【作者】 李文海；

【导师】 刘锟；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 博士

【摘要】 肺癌是一种严重威胁人类健康的恶性肿瘤,其发病率和死亡率呈上升趋势。在我国数个大中城市中,肺癌的发病率、死亡率均已占男性恶性肿瘤的首位,女性恶性肿瘤的第二位。尽管手术、联合化疗及放疗在肺癌的治疗中起到了积极的作用,但因缺乏有效的早期诊断方法,70-80%的患者在确诊时已失去早期根治性切除的时机。手术及放化疗复发率高、易出现耐药,故患者的5年生存率长期停留在20-30%左右。中国传统医药在肿瘤的治疗方面有其独特的理论。中医药治疗肺癌的有关资料表明:中医药不仅可以延长肺癌患者的生存期,而且能提高他们的生存质量。中药配合放疗、化疗比单纯使用放疗、化疗的疗效更好。中药既可增加化疗药及放射线的敏感性、又有放射防护作用,可减轻化疗放疗的副作用,起到抑瘤增效作用或放射增敏作用。天然中药以其疗效广泛、确切、毒性低,受到国内外广泛的关注。本课题我们取多种中药提取物或单体,通过体外实验,筛选出能够以较低浓度(微克/毫升水平)抑制肺癌生长或增殖的药物。根据上述实验结果,选择作用效果最明显的中药提取物或单体,研究其抑制肺癌细胞生长或增殖与相关信号转导通路的关系,以及对肺癌细胞化疗敏感性的影响。这些研究将为治疗肺癌新型药物的研制开发提供新思路,为我国传统中医药进一步用于肿瘤的治疗提供实验依据。基于此,本课题从以下四个方面进行了研究。1、抗肺癌中药单体的筛选:我们通过光学显微镜初步筛选了25种中药单体或粗提产物中能有效抑制肺癌细胞增殖的药物,发现可能有抑制肿瘤作用的(镜下观察有明显细胞形态改变的,如细胞皱缩,破碎,漂浮等)有12种,姜黄素、水飞蓟素、大黄素、茶多酚、松多酚、大豆异黄酮、葛根异黄酮、染料木素、生姜提取物、鬼臼素、贯叶连翘提取物、桫椤子提取物等。在此基础上,针对其中4种纯度较高(>80%)且抑制肿瘤细胞增殖明显的药物,姜黄素、大黄素、水飞蓟素、大豆异黄酮,进一步采用MTT试验验证其对肺癌细胞系的抑制效果,结果显示:SM对几种细胞均有较强的抑制作用;大黄素对Anip973和A549细胞有作用,但对NCI-H292和PLA-801细胞作用较弱;姜黄素对A549和NCI-H292细胞有作用,但对Anip973和PLA-801细胞作用较弱;大豆异黄酮对Anip973和PLA-801细胞有作用,但对A549和NCI-H292细胞作用较弱。这些结果表明药物对于不同的细胞作用并不完全一样,造成这一现象的机理有待于更深入的研究。2、水飞蓟素诱导高转移表型肺癌细胞系(Anip973)细胞凋亡的分子机制研究:本实验以肺癌细胞系Anip973细胞为研究对象,探讨分别给予不同剂量和不同时间点的水飞蓟素处理后的肺癌细胞的凋亡情况,通过形态学及流式细胞仪我们观察到水飞蓟素可诱导肺癌细胞系Anip973细胞产生凋亡,且具有时间和浓度依赖性。在凋亡发生过程中,半胱天冬酶( caspase)家族和Bcl-2家族发挥重要作用。进而通过免疫印记法检测发现SM作用于人高转移肺癌细胞系Anip973细胞后,抗凋亡蛋白Bcl-2表达明显降低,而凋亡蛋白Bax的表达明显增加,说明水飞蓟素的促凋亡机制与降低Bcl-2/Bax的比例有关。3、水飞蓟素通过MAPK信号转导通路逆转A549/DDP细胞对顺铂的耐药性:我们通过MTT法检测到水飞蓟素可以逆转肺癌A549/DDP细胞的耐药性,并呈时间依赖性;流式细胞仪分析结果提示水飞蓟素和顺铂联用可以引起A549/DDP细胞凋亡或死亡,并呈时间依赖性。为了进一步探讨水飞蓟素诱导A549/DDP细胞凋亡的机制,我们采用Western blot方法,应用针对MAPK家族成员JNK、ERK、p38磷酸化形式及非磷酸化形式的特异性抗体,检测了SM作用后以上激酶的表达水平及活化程度的改变。在不同浓度药物作用24hr后,这几种激酶的表达水平都没有明显的改变,而它们磷酸化的程度与加入DMSO的阴性对照组相比则有显著的改变。其中ERK的磷酸化受到明显的抑制,而JNK、p38MAPK的磷酸化水平有一定的增加。4、水飞蓟素对肺癌细胞Anip973裸鼠体内成瘤、增殖、凋亡以及血管形成的影响:本研究将Anip973细胞接种于裸鼠背部皮下,建立人肺癌裸鼠皮下移植瘤模型。建立模型后,给其腹腔注射SM结果显示,与未处理组的裸鼠相比,注射水飞蓟素的裸鼠肿瘤形成时间延迟,肿瘤生长缓慢,肿瘤体积及瘤重均明显减小。各组肿瘤标本HE染色结果显示,肿瘤细胞异形性明显,细胞大小不一,形状各异,瘤细胞生长致密。未处理组肿瘤标本凋亡细胞少见,而水飞蓟素组肿瘤标本提示凋亡细胞增多,凋亡细胞表现为细胞变圆、固缩、胞膜出泡、染色质浓集、核深染。综上所述,我们认为水飞蓟素具有通过调节半胱天冬酶( Caspase)家族和Bcl-2家族发挥其抑制肺癌细胞增殖及促凋亡的作用;另外水飞蓟素可以通过调节MAPK信号转导通路逆转肺癌A549/DDP细胞的耐药性,并能增加顺铂化疗的敏感性。以上研究将为治疗肺癌新型药物的研制开发提供新思路,为我国传统中医药进一步用于肿瘤的治疗提供实验依据。更多还原

【Abstract】 Lung cancer is a kind of malignant tumor with increasing risk of the morbidity and mortality, which is severely threatening human health. In many big cities in China, lung cancer is becoming the main cause for the death and morbidity of patients with malignant tumor in male, and the second cause in female. Although operation, combined chemotherapy and radiotherapy are playing an active role in the treatment of lung cancer, 70-80% patients have lost the chance of early radical resection due to the lack of effective methods for early diagnosis. The recurrence rate after the operation, radiotherapy and chemotherapy is high with resistance so that the 5-year survival rate of the patients has stayed at 20-30% for a long time.The traditional Chinese medicine has unique theories on the treatment of tumors. The relevant reports about the treatment of lung cancer with traditional Chinese medicines indicated that the traditional Chinese medicine could prolong the life span of the patients with lung cancer as well as increase their quality of life. The curative effect of combination of traditional Chinese medicine with radiotherapy and chemotherapy is better than that of singly application of chemotherapy or radiotherapy. The traditional Chinese medicine could increase the sensitivity to chemotherapy and radiotherapy, and reduce the adverse effects of chemotherapy and radiotherapy, so that it has the functions of inhibiting tumors and synergia. The natural Chinese medicine has raised broad interests of worldwide researchers due to its extensive and certain curative effect and low toxicity.In this study, we selected many extracts or monomer of the traditional Chinese medicines to screen the one(s) that can inhibit the growth or proliferation of lung cancer cells at low concentration (the level ofμg/mL) through in vitor experiments. Based on these results, we choose the extract or monomer of the traditional Chinese medicine with the most significant efficiency and studied the relation between its effect of inhibiting the growth or proliferation of lung cancer cells and the relevant signal pathways as well as its effect on the sensitivity of lung cancer cells to chemotherapy. These studies will provide the possibility of finding some new ways in the development of new drugs for the treatment of lung cancer and provide experimental bases for the further application of traditional Chinese medicines in the treatment of tumors. Based on the above mentioned contents, we carried out the study from the following four aspects:1. The screening of Chinese herb with anti-lung cancer effect: we preliminarily screened 25 kinds of monomer or crude extracts of the traditional Chinese medicines which can effectively inhibit the proliferation of lung cancer cells as shown under the light microscope and found that 12 kinds of them had the effect of inhibiting the tumor growth (there were obvious morphological changes of the cell, such as cell shrinkage, disruption, flotation, etc), which include curcumine, silymarin, emodin, tea polyphenols, pine polyphenols, soybean isoflavone, pueraria isoflavone, genistein, ginger extract, podophyllotoxin, forsythia extract and cyathea spinulosa wall extract. Based on these results, we choose 4 kinds of drugs, including curcumine, emodin, silymarin and soybean isoflavone, with high purity (>80%) and significant inhibitory effect on the growth of tumor cells to further confirm their inhibitory effects on the lung cancer cell lines through the MTT test, our results showed that silymarin had strong inhibitory effect on all tested cell lines; emodin had inhibited the growth of Anip973 and A549, and it had only weak effects on NCI-H292 and PLA-801; curcumine had effects on A549 and NCI-H292, and it only had weak effects on Anip973 and PLA-801; soybean isoflavone affected on Anip973 and PLA-801, and it had only weak effects on A549 and NCI-H292. These results suggested that the drugs had different effects on various cell lines and the mechanism leading to this phenomenon needed to be further studied.2. The study of the mechanism of cell apoptosis of the lung cancer cell line with high-metastatic phenotype (Anip973) induced by silymarin: In this experiment, we took lung cancer cell line Anip973 to study the apoptosis of the lung cancer cells at different time points treated with silymarin of different concentrations, and we found that silymarin could induce apoptosis of Anip973 cells through the morphological observations and flow cytometer analysis the apoptosis is time-dependent and concentration-dependent. The caspase family and Bcl-2 family play important roles in the process of apoptosis. Moreover, the results of Western blot indicated that, when Anip973 cells with high-metastatic phenotype were treated by silymarin, the expression of the anti-apoptosis protein Bcl-2 decreased significantly, in the meanwhile the expression of the apoptosis protein Bax increases significantly, which suggested that the promotion of apoptosis induced by silymarin was relevant to the decreased ratio of Bcl-2/Bax.3. Silymarin reversed the drug resistance of A549/DDP cells to cisplatin through the MAPK signal pathway: We found that silymarin could reverse the drug resistance of A549/DDP cells through the MTT test and the effect was time-dependent. The analysis results of flow cytometer indicated that the combination of silymarin and cisplatin could induce the apoptosis or death of A549/DDP cells and the effect was time-dependent. To further study the mechanism of the apoptosis of A549/DDP induced by silymarin, we detected the changes of the expression levels and the degrees of activation of MAPK family members including JNK, ERK and p38 through the Western blot with the application of specific antibodies of the phosphorylated and non-phosphorylated forms of those above mentioned kinases. There was no significant change on the expression levels of these kinases when the cells were treated by silymarin of different concentrations for 24h, however, their phosphorylation degrees were significantly changed compared with the negative control group treated by DMSO. The phosphorylation of ERK was significantly inhibited, but the phosphorylation levels of JNK and p38MAPK increased.4. The effects of silymarin on the tumorigenicity in nude mice, proliferation and apoptosis of the lung cancer cell line Anip973 cells and angiogene: In this study, the Anip973 cells were subcutaneously inoculated on the back of the nude mice to establish the subcutaneous xenotransplanted tumor Model of human lung cancer in nude mice. The results indicated that, in the established models treated by intraperitoneal injection of silymarin, the tumor formation was delayed, the tumor growth was slower and the volume and weight of tumors were significantly decreased compared with the nude mice without treatment. The HE staining of the tumor specimens of each group indicated that the heteromorphism of the cancer cells were obvious, the sizes and shapes of cells are different and the cancer cells grow densely. Cell apoptosis was scarcely observed in the untreated tumor specimens, however, cell apoptosis increased in the tumor specimens treated by silymarin, and the apoptosis cells presented cell rounding, pyknosis, cell membrane blebbing, chromatin condensation and deep staining of nucleolus.In conclusion, it is suggested that silymarin had the function of inhibiting the proliferation of lung cancer cells and promoting apoptosis by regulating the caspase family and Bcl-2 family. Meanwhile silymarin could reverse the drug resistance of lung cancer cell line A549/DDP cells by regulating MAPK signal pathway and increase the sensitivity of the cells to the chemotherapy with cisplatin. This study will give new light for the development of new drugs for the treatment of lung cancer and provide experimental bases for the further application of traditional Chinese medicines in the treatment of tumors.更多还原