【作者】 唐晓勇；

【导师】 唐迎雪；

【作者基本信息】 山东中医药大学 ， 中药学， 2012， 博士

【摘要】 目的：1.探讨“痰邪致病”与肺癌多药耐药相关性的中医理论。2.探讨肺癌中医证候与化疗疗效的相关性。3.探讨浙贝母碱逆转A549/DDP肺癌细胞株耐药的作用及可能的机理。方法：1.梳理中医学关于肺癌的古今文献资料，采用“取象比类”的方法将“痰邪致病”与肺癌耐药特点进行类比。2.回顾性分析282例肺癌患者中医证候类型与化疗疗效的相关性，作为本论文的临床工作基础研究。3.以理论和临床研究为基础，展开实验研究，以A549/DDP耐药肺癌细胞株为研究对象，以川芎嗪做对照药物，采用MTT法检测细胞耐药倍数、浙贝母碱的最佳逆转耐药浓度及逆转耐药倍数；用流式细胞仪检测耐药细胞凋亡率，细胞免疫荧光法检测L-RP蛋白表达，RT-PCR检测细胞ERCC1-mRNA表达。结果：1.通过文献整理及“取象比类”的方法，论文提出“痰邪是肺癌多药耐药的重要病因，化痰散结法是治疗肺癌多药耐药的基本治法”的假说。2.临床研究显示：正气亏虚证型组较气虚痰湿型组、气血瘀滞型组和阴虚热毒型组肺癌对化疗有效率高（64.1%），治疗期间患者更少的出现疾病进展（19.6%）。正气亏虚型与气虚痰湿型两组相比，气虚痰湿型肺癌化疗有效率低，更多的患者出现进展。中位肿瘤进展时间和总生存时间单纯正气亏虚证型组优于其他各组。3.实验研究显示：A549/DDP耐药倍数为13.08倍，细胞高度耐药。空白对照组、DDP组、川芎嗪实验组、浙贝母碱实验组各组随DDP浓度的增加，药物对A549/DDP细胞的抑制率不断增加。川芎嗪实验组逆转倍数3.62，浙贝母碱实验组逆转倍数3.73。流式细胞仪检测凋亡发生率各组分别为2.56±0.44、13.5±1.42、33.82±4.56、38.16±2.25，川芎嗪实验组和浙贝母碱实验组与空白对照组和DDP组比较差异有统计学意义（p <0.05）。细胞免疫荧光测定的结果显示，川芎嗪实验组和浙贝母碱实验组较空白对照组和DDP组的L-RP表达明显降低（p <0.05）。各组ERCC1mRNA表达分别为0.62±0.04、0.56±0.03、0.36±0.07、0.32±0.08，川芎嗪实验组和浙贝母碱实验组与空白对照组和DDP组比较显著降低了ERCC1mRNA的表达，差异有统计学意义（p <0.05）。结论：1.本论文理论研究部分提出假说，通过文献整理及“取象比类”为假说提供了理论依据。理论研究的内容丰富了中医学对肺癌多药耐药的认识。2.临床研究提示正气亏虚型是晚期肺癌中预后较好的分型，气虚痰湿型、气血瘀滞型和阴虚热毒型是晚期肺癌中预后较差的分型，推测肺癌的发生、发展可能遵从先出现正气亏虚，而后合并出现痰阻、瘀血、热毒致虚实夹杂，最后出现邪盛正衰的病机演变规律，而合并出现痰浊、瘀血、热毒等实邪时化疗有效率降低、生存期缩短、疾病进展迅速可能与对化疗药物的多药耐药有关。这为假说提供了临床方面的证据支持。3.实验研究提示浙贝母碱可逆转肺癌A549/DDP细胞株的多药耐药，其机理与促进细胞凋亡、下调L-RP蛋白表达、抑制ERCC1mRNA表达，从而降低DNA的修复能力有关。这为假说提供了实验方面的证据支持。更多还原

【Abstract】 Objective:1.To study theory of Traditional Chinese Medicine（TCM）between sputum evil andLung Cancer multi-drug resistance(MDR).2.To explore correlation between syndromes of TCM and effect of chemotherapyabout Lung Cancer.3.To explore effect and mechanism of MDR in adenocarcinoma line A549/DDPreversed by peimine.Methods:1. Through reviewing literature of TCM about Lung Cancer, contrasting featuresof sputum evil and Lung Cancer MDR,dissertation put forward a hypothesis.2.On base of clinical practice,the dissertation supported hypothesis byretrospective analysis in282Lung Cancer patients.3.On base of theoretical and clinical pratice,the dissertation developed experimentto assure the hepothesis.MTT assay was employed to measure cell resistant index andreversing effect, flow cytometry was used to analyse apoptosis, cell immunofluorescencetechnique was used to measure expression of resistance associated protein LRP, RT-PCRwas employed to evaluate expression of ERCC1-mRNA.Results:1.The dissertation put forward a hypothesis that sputum evil might be importantreason of MDR,Huatan Sanjie might be basic law of reversing Lung Cancer MDR.2.The clinical study showed that RR,TTP,OS in team of Vital qi deficiency bemuch better than in the other groups. 3.Resistant index in A549/DDP line was13.08.There were common feature that cellinhibitory rate increased with the increase of DDP concertration among blank contronlgroup,DDP contronl group,Tetramethylpyrazine experiment group and peimine experimentgroup.Reverse index of Tetramethylpyrazine experiment group was3.62,thus that ofpeimine experiment group was3.73.Apoptosis rate of the four group was respectively2.56±0.44、13.5±1.42、33.82±4.56、38.16±2.25,in which that of the experiment group wasmuch more than that of the control group.Cell immunofluorescence technique showed thatthe expression of L-RP in experiment group was much lower than that in the control group（p <0.05）.The expression of ERCC1mRNAwas respectively0.62±0.04、0.56±0.03、0.36±0.07、0.32±0.08,the difference between the experiment group and the control groupwas statistically significant（p <0.05）.Conclusions:1.On the base of the literatural study and analogy,the doctoral dissertation putforward a hypothesis,and provided it by lots of evidence.2.Vital qi deficiency was the most better prognosis of all syndromes. RR,TTP,OSwould decrease when sputum evil,blood stasis,heat evil appeared.The phenomenonwould be the result of MDR.3.Peimine could reverse MDR in A549/DDP cell line.Inducing apoptosis, down-regulating L-RP and ERCC1mRNA express were the possible mechanisms.更多还原