【作者】 谢慧珺；

【导师】 蒋时红；

【作者基本信息】 河南中医学院 ， 中西医结合基础， 2009， 硕士

【摘要】 研究背景原发性肝癌是全球高发的恶性肿瘤,据国际癌症研究中心估计,2000年全球肝癌发病人数56.4万,肝癌死亡54.9万;其中中国肝癌发病人数30.6万,死亡30.0万,中国的发病（死亡）数占全世界肝癌的一半,由此可见,原发性肝癌是威胁人类健康的主要疾病之一。我国现有癌症病人约250万人,平均每死亡5个人中就有一个死于肝癌,每年因治疗而支付的医疗费用达300多亿元,间接损失更高达上千亿元。目前临床上的治疗一般视手术、放疗、化疗为三大支柱,但由于肝癌起病隐匿,恶性程度高,发展变化快,患者就诊时往往已进入中晚期,失去了最佳手术时机,而大多数化疗药物虽可抑制肿瘤细胞过度增生,但毒副作用大,对正常组织细胞也有很强的杀伤作用,部分病人因身体极度虚弱已不能耐受西医西药的治疗。而中医药在治疗肝癌方面因其独特的优势而被临床所广泛采纳,在治疗局部的同时,重视全身治疗,改善和提高病人的生活质量,延长生命周期,并且副作用小,因此,发挥我国中医药的优势,为肝癌的治疗寻求高效低毒、价廉质优的中药复方,并探讨其作用机理,对于提高肝癌患者生存质量,降低肝癌治疗费用,扩展中药复方应用范围,具有十分重要的社会效益,显著的经济效益和学术价值。中医理论认为,肝癌的发病是在内外因共同作用下,机体阴阳失调,脏腑功能障碍,经络阻塞,气血运行失常,气滞血瘀,湿聚痰凝,郁久化火,终致气、血、湿、热、瘀、毒互结而发病。关于本病的辩证施治,临床治疗常选用活血化瘀、清热祛湿以及养阴柔肝等治法组方。但本病病机复杂,病症危重,往往多型并见,治疗本病较为棘手。目前研究认为,中药复方治疗肝癌机理主要体现在以下几个方面:直接杀伤肿瘤细胞、抑制肿瘤血管生成、抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡分化、调节机体免疫功能以及影响癌基因表达等,但对以上三种不同治法中药复方治疗肝癌机理的比较研究还少有报道。目的通过本课题的研究,探讨中医活血化瘀、清热祛湿、养阴柔肝三种治法治疗肝癌的作用机制,比较其不同作用靶点,为中药复方治疗肝癌提供理论依据。方法1.无菌抽取H₂₂瘤细胞(浓度为1×10⁷ mL^-1),小鼠右侧腋下消毒后接种0.2 mL。2.以活血化瘀、清热祛湿、养阴柔肝三方最佳浓度（由预实验结果确定）中药水煎液,灌胃H₂₂荷瘤小鼠,观察其一般情况（包括毛发色泽、饮食情况、活动度等）。3.称量各组瘤体重量,计算抑瘤率,分析三种治法方药对肿瘤细胞生长的抑制作用。4.显微镜下观察瘤体组织细胞形态学变化。5.称量各组脾脏重量,计算脾指数,分析三种治法方药对荷瘤小鼠免疫功能的影响。6.应用TUNEL技术,检测瘤体细胞凋亡情况,计算凋亡指数。7.应用免疫组化技术,对细胞内细胞周期及凋亡细胞相关蛋白（CyclinD1、PCNA、Bcl-2及Bax）表达水平进行分析,探讨三种治法方药抑制瘤体细胞增殖、诱导细胞凋亡的不同作用机制。结果1.小鼠移植性肝癌(H₂₂)模型复制成功,4天后肉眼可见皮下实体瘤,成功率100%。2.活血化瘀、清热祛湿、养阴柔肝组抑瘤率分别为:43.64%、50.91%、45.45%。3.活血化瘀、清热祛湿、养阴柔肝方对H₂₂小鼠脾重无显著影响（P＞0.05）。4.活血化瘀、清热祛湿、养阴柔肝方对肝癌细胞增殖影响显著（P＜0.05）。5.活血化瘀、清热祛湿、养阴柔肝方对肝癌细胞凋亡影响显著（P＜0.05）。6.免疫组化显示:活血化瘀、清热祛湿、养阴柔肝方作用后,肝癌细胞PCNA、CyclinD1、Bcl-2表达量减少,Bax表达量增多,与模型对照组比较有显著性差异（P＜0.05）。结论1.三种治法均具有抑制肝癌瘤体生长的作用,其中清热祛湿方的抑瘤作用最明显。2.三种治法均可以降低PCNA在H₂₂肝癌细胞内的表达量,抑制细胞内DNA合成进程,进而抑制肝癌细胞的增殖。3.三种治法方药均可以明显抑制肝癌细胞CyclinD1表达,推断三种治法能够抑制肝癌细胞增殖活性的分子机制,可能是通过降低肝癌细胞内CyclinD1的阳性表达,阻断CyclinD1-CDK4-Rb通路,降低细胞周期转换速度实现的。4.三种治法均可诱导肿瘤细胞发生凋亡;相关蛋白Bcl-2、Bax可能是三种治法诱导肝癌细胞凋亡的重要调控因子。5.活血化瘀方法主要通过抑制肝癌细胞增殖发挥抑癌作用。6.清热祛湿、养阴柔肝方法主要通过诱导肿瘤细胞凋亡发挥抑癌作用,二者作用结果相似。更多还原

【Abstract】 BackgroundPrimary hepatic carcinoma（PHC） is a high occured rate disease all over the world,IARC estimated that there are 564,000 people suffer from PHC and 549,000 are died;In China,there are 306,000 patients,and 300,000 are died,which is a half of total caicinomatous and death number,so PHC is one of the main diseases that threatens human’s life in our country.There are 2,500,000 cancer patients in China,and one fifth is died from it,the medical care expense on cancer amounts to over￥30 billions annually,the indirect loss is higher than hundreds of billion yuan.Now the main therapies are opration,radiotherapy and chemo,but lots of patient don’t suit for opration at advanced stage when they go to hospital as the hide,malignant and growth nature of PHC,most chemotherapy drugs can restrain excess of proliferation,but side effects are so severe that patients can’t afford by their weak bodys.However,Chinese patent medicine are extensive absorbed by advantages of comprehensive therapy,enhance the quality and prolong life and little side effect in clinic.Therefor,it is overwhelmingly significant in social benefit,economic benefit and especially academical values to exert the advantage of Chinese patent medicine,seek for some kind of high-effective and low-cytotoxic Chinese patent medicine,study the mechanism of it,to improve survival probability,reduce therapy expence and widen the range of Chinese patent medicine’s application.PHC is a diease which is under mutual effect of intra and external factors,can lead to imbalance of body’s Yin and Yang,disfunction of viscera,choken meridian,abnormal operation of Qi and blood,stagnation of Qi and blood,accumulation of wetness and phlegm, reduction evil fire,finally to fall ill combined with Qi,blood,wetness,heat,Liver Qi depression and pathogenic fire and toxin,we always choose promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy to treat the disease in clinic.But PHC is a thorny problem in clinic work since complicated pathogenesis,critical symptoms and contemporaneous syndrome types. The modern medicine shows that the mechanism of Chinese patent medicine cured PHC are various,embodied by:kill the cancer cells directly,restrain to form of cancer’s blood vessel, restrain to proliferation of cancer cell,inducement apoptosis of cancer cell,regulate the function of body’s immunity and influence gene’s expression,but we haven’t found the mechanism and relative study of promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy yet.ObjectiveTo study the therapy mechanism of PHC by promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy,compare the function targets,provide experimental evidence on the treatment of PHC with Chinese patent medicine.Methods1.Exsuct asepsis H₂₂ tumor cell which concentrated to 1×10⁷mL^-1,hypodermic inject 0.2mL at right oxter of mice after antisepsis.2.Repectively fed H₂₂ tumor-bearing mice by the water-solubility liquid of promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy, nourishing Yin of liver and kidney therapy,whose best concentration of Chinese patent medicine have been confirmed by fore-experiment,observed their common situations（included by fur’s colour,appetite situation,activity limit and so on）.3.Weighed tumor up,accounted rate of anti-tumor,analysed inhibiting of growth of tumor cell treated with Chinese patent medicine.4.Observed shape’s change of tumor cells by microscope.5.Weighed spleen up,accounted rate of its exponential,analysed immunity effect of tumor-bearing mice treated with Chinese patent medicine.6.TUNEL was applied to test the situation of apoptosis.7.Immunohistochemistry was applied to analyze the expression level of cell cycle and apoptosis cell of CyclinD1、PCNA、Bcl-2 and Bax proteins in the cell,to research the relationship between the mechanism of proliferating,apoptosis,the regulation and control of gene expression.Results1.H₂₂ tumor-bearing mice model was established successfully,,visible hypodermal tumor after 4 days,the rate of successful model is 100%.2.Rate of anti-tumor of promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy groups are 43.64%、50.91%and 45.45%.3.The effect of splenic exponential was not obviously treated with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy, nourishing Yin of liver and kidney therapy（P＞0.05）.4.The effect of proliferating was significantly different with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy（P＜0.05）.5.The effect of apoptosis was significantly different with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy（P＜0.05）.6.The data of immunohistochemistry indicated:compared with the model group,the expression of PCNA、CyclinD1 and Bcl-2 were reduced,Bax was increased remarkably after treated with promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy （p＜0.05）.Conclusion1.Promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy all can resist proliferating of carcinoma cell,especial for heat clearing and wetness removing therapy.2.Promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy all can reduce cell’s expression of PCNA,resist compose progress of DNA,then resist proliferating of carcinoma cell.3.Promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy all can resist proliferating of carcinoma cell,which mechanism may reduced expression of CyclinD1,blocked the way of CyclinD1-CDK4-Rb.4.The apoptosis-associated proteins Bcl-2 and Bax were probably significant regulation factors of apoptosis-induced by promoting blood circulation and removing blood stasis therapy,heat clearing and wetness removing therapy,nourishing Yin of liver and kidney therapy.5.The result of experiment indicated that promoting blood circulation and removing blood stasis therapy can resist the growth of PHC mostly by resisted proliferating of carcinoma cell.6.The result of experiment indicated that heat clearing and wetness removing therapy and nourishing Yin of liver and kidney therapy can resist the growth of PHC mostly by abducted apoptosis of carcinoma cell.更多还原