【作者】 彭新；

【导师】 顾立刚；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2009， 博士

【摘要】 肿瘤是机体在各种致瘤因素的作用下,局部组织的细胞在基因水平上失去对其生长的正常调控,导致克隆性异常增生或凋亡不足而形成的新生物,这种新生物往往形成局部肿块。肿瘤不管是良性还是恶性,也不管是上皮组织来源还是间叶组织来源,本质上都表现为细胞失去控制的异常增殖,这种异常生长的能力除了表现为肿瘤本身的持续生长之外,在恶性肿瘤还表现为对邻近正常组织的侵犯以及经血管、淋巴管和体腔转移到身体其它部位,而这往往是肿瘤致死的原因。祖国传统医学关于肿瘤发生的病机,历代医家的论述主要包括正气内虚、血瘀痰凝、邪毒内攻等。根据肿瘤患者病理性肿块、疼痛、出血、舌质瘀紫或有瘀点瘀斑,以及晚期面色黧黑、肌肤甲错等临床表现,认为血瘀症与肿瘤关系密切。近年来,随着分子生物学理论不断深入研究,中医药治疗肿瘤方面的研究进展取得了较为长足的进步,使我们对中药在抑制肿瘤生长,提高机体免疫功能,防止肿瘤转移的分子机制有了初步的认识。中医药对肿瘤的影响也从最初的免疫功能调节研究,发展到了今天的抗肿瘤血管生成研究,诱导细胞凋亡,抑制端粒酶活性等研究。苏木(Caesalpinia Sappan L,CAE)为豆科云实属植物的干燥心材。有行血祛瘀,消肿止痛之功效,中医临床常用于经痛、风湿痛、跌打瘀痛等症。临床上也用于肿瘤的治疗,现代药理学研究表明其具有抗肿瘤、免疫抑制等作用。但其具体的抗癌机制目前仍未知。为此我们用现代医学测试技术,测定苏木醇提取物免疫抑制与对抗肿瘤的作用机制。本研究的第一部分主要观察苏木不同提取物对小鼠胸腺组织、外周T细胞增殖和产生INF-γ的影响。通过制备苏木不同提取物分别给予灌胃小鼠,取小鼠胸腺称重计算胸腺指数;采用MTT法测定小鼠脾T细胞的增殖能力;用双抗体夹心ELISA法测定T细胞培养上清中产生INF-γ的含量。结果发现苏木不同提取物不仅对胸腺重量具有明显的抑制作用,而且还对T细胞的增殖和产生INF-γ的能力具有明显的抑制作用。其中苏木醇提取物和正丁醇提取物对T细胞的抑制作用较强。鉴于醇类提取物作用较强,在研究的第二部分主要观察苏木醇提物对人肺癌A549细胞的细胞毒作用和诱导凋亡作用。将苏木醇提物按不同浓度分别同人肺癌A549细胞共同培养,MTT法检测对细胞增殖作用的影响,光学显微镜、细胞膜磷脂酰丝氨酸外翻分析、细胞周期分析观察不同浓度苏木醇提物对人肺癌A549细胞的细胞毒和调亡作用。发现苏木醇提物对人肺癌A549细胞有抑制作用,并呈浓度依赖性;苏木醇提物在诱导细胞发生凋亡外还存在其它形式的细胞死亡方式并对细胞各期均有影响。经过第二部分体外抗肿瘤实验后,进行体内抗瘤实验。第三部分研究主要观察苏木醇提物对H22腹水瘤细胞抑瘤作用,通过皮下接种H22腹水瘤细胞的方法建立荷瘤惺竽Ｐ?用苏木醇提物灌胃处理,观察各组荷瘤小鼠的肿瘤生长情况,采用缺口末端标记法测定肿瘤细胞凋亡指数,免疫组织化学方法测定凋亡蛋白Bcl-2、Bax的表达情况。发现苏木醇提物可抑制H22移植瘤生长,促进瘤体细胞凋亡,降低瘤体细胞Bcl-2/Bax比值;其机制与通过调控凋亡相关蛋白Bcl-2、Bax的表达诱导肿瘤细胞凋亡有关。更多还原

【Abstract】 With the effects of various tumorigenic factors, cells of local tissues will lose control on their growth in the level of genes, which leads to clonal hyperplasia or insufficient apoptosis and results in a neoformation called tumor, often existing in the form of local mass. Whether the tumor is benign or malignant, or has its origins in epithelial tissue or mesenchymal tissue, it manifests abnormal proliferation of cells losing control. This abnormal growing ability can not only be seen from tumor’s continuous growth but also from aggression to its adjacent normal tissues and transfer to other parts through blood vessels, lymphatic vessels and body cavity especially for malignant tumor, which is always the cause of death.In traditional Chinese medicine, physicians in past dynasties regarded that the pathogenesis of tumor mainly contained insufficient healthy qi, blood stasis and phlegm stagnation and invasion of pathogenic toxins etc. They thought tumor is closely related with blood stasis syndrome based on the clinical manifestations of tumor patients such as pathologic tumor mass, pain, hemorrhage, purple tongue with stasis or ecchymosis or blood spots, swarthy complexion and scaly skin in the advanced stage.In recent years, with constant study on molecular biology theory, the study of treating tumor with traditional Chinese medicine has made great progress, giving us a preliminary understanding of the molecule mechanism of inhibiting tumor growth, enhancing immunity and preventing the transfer of tumor with Chinese herbs. The study of traditional Chinese medicine’s effects on tumor has developed from the preliminary study on immune function regulation to the current anti-tumor angiogenesis research and apoptosis induction and telomerase activity inhibition research.Hematoxylon (Caesalpinia Sappan L, CAE) is the dry heartwood of Leguminosae Caesalpinia which has the effect of promoting blood flow to eliminate stasis and dissolving the swelling to kill the pain. Caesalpinia Sappan L is used to treat dysmenorrheal, rheumatic pain and traumatic injuries and pains etc in Chinese medicine. It can also be used to treat tumor, as the modern pharmacological research shows that it has anti-tumor and immunosuppressive functions, although its specific anti-tumor mechanism is still unknown. Therefore, we can use modern medical testing technology to study the anti-tumor and immunosuppressive mechanism of Hematoxylin alcohol extracts.In the first part, the main purpose is to observe the effect of different extracts of Hematoxylon in mouse thymus, peripheral T cell proliferation and INF-γgeneration. Different extracts prepared from Caesalpinia Sappan L were lavaged to the mice in order to count thymus index by taking out their thymus, to calculate the proliferation capacity of spleen T cells by using MTT method, to measure the INF-γcontent generated by T cell culture supernatant by using double-antibody sandwich ELISA method. The results showed that different extracts of Hematoxylon not only had remarkable inhibition effect in thymus weight but also in T cell’s proliferation and INF-γgeneration abilities. Hematoxylin alcohol and n-butanol extracts had stronger inhibiting effects on T cells.Given the strong effects of alcohol extract, in the second part of the study, the main purpose is to observe Hematoxylin alcoholl extract’s cytotoxicity and apoptosis inducing effects on A549 cell of lung caner. Hematoxylin alcoholl extracts with different concentration were co-cultured with lung cancer A549 cells in order to observe effects on cell proliferation by using MTT method, study the effects on cytotoxicity and apoptosis of lung cancer A549 cells using optical microscope, phosphatidylserine ecstrophy analysis and cell period analysis. The results showed that Hematoxylin alcohol extracts had inhibiting effects on human lung cancer A549 cells in a dose-dependent manner; besides the cell apoptosis induced by these extracts, there existed other forms of cell death and affected cells in every period.Following the second part of anti-tumor experiment outside, here comes the internal one. The third part is mainly to observe the extract’s anti-tumor effect on H22 ascites tumor cells. Tumor-bearing rat model was built by subcutaneous inoculation of H22 ascites tumor cells and lavaged with Hematoxylin alcohol extracts in order to observe the tumor growth conditionsmof tumor-bearing mice in each group, measure the tumor cell apoptotic index by using the nick end labeling method and apoptotic protein Bcl-2, Bax expression by using immunohistochemical technology. The results showed that Hematoxylin alcohol extract can inhibit the H22 transplantation tumor growth, facilitate tumor cell apoptosis, reduce Bcl-2/Bax ratio of tumor cells and the mechanism was related with the induction of tumor cell apoptosis by regulating the expression of apoptosis-related protein Bcl-2/Bax.更多还原