【作者】 王琦；

【导师】 郑燕芳；

【作者基本信息】 南方医科大学 ， 肿瘤学（专业学位）， 2019， 硕士

【摘要】 一、研究背景八宝丹是一种名贵的中药,由牛黄、麝香、三七、羚羊角及珍珠等组成,由厦门中药有限公司生产,具有清利湿热、祛黄解毒、活血化瘀等功效,能够治疗病毒性肝炎、急性泌尿系统感染等疾病。肺癌是全世界发病率和死亡率第一的恶性肿瘤,针对肺癌探索有效的治疗措施意义重大。八宝丹,作为一种自明朝时期即被人类广泛使用的传统中药,其在肿瘤中的应用与机制研究较少;在对肺癌的作用与机制方面,尚无研究报道。在长期临床观察中我们发现,对于原发性肝癌和肺癌的患者,给予化疗药物的同时配合使用中药八宝丹,可增强化疗的效果,降低化疗的毒副作用。因此我们推测,八宝丹可能具有一定的抗癌作用,并增加化疗药物的敏感性。初步的前期预实验证实了我们的推测。本研究拟在细胞株和动物体内,研究八宝丹对非小细胞肺癌的作用、对肿瘤细胞顺铂耐药的影响,并深入探讨其分子机制。二、研究方法及内容①通过CCK8实验和平板克隆实验分析单药八宝丹对肺腺癌细胞生长的影响及联合顺铂对细胞抑制率的影响;利用自噬抑制剂3-MA预处理,检测细胞活力并对比。② 利用蛋白质免疫印迹法,八宝丹处理细胞后检测自噬相关蛋白(LC3-II)及PI3K/AKT/mTOR通路相关蛋白的表达,利用电镜观察自噬体,明确八宝丹对肺腺癌A549和A549/DDP细胞自噬的影响。③利用细胞划痕实验和Transwell实验,分析八宝丹联合顺铂对细胞侵袭迁移的影响。④利用实时荧光定量PCR和蛋白质免疫印迹法,检测八宝丹对A549和A549/DDP细胞多药耐药基因(MDR1)及药物转运蛋白P-gp的影响。⑤利用裸鼠体内移植瘤模型,体内验证八宝丹对肿瘤生长的影响及对顺铂的增敏作用。三、研究结果①八宝丹时间—浓度依赖性的抑制肺腺癌A549和A549/DDP细胞的活力随着八宝丹作用时间和作用浓度的增加,肺腺癌A549和A549/DDP细胞的活力和增值能力逐渐减弱。②八宝丹通过促进细胞自噬水平发挥抑制肿瘤细胞生长作用浓度递增的八宝丹并不引起细胞凋亡相关蛋白的变化,而是通过增加自噬流,使自噬特异性蛋白LC3-Ⅱ表达升高,使细胞内自噬小体数目增多来抑制细胞生长。且抑制自噬,可见八宝丹对A549和A549/DDP细胞的抑制作用同样被削弱。③八宝丹通过抑制PI3K/AKT/mTOR通路增加细胞自噬水平随着八宝丹浓度的增加,我们发现p-PI3K、p-AKT、p-mTOR蛋白表达减少,LC3-Ⅱ蛋白增加;而联合使用PI3K通路激活剂IGF-1,八宝丹引起LC3-Ⅱ的增加被一定程度逆转。由此可得,八宝丹是通过抑制PI3K/AKT/mTOR通路增加自噬的。④八宝丹促进顺铂杀肿瘤作用相对于顺铂单药组,一定浓度的八宝丹联合顺铂同时作用于细胞,更能有效抑制A549和A549/DDP的活力及迁移侵袭能力。⑤裸鼠移植瘤模型体内验证八宝丹对肿瘤的抑制作用通过裸鼠皮下成瘤实验,测量肿瘤体积和重量可得八宝丹能够抑制肿瘤细胞生长,增加顺铂的抗肿瘤作用,且用药过程并未产生明显的毒副作用。四、结论我们通过体内和体外实验首次进行了八宝丹在肺腺癌中的作用及机制研究,并证明其能够通过抑制PI3K/AKT/mTOR信号通路促进A549和A549/DDP细胞的自噬水平,从而抑制细胞生长。同时,降低多药耐药基因MDR1的表达和药物转运蛋白P-gp的水平,增加顺铂敏感性。这对于八宝丹在肺癌及其他肿瘤应用潜力的挖掘中具有重要意义。更多还原

【Abstract】 BACKGROU:NDLung cancer is the most frequent malignancy all over the world,and the main cancer-related cause of mortality worldwide in both males and females combined[2].In China,there were an nearly 343,991 new cases of lung cancer(19.30%of total cancer incidence cases)and 286,978 lung cancer-related deaths(25.54%of total cancer mortality cases)during 2008-2012,which made lung cancer rapidly became first in the malignant neoplasm for both incidence and mortality[3].Unfortunately,a great many lung cancers were confirmed in its advanced stage and became unresectable.For these patients,chemotherapy became the main treatment.Cisplatin,an effective agent,using worldwide for the treatment of multiple cancers,including lung cancer.To reach the best treatment outcome,it has to be administrated repetitively.However,its therapeutic efficacy is limited due to the development of acquired resistance,and reduced the survival rate of patients.Clinical tumour resistance to chemotherapy could be intrinsic or acquired.Acquired resistance occurs in tumours which are often highly responsive to initial treatment,but on tumour recurrence,exhibit an absolutely different phenotype,and become resistant to both primarily used drugs,and new agents with extremely different structures and mechanisms of action.Clinical resistance to various anticancer agents should not be confused with’multidrug resistance’,which in experimental models is related to increased expression of P-glycoprotein(P-gp).Experimental models prove that multidrug resistance can be induced by increased expression of adenosine triphosphate(ATP)-dependent efflux pumps[4].These pumps actively transport chemotherapy drugs out of the cell,The ABCl multi-drug resistance(MDR1)gene generates the transporter P-gp[5],Which is overexpressed in many multidrug resistant cells[6-12].Autophagy is a lysosomal degradation approach that is essential for survival,differentiation,development,and homeostasis.Autophagy primarily serves an adaptive role to protect organisms against various diseases,including infections,cancer,neurodegeneration and heart disease.However,in certain experimental disease models,paradoxically,even the prosurvival functions of autophagy may be detrimental.Excessive activation of autophagy could lead to cell death by destroying major proportions of cytoplasm[13].Thus,autophagic cell death,which would kill cells as an alternative pathway except for apoptosis,represents a new approach to inhibit the growth of apoptosis-resistant cancer cells[14-19].It’s believed that diverse Chinese traditional medicine closely related to anti-NSCLC activities,and that even could impair the acquired multiple drug resistance(MDR).What’s more,autophagy might be the potential mechanisms which would play a role as a candidate targets of natural active compounds.Recent studies of terpenoids,alkaloid,dietary polyphenols and other active constituents that extracted from various herbs indicates that different natural compounds could either regulate the activity of pro-death autophagy or affect the level of protective autophagy of NSCLC cells,that changing their drug sensitivity and cell viability[20-27].MethodsCell viability was measured by CCK8 assay.Cell proliferation was determined by colony formation assay.Cell invasion and migration ability were measured bywound healing assay and transwell assay.The expression of MDRlgene was valued by qPCR.Protein levels were analyzed by Western-blot analysis.The formation of autophagosome and autolysosome were visualized by electron microscope.Tumor xenograft experiment was established to estimate the effect in vivo.All the experiments were performed three times in triplicates,and results were expressed as mean± SEM.Differences between two groups were assessed by Student t test,and multiple comparisons were conducted using one way ANOVA through SPSS 16 software.Differences were considered statistically significant when P<0.01 or P<0.05.Results1.BBD inhibits the viability of A549 and A549/DDP cells in a dose-time dependent mannerWith the increase of the action time and concentration of Babaodan,the viability and value-adding ability of lung adenocarcinoma A549 and A549/DDP cells gradually weakened.2.BBD suppresses tumour growth via promoting the activity of pro-death autophagyThe increasing concentration of Babaodan does not cause changes in apoptosis-related proteins,but increases the autophagic flow,increases the expression of autophagy-specific protein LC3-Ⅱ,and increases the number of autophagosomes in the cells to inhibit cells.Growing.And inhibition of autophagy,it can be seen that the inhibition of Babaodan on A549 and A549/DDP cells is also weakened.3.BBD increases cell autophagy through downregulating the PI3K/AKT/mTOR signaling pathwayWith the increase of the concentration of Babaodan,we found that the expression of p-PI3K,p-AKT,p-mTOR protein decreased,LC3-Ⅱ protein increased;and the combination of PI3K pathway activator IGF-1,Babaodan caused LC3-II The increase was reversed to some extent.Thus,Babaodan increases autophagy by inhibiting the PI3K/AKT/mTOR pathway.4.BBD facilates the antitumour effects of cisplatin Compared with the cisplatin monotherapy group,a certain concentration of Babaodan combined with cisplatin acts on the cells at the same time,which can effectively inhibit the viability and migration invasive ability of A549 and A549/DDP.5.Tumor xenograft experiments demenstrates that BBD inhibits tumour growth and improves cisplatin sensitivityThrough the subcutaneous tumor formation experiment in nude mice,measuring the tumor volume and weight can make Babaodan inhibit tumor cell growth,increase the anti-tumor effect of cisplatin,and the drug process does not produce obvious toxic side effects.ConclusionWe conducted the first study on the role and mechanism of Babaodan:in lung adenocarcinoma by in vivo and in vitro experiments,and demonstrated that it can inhibit the autophagy level of A549 and A549/DDP cells by inhibiting PI3K/AKT/mTOR signaling pathway.Cell growth.At the same time,the expression of the multidrug resistance gene MDR1 and the level of the drug transporter P-gp were reduced,and cisplatin sensitivity was increased.This is of great significance for the exploration of the potential of Babaodan in the application of lung cancer and other tumors.更多还原