased before the entrance of3H-TdR and addition of VSMC’s number. That’s to say, ET can promote VSMCs’proliferation by increasing some oncogenes’expression. Thus we investigate the levels of c-myc and ET mRNA in the cultured VSMCs. In addition, As is a generalized disease in Traditional Chinese Medicine(TCM), belonging to Root vacuity and Manifestation excess. Heart, spleen and kidney are 2 the Root, qi stagnation, blood stasis and phlegm accumulation are the Manifestation. Treating the Root involves fortifying the spleen and boosting qi, enriching yin and nourishing the blood, supplementing and boosting the liver and kidney; treating the Manifestation involves quickening the blood and transforming stasis, softening hardness and dissipating binds, dispersing food and transforming phlegm, freeing the bowels and transforming turbidity. Herbs selected according to these principles can resist As through clinical and experimental studies. For example, Xue Guang Tong can inhabit the proliferation of VSMCs by influencing the expression of c-myc mRNA. In our test, we select Sodium Tanshinone Ⅱ a Sulfonate (TN Ⅱa), Chuanxiongzine hydrochloride and the extraction of Ganoderma, lucidum (triterpene) to observe their effects. During the experiment, the models of cultured VSMCs induced by Angeotensin Ⅱ (107mol/L) were established. The cells Were randomly divided into six groups. After giving them suitable treatment, extract total RNA, use RT-PCR to analysis the results. The results are: all of the three herbs can inhabit the excessive expression of c- myc, ET mRNA in the VSMCs, but can make them return to normal.更多还原