【作者】 施华平；

【导师】 马健；

【作者基本信息】 南京中医药大学 ， 中医临床基础， 2010， 博士

【摘要】 卫生部全国第三次死亡原因调查结果显示,恶性肿瘤仅次于脑血管病成为我国城乡居民因病死亡的主要疾病,与之前调查结果相比,恶性肿瘤死亡率比上世纪70年代中期增加了83.1%。现代医学对肿瘤的治疗历经手术、放疗、化疗及生物治疗阶段,近来众多学者又提出肿瘤综合治疗的概念,虽然与过去相比不断得到完善,但在诊断治疗上还存在不少缺陷。如早期肿瘤未转移者；不适于手术、放疗、化疗及患者不愿意西医治疗者；晚期癌痛西药无效者；已经接受手术、放疗、化疗需要中医减轻并发症及辅助治疗的患者等,这些缺陷正是现代医学亟待研究的课题。而传统中医药在某些方面却有着一定的优势,如何充分发挥这些优势,为恶性肿瘤的治疗找到一条新的有效的途径,积极研究中医药抗肿瘤的组方并探索其配伍规律,通过在实验研究的基础上改良或创制新的有效处方为广大肿瘤患者提供中医药服务,正是我们研究的出发点和目的之所在。恶性肿瘤在古代中医文献中早有很多记载,属于癥瘕积聚范畴,其产生的病因病机比较复杂,临床表现多有瘀热之证,瘀热作为一种新的致病因素越来越受到业界的重视,查阅文献资料发现,瘀热互结是恶性肿瘤的一个重要病机。现代医学也证实血瘀是恶性肿瘤形成发展的主要病理机制,而且出现在恶性肿瘤的生长期、浸润期和转移期各个阶段,肿瘤患者同时存在着血液粘滞度增高、血液流变学异常及微循环障碍等病理性改变。活血化瘀祛邪成为肿瘤治疗中广泛采用的重要手段之一,同时在疾病的不同阶段兼顾扶正补虚。抵当汤是张仲景活血化瘀的经典方,主要用于太阳阳明蓄血证及妇人经不不利下之闭经,由水蛭、虻虫、大黄、桃仁四味药物组成,具有破血逐瘀,软坚散结之功,文献报道抵当汤有抗肿瘤作用。根据中医理论的指导和对历代活血化瘀方配伍规律的研究,我们在抵当汤原方基础上分别加上行气药柴胡、温经药桂枝和补气药黄芪,再进行实验比较研究。本文观察了抵当汤对疼痛炎症的影响；观察了抵当汤及其加味方对人乳腺癌MCF-7细胞,人结肠癌HCT-28细胞,人肝癌HepG-2细胞的影响；研究了其对S180荷瘤小鼠的肿瘤生长、机体免疫状态、肿瘤局部及全身血液循环、外周血液中的白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)的影响。以期通过系列实验明确抵当汤及其加味方的作用,并在此基础上分析抵当汤及其加味方的组成,在中医理论的指导下,探求其组方的原则和规律。1.抵当汤镇痛抗炎作用：以耳肿法和醋酸扭体法观察抵当汤对炎症疼痛的影响,结果表明抵当汤低、中、高剂量组均可明显对抗二甲苯所致小鼠耳肿胀和醋酸引起小鼠扭体反应,且存在一定的量效关系；2.抵当汤体外抑制肿瘤细胞的生长：采用MTT法观察抵当汤对人MCF-7、HCT-28、HepG-2增殖的影响,结果表明抵当汤对人MCF-7、HCT-28、HepG-2细胞均有一定的杀伤能力,其中人MCF-7和HepG-2细胞对其更为敏感；3.抵当汤对S180荷瘤小鼠的抑瘤作用：以S180荷瘤小鼠为模型,观察抵当汤对瘤重、免疫器官脏器指数和白细胞计数的影响,结果表明抵当汤中、高剂量组可明显抑制S180荷瘤小鼠的瘤重、提高S180荷瘤小鼠脾指数和胸腺指数及外周血白细胞数量；4.抵当汤及其加味方对体外肿瘤细胞生长的影响：采用MTT法观察抵当汤及各加味汤对人MCF-7、HCT-28、HepG-2生长的影响并计算IC50,结果表明各加味汤均能抑制癌细胞的增殖,且较抵当汤明显,抵当汤加柴胡组优于抵当汤加桂枝组而又优于抵当汤加黄芪组；5.抵当汤加味方对S180荷瘤小鼠的抑瘤影响：以S180荷瘤小鼠为模型,观察各方对抑瘤率、机体免疫状况、血液指标、肿瘤局部血管及血液循环及外周血白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)的影响,结果表明抵当汤组、抵当汤加柴胡组、抵当汤加桂枝组均可明显抑制肿瘤的生长,抵当汤加用柴胡和桂枝后,抑瘤率有进一步增加,其中加用柴胡组与单用抵当汤相比抑瘤率有明显增加,提示加用柴胡和桂枝可加强抵当汤的抑瘤效应。抵当汤及分别加入柴胡、桂枝、黄芪三个加味方都有促进外周血液循环的作用,其中以抵当汤加桂枝组可明显提高小鼠耳廓微循环；抵当汤及分别加入柴胡、桂枝、黄芪三个加味方都有抑制S180荷瘤小鼠肿瘤局部微循环,其中以抵当汤加黄芪组作用最为明显。抵当汤及加味方对外周血白细胞介素-8(IL-8)和肿瘤坏死因子-α(TNF-α)与对照比有差异,其中加入柴胡组有明显差异。综上所述,1、抵当汤及其加味方有抗肿瘤作用,而分别加入柴胡、桂枝的加味方比加入黄芪和原抵当汤的作用显著,其中加入柴胡组有明显差异；2、抵当汤及加味方能促进外周血液循环而抑制肿瘤局部血液循环；3、抵当汤对体外肿瘤细胞有活性作用,而加入柴胡、桂枝、黄芪的三个加味方活性作用均大于原方抵当汤活性作用,其中加入柴胡、桂枝的活性作用相比黄芪又更为明显,抵当汤及加味方对人MCF-7细胞的生长抑制最为明显。本文通过对抵当汤及加味方组成研究将有助于对中药复方配伍规律科学性的理解,进一步拓宽仲景活血化瘀组方思路,找出活血化瘀配伍的内在规律,为我们组成新的方剂提供实验依据。更多还原

【Abstract】 According to Ministry of Health’s third time survey results on the cause of death:after cerebrovascular disease, malignant cancer is the second main disease causing death in China’s urban and rural area; and compared to the survey on the death rate caused by the malignant tumor in the middle of 1970s, it is increased by 83.1%today. After surgery, radiotherapy, chemotherapy and biological treatment stage, many scholars have recently proposed the concept of comprehensive cancer treatment, which has been refined than in the past, but there are still many deficiencies on the diagnosis and treatment. Such as patient of early cancer without metastasis; patient not suit to surgery, radiotherapy, chemotherapy or do not accept the Western Medicine treatment; medicine is ineffective in advanced cancer patient; patient who has already receive surgery, radiotherapy and chemotherapy needs to receive Traditional Chinese Medicine as adjunctive therapy or reducing the complications, etc.. All these defects are urgent problems need to be resolved by the modern medicine. The traditional Chinese Medicine in some ways has a certain advantage. Our research aims are:How to fully exploit these advantages to find a new effective way for the treatment of the tumor, research Traditional Chinese Medicine prescriptions on tumor and explore the law of compatibility, and based on experimental study to improve or create new effective prescription for the majority of cancer patientsMalignant tumor has been recorded numerously in the ancient literature, which belonged to the sphere of aggregation-accumulation. Its etiological factor and pathogenesis is complicated, and mostly clinical manifestation is heat stasis, which becomes to a new disease factor and more and more highlighted by the medical area. By the literature information, stasis and heat complex is an important pathogenesis inviting the malignant tumor. Modern medicine also proved that blood stasis is a main pathogenesis to cause malignant tumor, and appeared in the growth period, infiltrated period and extension period. The patient of tumor always goes through the pathologic changes like increasing of blood viscosity, abnormality of blood rheological characteristic and disorder of microcirculation. Activate blood, dissipate stasis and eliminate the pathogenic factors are the important methods applied universally in the treatment of tumor, meantime in different phases reinforce the healthy Qi and tonify the deficiency are necessary. Di dang decoction is the classic prescription to activate blood and dissipate stasis created by Zhang Zhong-jing, which is usually applied in great yang blood-retention syndrome or yang brightness blood-retention pattern and aggregation-accumulation of women. Di dang decoction is composed by leech, gadfly, pieplant and persicae, with the power to break blood and expel stasis, and soften hardness and dissipate binds. Besides, it is said that Di dang decoction has the function of anti-tumor. Under the guidance of TCM theory, and the research on compatibility of prescription of blood activation and stasis dissipation, we added bupleuri to move Qi, Gui-zhi to warm meridians and collaterals, and astragali to tonify Qi, and then make comparison research through the experiment.We observed the influence of Di dang decoction on the pain and inflammation, observed the influence of Di dang decoction and its modified formula to breast cancer MCF-7 cell, colon cancer HCT-28 cell, and liver cancer HepG-2 cell; observed the influence of decoction to S180 tumor-bearing mice’s tumor growth, organism immunity, region of tumor and blood circulation all over the whole body, periphery blood interleukin-8, tumor necrosis factor-α. Though the series of experiments, we would like to identify the function of Di dang decoction and its modified formula, then under the direction of TCM, we will give the further analysis to its principle of composition.The anti-inflammatory analgesic function of Di dang decoction:Through the ear swelling method and acetic acid writhing method to observe the impact of Di dang decoction on inflammatory pain, results showed that Di dang decoction in low, middle or high dose group could significantly against the ear swelling and acetic acid writhing response induced by xylene in mice, and there was a certain dose-effect relationship;Extraorgan inhibition of growth of tumor cells of Di dang decoction:MTT was used to observe the influence of Di dang decoction on MCF-7, HCT-28, HepG-2 proliferation in human body, the results showed that Di dang decoction had the certain destruction ability on MCF-7, HCT-28, HepG-2 cells, MCF-7 and HepG-2 cells of which were more sensitive to Di dang decoction;Tumor inhibition on the S180 tumor-bearing mice of Di dang decoction:S180 tumor-bearing mice as a model to observe the effect of Di dang decoction on tumor weight, organ index of immune organs and leukocyte counts, the results showed that when Di dang decoction in middle or high dose group, the tumor weight of S180 tumor-bearing mice was significantly inhibited, and index of spleen and thymus are obviously improved, as well as the number of peripheral blood leucocyte.Impact of Di dang decoction and its modified formula on the growth of extraorgan tumor cells growth:MTT was applied to observe the impact on MCF-7、HCT-28、HepG-2 cells growth and calculate IC50, we found that Di dang decoctions added with different herb medicines could inhibit the proliferation of cancer cells, the effect being superior to Di dang decoction itself. In three different types of its modified formula, bupleuri-added decoction was superior than Gui zhi-added decoction, astragali-added decoction was the last one of three;Tumor inhibition of Di dang decoction’s modified formula on S180 tumor-bearing mice:S180 tumor-bearing mice as a model to observe the effect on the rate of inhibition of tumor, the organism immunity situation, blood index, the regional blood vessel of tumor, blood circulation and IL-8, and TNF-a, and the results were Di dang decoction, Di dang decoction plus bupleuri and Di dang decoction plus Guizhi inhibited the growth of tumor significantly; the function of inhibition of tumor growth enhanced when Di dang decoction plus bupleuri or Guizi. Separately, the function of tumor growth inhibition of Di dang decoction plus bupleuri was much more significant than Di dang decoction itself only, implying that the tumor inhibition effect was enhanced when the Di dang decoction plus bupleuri or Guizi. Di dang decoction and its prescrption separately added bupleuri, Guizhi or astragali could stimulate the peripheral blood circulation, of which Guizhi-plus group could improve auricle microcirculation of mice notably. Di dang decoction and Di dang decoction separately added bupleuri, Guizi or astragali could inhibit the regional blood circulation around the tumor of S180 tumor-bearing mice, of which astragali-plus prescription was the most effective one. The function on IL-8 and TNF-a of Di dang decoction and its modified formula distinguished from control group, especially the bupleuri-plus group.In summary:1.Di dang decoction and its modified formula has the anti-tumor function, and anti-tumor function in Bupleurum-modified and Guizhi-modified prescription are much more effective than astragalus group and original decoction, Bupleurum group was significantly different; 2. Di dang decoction and its modified prescription can promote peripheral blood circulation and inhibit local blood circulation around tumor;3. Di dang decoction has the activity function to extraorgan tumor cells. While adding Bupleurum, Guizi and Astragalus, the activity function of these improved decoctions are greater than the original decoction, in addition, Bupleurum and Guizhi group are more obvious than Astragalus group. Didangtang decoction and its improved prescription inhibit the growth of breast cancer MCF-7 significantly. The research on the composition of Di dang decoction and its improved prescription will contribute to the scientific understanding to the TCM compound compatibility law; find out the inherent rule of activating blood and dissipate stasis compatibility, which provide experimental evidence as we form a new prescription, to develop Zhongjing’s recipe idea of activating blood and dissipate stasis.更多还原