【作者】 张磊；

【导师】 杨岚；

【作者基本信息】 成都中医药大学 ， 人体解剖与组织胚胎学， 2017， 硕士

【摘要】 实验背景和目标肺癌是临床最常见的恶性肿瘤之一,其发病率和死亡率在世界上所有肿瘤中最高^[1]。非小细胞肺癌（Non-small cell lung cancer,NSCLC）约占肺癌总数的85%,由于临床症状出现较晚且不特异,早期诊断困难。晚期不能手术的患者通常只能接受毒副作用大的常规化疗或放疗,疗效很不理想。肿瘤坏死因子诱导凋亡配体（TRAIL）是肿瘤坏死因子（TNF）家族的成员。是一种安全且极具潜力的抗肿瘤新型药物。临床非小细胞肺癌常对TRAIL耐药,耐药机理尚未完全阐明。黄芩苷在抗氧化,抗病毒,抗血栓形成,抗炎症等方面表现出良好的作用,是增强TRAIL的抗肿瘤作用的极佳候选药物。此次研究主要是将TRAIL和黄芩苷联合应用于体外培养的人肺腺癌细胞株,确认其抗肿瘤效应并初步探索其机制。实验方法采用肿瘤细胞体外培养,LDH释放检测,免疫蛋白印迹技术（Western blot,WB）,活性氧的检测（Detection of ROS）和流式细胞仪（Flow cytometry）等实验方法,检测细胞死亡及其分子机制。实验结果在两种不同的肺癌细胞株A549、H2009中,倒置相差显微镜下观察及LDH释放试验检测得出:不同浓度的黄芩苷与不同浓度的TRAIL联用均有显著的杀伤细胞的作用,细胞死亡率较单药组显著增加,并呈剂量依赖性。通过流式细胞检测凋亡实验得出:黄芩苷增敏TRAIL是通过诱导细胞凋亡实现的;LDH释放实验显示:加入泛Caspase抑制剂z-VAD-fmk后合药组细胞死亡率明显降低;Western Blot检测凋亡蛋白PARP显示合药组PARP被剪切,均表明黄芩苷增敏TRAIL是通过凋亡通路进行的;P38抑制剂SB203580预处理后,合药组细胞死亡率明显降低。合药组细胞在短时间内P-p38表达增强,P38信号通路较单药组显著激活。ROS抑制剂NAC、BHA预处理后,合药组细胞死亡率明显降低,合药组细胞ROS在3 h处表达增强,ROS信号通路较单药组显著激活。实验结论黄芩苷增敏TRAIL的抗肺癌作用,其机制主要通过激活Caspase信号通路诱导细胞凋亡同时激活P38信号通路和ROS信号通路,P38信号通路与ROS信号通路激活具有促进细胞死亡的作用。本研究为非小细胞肺癌的治疗提供新思路,为天然药物联合分子靶向药物治疗肿瘤提供一定理论依据。更多还原

【Abstract】 Background and purpose: Lung cancer is one of the most common malignant tumor in the world.Non-small cell lung cancer(NSCLC)account for 85 % of lung cancer.Due to the later appearance of clinical symptoms and presence with non-specific clinical signs,it is hard to diagnose NSCLC at the early stage.Patients with later period of lung cancer have to undergoing chemotherapy or radiotherapy which with severe toxic and side effects but lacking of significant curative effect.Tumor necrosis factor related apoptosis ligand(TRAIL)is a member of tumor necrosis factor(TNF)family.As a new profound potential medicine.TRAIL is often resistanced by NSCLC,the mechanism is still unknown.Baicalin had good therapeutic effects in anti-oxidant,anti-viral,anti-thrombosis,inflammation and so on,which make it an excellent candidate drug for enhancing the anti-tumor effect of TRAIL.This research combined TRAIL and baicalin and applied to the human lung adenocarcinoma cells in vitro,in order to detect the the mechanism of anti-tumor effect.Experimental approach: Cell culture,LDH release assay,immunoblot analysis,detection of ROS and flow cytometry.Key results: Cell death both in NSCLC cells A549 and H2009 were observed under inverted phase contrast microscope and measured by LDH releasing assay.The results showed that different concentrations of Baicalin and TRAIL had more significant synergistic cell death rates than two single drugs with a dose dependent.Flow cytometry analysis showed that Baicalin sensitized TRAIL through apoptosis.LDH releasing assay showed that cell death rates were dropped significantly after treatment with z-VAD-fmk(general caspase inhibitor).Western Blot showed that PARP of combination group was cleavaged.These results indicated that Baicalin sensitized TRAIL through apoptosis pathway.SB203580(P38 inhibitor)pretreatment could distinctly suppress the cell death rate of combined drug group.After 2 and 4 hours treatment,p38 was significantly activated after treatment withTRAIL and baicalin.ROS scavengers BHA and NAC pretreatment significantly suppress the cell death of combined drug group.Meanwhile,ROS signal pathway was activated after 3 hours treatment with TRAIL and baicalin.Conclusions and Implications: In this study,we found that by inducing P38 and ROS signal pathway and activating caspase signal pathway,baicalin greatly sensitized TRAIL induced apoptosis in human NSCLC cell.Our research might provide new insights into lung carcinoma therapy,and find a strategic of the combination of traditional Chinese medicine and targeting drugs.更多还原