平肺复方对人肺腺癌A549细胞的增殖抑制及作用机制

【作者】 王玥姣；

【导师】 贾立群；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2014， 博士

【摘要】 本论文包括文献综述和实验研究两个部分。文献综述一回顾了肺癌的中医药治疗进展,主要从病因病机、辨证分型、中医治法研究、中西医结合辨证论治、中医药维持治疗晚期肺癌、中医药治疗肺癌的机制研究六个方面进行论述。文献回顾表明中西医结合治疗肺癌注重辨证施治,标本兼顾,扶正与祛邪相结合,在改善临床症状、减轻放化疗毒副反应、放化疗增敏、提高生活质量、延长生存时间等方面取得了良好的临床疗效。文献综述二总结了目前对PPAR-γ信号通路的研究,主要从蛋白结构和传导途径、与肿瘤的关系、对其抗肿瘤机理研究进展等几个方面进行论述,有部分文献表明天然药物可能从PPAR-γ信号通路发挥抗肿瘤作用。中医药治疗肿瘤的部分机制不明,在机理方面一直缺乏突破性研究,相关信号通路与药物靶标不明,使中医药走向国际受到了一定阻碍。为探讨中药复方的可能作用靶点,本实验研究采取血清药理学、流式细胞学、蛋白质免疫印迹等方法,观察了平肺复方对人肺腺癌A549细胞的增殖抑制及作用机制。目的临床研究发现以养阴清肺为治法的平肺复方是延长肺癌患者生存期的独立预后因素之一,但缺乏深入研究。本实验是建立在以往相关研究的基础上,初步探讨了平肺复方生药及其含药血清对人肺腺癌A549细胞增殖的抑制作用,并观察了平肺复方对A549细胞凋亡及Ca spase-3蛋白表达水平的影响,为之后从细胞、分子角度证明平肺复方治疗肺癌的作用机理,是否影响PPAR-γ信号传导通路奠定基础,揭示中药与化疗药物治疗肿瘤的疗效差异和可能机理。方法①平肺复方制剂20g/kg/d灌服大鼠,取含药血清培养A549细胞,设5%、15%和25%3个不同浓度正常血清组和5%、15%和25%3个不同浓度含药血清组,观察培养至0h、24h.48h.72h.96h各组细胞存活率；②将平肺复方生药直接添加入含10%胎牛血清的高糖DMEM,设1mg/ml.5mg/ml和10mg/ml3个不同浓度平肺复方生药组,观察培养24h、48h、72h各组细胞存活率；③将PPAR-γ特异性阻断剂GW9662与10mg/ml平肺复方生药同时作用于A549细胞培养体系,观察PPAR-γ通路阻断后平肺复方对A549细胞生长的影响；④将1mg/m1.5mg/ml和10mg/ml3个不同浓度平肺复方生药作用于A549细胞,应用磷脂酰丝氨酸外翻分析法,于流式细胞仪检测细胞凋亡情况。⑤将10mg/ml平肺复方生药作用于人肺腺癌A549细胞,蛋白质免疫印迹法检测培养24h.48h和72h后A549细胞中Caspase-3蛋白的表达水平,β-actin作内参。数据录入Excel软件进行保存,使用SPSS17.0软件进行分析,多个样本均数的比较采用t检验。结果①在常规培养体系中添加5%-25%正常大鼠血清,对A549细胞生长存在明显影响,表现为生长抑制作用；但这种生长抑制作用与添加大鼠血清的浓度关系不大。在常规培养体系中添加25%含药大鼠血清培养至48h时即对A549细胞增殖存在影响,表现为生长抑制作用,与同浓度血清对照组的细胞存活率相比存在统计学差异(P<0.05)；这种抑制作用随时间增长而增强,培养至72h、96h与对照组相比存在明显差异(P<0.01)。②应用CCK-8法检测人肺腺癌A549细胞的增殖显示,平肺复方生药1mg/ml在体外作用72h内对细胞生长没有明显影响,平肺复方生药5mg/ml和10mg/ml在作用24h、48h和72h表现出对A549细胞增殖的抑制作用,与空白对照组相比存在统计学差异(P<0.05)。③平肺复方生药在体外作用24h后即显现对A549细胞的生长抑制作用,这种抑制作用随时间的延长而增强,与对照组相比具有明显差异(P<0.01)；单纯加入GW9662对细胞的生长并未产生影响；平肺+GW组在培养至24h、48h、72h均对细胞生长产生抑制作用,与对照组相比具有统计学差异(P<0.05),但这种抑制作用较平肺组弱(P<0.05)。④平肺复方10mg/ml作用17h可引起A549细胞发生早期凋亡及死亡,与空白对照组相比存在统计学意义。⑤将生药终浓度为10mg/ml的平肺复方生药作用于人肺腺癌A549细胞培养体系,可以提高肺癌细胞中Caspase-3蛋白的表达,这种作用在体外24h后即能显现,与对照组相比具有统计学意义(P<0.05)；Caspase-3蛋白的表达随时间的延长有所增强,培养至72h Caspase-3蛋白的表达较24h明显增多(P<0.05)。结论平肺复方生药及其含药血清均可抑制人肺腺癌A549细胞的增殖；特异性拮抗剂GW9662可部分逆转平肺复方对人肺腺癌A549细胞的增殖抑制作用,平肺复方的抗肿瘤作用机制可能部分涉及到PPAR-γ信号通路,但并不完全是通过这一条信号通路在发挥作用；平肺复方可明显上调Caspase-3蛋白的表达水平,并诱导肿瘤细胞的早期凋亡。更多还原

【Abstract】 This thesis includes two parts:two literature reviews and an experimental study.The first part of the literature review was the progression of the TCM therapy of lung cancer in recent years. It was including six parts:etiology and pathogenesis, syndrome differentiation, the TCM treatment research, the syndrome differentiation and treatment of integrated traditional Chinese and western medicine, the TCM maintenance therapy in advanced lung cancer, and the mechanism of Chinese medical treatment for lung cancer. This literature review showed that integration of traditional and western medicine treatment of lung cancer emphasizes the syndrome differentiation, specimens consideration, integration of strengthening healthy energy and eliminating evil. And this kind of treatment obtained satisfied clinical effect on improving clinical symptoms, reducing chemoradiotherapy adverse reaction, enhancing the quality of life, prolonging the survival time.The second part of the literature review summarized the research in PPAR-γ signal path at present from following several aspects:protein structure and pathway, the connection with carcinoma, the investigation progress of ant i-tumor mechanism. Some of papers showed that the natural medicine may could against the cancer cells through the PPAR-γ signal path.But part of mechanism of TCM therapy for cancer is still unknown. This would be likely to create difficulties on globalizing. In order to found the potential mechanism of the Pingfei Compound, this research observed the inhibitory action of the medicinal serum/apozem of the compound to the proliferation of the human lung adenocarcinoma cell line A549, and the apoptosis of the tumor cells, the expression of Caspase-3.Purpose This research discussed the inhibitory action of the medicinal serum/apozem of the Pingfei Compound to the proliferation of the human lung adenocarcinoma cell line A549, the apoptosis and the expression of Caspase-3, through serum pharmacology methods, flow cytometer, western blot, on the basis of previous studies.Methods①erum containing Pingfei Compound was prepared by serologic pharmacology method. Pingfei Compound was administered orally to SD rats at doses of20g/kg/d. The proliferation of A549cells after exposure to the serum was measured by CCK-8assay at Oh,24h,48h,72h and96h.②Groups were blank control, liquid extract of Pingfei prescription1mg/mL,5mg/mL and10mg/mL group. Prol iferat ion of A549cells after exposure to the apozem was measured by CCK-8assay at24h,48h, and72h.③The PPAR-y specificity blocker GW9662and the Pingfei Compound were added in the cell cultures. Observe if the Pingfei Compound could influence the growth of the A549cells or not.④For apoptosis study, it was detected by analyzing plasma membrane translocation of phosphatidylserine, using FACS.⑤Caspase-3in A549cells was measured with Western Blot. Statistic analysis for quantitative data is student t-test.Results⑥The growth of A549could be suppressed at the48h after exposure to the serum from rats (P<0.05). The inhibi t ion was enhanced over time. The results exists significant difference compared with the control group at the72h and96h (P<0.01)⑦5mg/mL and10mg/mL groups presented inhibitive effect on A549growth at24h,48h and72h time points{P<0.01).⑧GW9662reversed partly the effect of Pingfei Compound on the proliferations of A549cells.⑨10mg/ml group could induse the early and late apoptosis induction (P<0.01).⑩The expression of Caspase-3was significantly increased by Pingfei Compound.Conclusion Apozem of Pingfei Compound, a TCM formula, could suppress A549growth in vitro in dose and time dependent manner. This inhibitory effect was related to its apoptosis induction. Perhaps the increase of the expression of Caspase-3induced the apoptosis. The mechanism of Pingfei Compound may could be involved in PPAR-y signal pathway.更多还原