周仲瑛教授从痰瘀热毒辨治肺癌的临床经验及益肺解毒汤的抗肿瘤实验研究
发布时间：2019-06-12 06:52
【摘要】：研究目的从多层次、多角度对国医大师周仲瑛教授治疗的肺癌病案进行挖掘分析,基于病机辨证总结其治疗肺癌的临床经验和学术思想,形成周仲瑛教授病机辨治肺癌的基本方案;同时对周教授治疗肺癌的基本方(益肺解毒汤)进行体外抗肿瘤实验研究,探讨其抗肿瘤效果及作用机制。研究方法(1)"人机结合,以人为本",即在跟随周仲瑛教授门诊抄方学习的基础上,结合论著学习、病案研习、名医访谈等,收集、整理周教授近10年来的肺癌门诊病案,严格按照诊断标准、排除标准筛选出符合纳入标准的病案共646例(共计2451诊次),建立病案数据库,运用频数法、关联规则和系统聚类等方法挖掘分析病案中证候、病机、方药之间的规律,提炼总结周教授辨治肺癌的临证经验、辨治方案和学术思想。(2)实验部分以人肺腺癌A549细胞为研究对象,正常条件下培养,再用低、中、高浓度的益肺解毒汤进行干预,运用MTT法观察其对A549细胞的增殖抑制作用,流式细胞术检测细胞的凋亡情况及细胞周期,Westernblot方法观察A549细胞凋亡相关蛋白的表达;使A549细胞在缺氧条件下培养,再用益肺解毒汤干预,在显微镜下观察细胞形态学改变,运用Transwell实验观察细胞侵袭能力,细胞划痕实验检测细胞迁移能力,Western blot方法观察A549细胞发生上皮间充质转化(EMT)相关的蛋白表达。研究结果(1)一般情况:646例肺癌患者,共计2451诊次,其中男性332例,共1557诊次,女性患者314例,共计894诊次,年龄最大的87岁,最小26岁,平均年龄61.75±10.05岁。(2)证候分布情况:涉及的临床表现共324种,主要的临床表现有咳嗽、少痰、口干、胸闷、胸痛等;涉及舌、脉象共61种,主要的舌脉有:质暗紫、质暗红、有裂纹,苔黄薄腻、苔薄黄腻、苔淡黄腻,脉滑、细、弦。(3)病机分布情况:病案中病机描述共133种,主要的病机有气阴两伤、痰瘀热毒、癌毒走注、痰瘀郁毒。主要的病理因素是痰、瘀、癌毒、热、气郁,最常见的病性因素为阴虚、气虚,主要的病位因素有肺、胃、脾、肾、肝。(4)常用方药分布:药物总计涉及345种,主要的药物有北沙参、南沙参、麦冬、山慈菇、泽漆、仙鹤草、太子参、猫爪草、白花蛇舌草、僵蚕、肿节风、薏苡仁、制南星、鳖甲、半枝莲、法半夏,使用频次超过400次的药物有46味,药物分祛邪、扶正、调理3大类,祛邪以清热解毒,化痰散结,活血化瘀,祛风通络攻毒,清肺化痰类药物为主,分别占10.93%,12.74%,9.45%,7.86%,5.09%;扶正以益气养阴,补益气血,滋补肝肾类药物为主,分别占16.48%,6.54%,4.80%;调理以健脾助运药物为主。(5)K-均值聚类法:对总体肺癌病案中涉及的345种药物进行聚类分析,并结合周教授本人意见,得出肺癌基本方:炙鳖甲,南北沙参,天麦冬,太子参,仙鹤草,制南星,山慈菇,泽漆,猫爪草,炙僵蚕,白花蛇舌草,半枝莲等18味(暂时公开14味)。采用聚类法进行分析,在较低距离尺度水平,发现若干药物与药物的相关性药物组合,如黄芪+党参+灵芝+鸡血藤,地骨皮+知母+桑白皮,泽漆+山慈菇,狗舌草+漏芦。(6)关联分析结果:通过关联规则分析肺癌最核心的药物组合为南沙参、北沙参、麦冬、山慈菇、泽漆;运用关联规则分析常见病机、病理因素与药物的相关性,例如:与痰瘀热毒阻肺相关联的药物包括:山慈菇、猫爪草、泽漆、僵蚕、薏苡仁、露蜂房、白花蛇舌草、肿节风、漏芦、白毛夏枯草等;与痰相关联的药物为:法半夏、僵蚕、露蜂房、猫爪草、制南星、泽漆、山慈菇、薏苡仁、苏子、白芥子、贝母等。(7)实验研究结果:益肺解毒汤能抑制A549细胞增殖,且这种作用呈时间和剂量依赖性,可减少A549细胞DNA的合成,使细胞阻滞于G0/G1期,并且呈现剂量依赖性,可促使其调亡,调亡率分别为(11.6±3.07%,22.70±2.33%,26.67±3.69%vs 8.07±2.62%),cleaved-caspase3、cleaved-caspase9蛋白表达均出现浓度依赖性上调,高剂量组差异显著(P0.05),p-jnκ、p-p38MARK、p-erk蛋白表达亦成浓度依赖性上调,高剂量组差异显著(P0.05)。缺氧可诱导细胞发生形态改变;益肺解毒汤干预后可抑制缺氧诱导的细胞形态改变、抑制细胞侵袭和迁移能力;与缺氧组相比,缺氧+中药组可上调E-cadherin,P-gsk-3β蛋白表达(P0.05),下调 Vimentin,Snail,β-catenin,P-Smad2/3 蛋白的表达(P0.05)。研究结论(1)痰瘀热毒、气阴两伤为本病的基本病机;病理因素以痰、瘀、热、癌毒为主;病位在肺,涉及脾、胃、肝、肾等脏腑。(2)周教授治疗肺癌强调祛邪重于扶正,采用清热解毒,化痰散结,活血化瘀,益气养阴等复法组方。基本方为:炙鳖甲,南北沙参,天麦冬,太子参,仙鹤草,制南星,山慈菇,泽漆,猫爪草,炙僵蚕,白花蛇舌草,半枝莲等18味(暂时公开14味)。治疗以基本方为主,随兼夹病机、症状加减。(3)益肺解毒汤能抑制肺癌A549细胞增殖、促进其凋亡,并且能抑制缺氧诱导的肺癌A549细胞的形态改变、侵袭、迁移能力及上皮间充质转化(EMT),其机制可能与抑制Wnt/β-catenin 通路有关。
[Abstract]:The purpose of this study is to dig and analyze the medical records of lung cancer treated by Professor Zhou Zhongkai from multi-level and multi-angle, and to sum up the clinical experience and the academic thought of the treatment of lung cancer based on the syndrome differentiation, and to form the basic scheme for the pathogenesis and treatment of lung cancer by Professor Zhou Zhongkai. At the same time, we study the anti-tumor effect and the mechanism of anti-tumor of the basic prescription (Yifei Jiedu Decoction) for the treatment of lung cancer by Professor Zhou. The "Man-machine combination, people-oriented" of the research method (1) is to collect and arrange the medical records of lung cancer in the past 10 years, according to the diagnosis standard, on the basis of following the study of the outpatient paper of the Zhou Zhongkai University, combining with the study of the study, the study of the medical record, the interview of the famous doctor, etc., and collecting and arranging the medical records of the lung cancer in the past 10 years, A total of 646 cases (total of 2451 cases) were screened out according to the exclusion criteria, and the rules of the syndrome, the pathogenesis and the prescription of the medical records were analyzed by means of frequency method, association rule and system clustering. The experience of the treatment of lung cancer, the treatment plan and the academic thought are summarized and summarized. (2) In the experimental part, human lung adenocarcinoma A549 cells were cultured under normal conditions and then interfered with the low, medium and high concentration of Yifei Jiedu Decoction. The proliferation inhibition of A549 cells was observed by MTT method, and the apoptosis and cell cycle of the cells were detected by flow cytometry. The expression of apoptosis-related protein in A549 cells was observed by Western blot. A549 cells were cultured under the condition of hypoxia, and then the cells were treated with Yifei Jiedu Decoction. The morphological changes of the cells were observed under the microscope. Western blot was used to observe the expression of the expression of the epithelial mesenchymal transition (EMT) in A549 cells. Results (1) General cases:646 cases of lung cancer, total of 2451 cases, of which 332 were male,1557 for the total,314 in female,894 for total,87 for the maximum age,26 for the minimum, and 61.75 to 10.05 years for the average age. (2) The distribution of syndromes: There are 324 clinical manifestations involved. The main clinical manifestations include cough, less sputum, dry mouth, chest distress, chest pain, etc. The main tongue is: dark purple, dark red, cracked, yellow and greasy fur, thin and greasy fur, and light and greasy fur. The pulse is smooth, thin and string. (3) The distribution of pathogenesis: A total of 133 cases are described in the case of the medical record. The main pathogenesis is qi-yin, two-wound, phlegm-stasis, heat-toxic, cancer-related, and phlegm-stasis. The main pathological factors are phlegm, blood stasis, cancer, heat, and qi stagnation. The most common factors are yin deficiency and qi deficiency. The main factors are lung, stomach, spleen, kidney and liver. (4) the common formula distribution: the total amount of the medicine relates to 345 kinds, the main medicines include the radix glehniae, the radix adenophorae, the ophiopogon root, the mountain-type mushroom, the lacquer, the agrimony, the radix pseudostellariae, the cat claw grass, the oldenlandia diffusa, the Bombyx Batryticatus, the swelling and the wind, the coix seed kernel, the arisaea, the turtle shell, the half-branch lotus and the method of the pinellia ternate, The drug with the frequency of more than 400 times has 46 taste, and the medicine has the functions of eliminating pathogenic factors, strengthening body resistance, and regulating the three main categories, eliminating pathogenic factors, clearing away heat and toxic materials, eliminating phlegm and resolving hard mass, promoting blood circulation, removing blood stasis, dispelling pathogenic wind and dredging collaterals, and clearing lung-heat and eliminating phlegm, which accounts for 10.93%, 12.74%, 9.45%, 7.86% and 5.09%, respectively; It is mainly used for strengthening body resistance, invigorating qi, nourishing yin, invigorating qi and blood, and nourishing liver and kidney, accounting for 16.48%, 6.54% and 4.80%, respectively. (5) K-means clustering method: the cluster analysis of 345 drugs involved in the overall lung cancer medical record is carried out, and the basic prescription of the lung cancer is obtained by combining with the opinion of the professor Zhou, the basic prescription of the lung cancer is prepared by the following steps: 18-flavor (14-flavor) of Bombyx Batryticatus, Herba Hedyotidis Diffusae, and Herba Scutellariae Barbatae. The clustering method is used for the analysis, and at the lower distance scale level, a combination of several drugs and drugs is found, such as the yellow rice, the codonopsis pilosula, the lucid ganoderma and the suberect spatholobus, the cortex and the rhizoma anemarrhenae, the mulberry bark, the lacquer of the mountain, the mushroom, the doodoldenlandia and the unerutin. (6) The results of correlation analysis: The most important drug combination of lung cancer is analyzed by association rules, such as Radix Glehniae, Radix Glehniae, Radix Ophiopogonis, Sagittarius, and Zea; and using the association rules to analyze the correlation between the common machine, the pathological factors and the drug, such as: The medicine which is associated with the phlegm-stasis and heat-toxic drug-blocking lung comprises the following components in parts by weight: mountain-type mushroom, cat claw grass, lacquer, Bombyx Batryticatus, Coicis Semen, Nidus Vespae, Herba Hedyotidis Diffusae, Herba Pileae Scriptae, Herba Rubri, and Prunellae Spica; and the drug associated with the sputum is prepared from Rhizoma Pinelliae, Bombyx Batryticatus, Nidus Vespae, and Radix Araliae, Arisaema arisaematis, and Zea, Such as the mushroom, the coix seed, the perilla, the white mustard seed, the fritillary bulb, and the like. (7) The results of the experiment: Yifei Jiedu Decoction can inhibit the proliferation of A549 cells, and this effect is time-and dose-dependent, which can reduce the synthesis of A549 cell DNA, block the cell in G0/ G1 phase, and present dose-dependence, which can promote the apoptosis and the death rate of (11.6-3.07%). 22.70% (2.33%, 26.67% 3.69% vs 8.07% 2.62%), the expression of clear-caspase3 and clear-caspase3 showed a concentration-dependent increase, and the high-dose group was significantly different (P0.05), and the p-jn-, p-p38MARK, p-erk protein expression was up-regulated and the high-dose group was significantly different (P0.05). The changes of the morphological changes of the cells can be induced by hypoxia. After the intervention of the Yifei Jiedu Decoction, the changes of the cell morphology induced by hypoxia can be inhibited, the invasion and the migration ability of the cells can be inhibited. Compared with the hypoxia group, the hypoxia + traditional Chinese medicine group can increase the expression of E-cadherin and P-gsk-3 (P0.05), and down-regulate the Vimentin, Snail, HCO3-catenin, P-Smad2/3 protein expression (P0.05). The research conclusion (1) is the basic pathogenesis of the disease, such as phlegm, blood stasis, heat and cancer, and the disease is in the lung, involving the spleen, the stomach, the liver, the kidney and the like. (2) Professor Zhou's treatment of lung cancer emphasizes the importance of eliminating pathogenic factors and strengthening body resistance, and has the functions of clearing away heat and toxic materials, eliminating phlegm and resolving hard mass, promoting blood circulation, removing blood stasis, invigorating qi and nourishing yin, and the The basic method comprises the following raw materials in part by weight:18 Chinese-medicinal materials such as Carapax Trionycis, Radix Glehniae, Radix Ophiopogonis, Radix Pseudostellariae, Herba et Gemma Agrimoniae, Rhizoma Arisaematis, and Pseudosciatherae, and Herba Euphorbiae Laevigatae, Radix Arachiae, Bombyx Batryticatus, Herba Hedyotidis Diffusae, and Herba Scutellariae Barbatae. The treatment is based on the basic prescription, along with the pathogenesis and symptoms of the clip. (3) Yifei Jiedu Decoction can inhibit the proliferation of lung cancer A549 cells, promote its apoptosis, and can inhibit the morphological changes, invasion, migration and epithelial-mesenchymal transition (EMT) of lung cancer A549 cells induced by hypoxia, and the mechanism may be related to the inhibition of Wnt/ I-catenin pathway.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R249;R273
本文编号：2497825
[Abstract]:The purpose of this study is to dig and analyze the medical records of lung cancer treated by Professor Zhou Zhongkai from multi-level and multi-angle, and to sum up the clinical experience and the academic thought of the treatment of lung cancer based on the syndrome differentiation, and to form the basic scheme for the pathogenesis and treatment of lung cancer by Professor Zhou Zhongkai. At the same time, we study the anti-tumor effect and the mechanism of anti-tumor of the basic prescription (Yifei Jiedu Decoction) for the treatment of lung cancer by Professor Zhou. The "Man-machine combination, people-oriented" of the research method (1) is to collect and arrange the medical records of lung cancer in the past 10 years, according to the diagnosis standard, on the basis of following the study of the outpatient paper of the Zhou Zhongkai University, combining with the study of the study, the study of the medical record, the interview of the famous doctor, etc., and collecting and arranging the medical records of the lung cancer in the past 10 years, A total of 646 cases (total of 2451 cases) were screened out according to the exclusion criteria, and the rules of the syndrome, the pathogenesis and the prescription of the medical records were analyzed by means of frequency method, association rule and system clustering. The experience of the treatment of lung cancer, the treatment plan and the academic thought are summarized and summarized. (2) In the experimental part, human lung adenocarcinoma A549 cells were cultured under normal conditions and then interfered with the low, medium and high concentration of Yifei Jiedu Decoction. The proliferation inhibition of A549 cells was observed by MTT method, and the apoptosis and cell cycle of the cells were detected by flow cytometry. The expression of apoptosis-related protein in A549 cells was observed by Western blot. A549 cells were cultured under the condition of hypoxia, and then the cells were treated with Yifei Jiedu Decoction. The morphological changes of the cells were observed under the microscope. Western blot was used to observe the expression of the expression of the epithelial mesenchymal transition (EMT) in A549 cells. Results (1) General cases:646 cases of lung cancer, total of 2451 cases, of which 332 were male,1557 for the total,314 in female,894 for total,87 for the maximum age,26 for the minimum, and 61.75 to 10.05 years for the average age. (2) The distribution of syndromes: There are 324 clinical manifestations involved. The main clinical manifestations include cough, less sputum, dry mouth, chest distress, chest pain, etc. The main tongue is: dark purple, dark red, cracked, yellow and greasy fur, thin and greasy fur, and light and greasy fur. The pulse is smooth, thin and string. (3) The distribution of pathogenesis: A total of 133 cases are described in the case of the medical record. The main pathogenesis is qi-yin, two-wound, phlegm-stasis, heat-toxic, cancer-related, and phlegm-stasis. The main pathological factors are phlegm, blood stasis, cancer, heat, and qi stagnation. The most common factors are yin deficiency and qi deficiency. The main factors are lung, stomach, spleen, kidney and liver. (4) the common formula distribution: the total amount of the medicine relates to 345 kinds, the main medicines include the radix glehniae, the radix adenophorae, the ophiopogon root, the mountain-type mushroom, the lacquer, the agrimony, the radix pseudostellariae, the cat claw grass, the oldenlandia diffusa, the Bombyx Batryticatus, the swelling and the wind, the coix seed kernel, the arisaea, the turtle shell, the half-branch lotus and the method of the pinellia ternate, The drug with the frequency of more than 400 times has 46 taste, and the medicine has the functions of eliminating pathogenic factors, strengthening body resistance, and regulating the three main categories, eliminating pathogenic factors, clearing away heat and toxic materials, eliminating phlegm and resolving hard mass, promoting blood circulation, removing blood stasis, dispelling pathogenic wind and dredging collaterals, and clearing lung-heat and eliminating phlegm, which accounts for 10.93%, 12.74%, 9.45%, 7.86% and 5.09%, respectively; It is mainly used for strengthening body resistance, invigorating qi, nourishing yin, invigorating qi and blood, and nourishing liver and kidney, accounting for 16.48%, 6.54% and 4.80%, respectively. (5) K-means clustering method: the cluster analysis of 345 drugs involved in the overall lung cancer medical record is carried out, and the basic prescription of the lung cancer is obtained by combining with the opinion of the professor Zhou, the basic prescription of the lung cancer is prepared by the following steps: 18-flavor (14-flavor) of Bombyx Batryticatus, Herba Hedyotidis Diffusae, and Herba Scutellariae Barbatae. The clustering method is used for the analysis, and at the lower distance scale level, a combination of several drugs and drugs is found, such as the yellow rice, the codonopsis pilosula, the lucid ganoderma and the suberect spatholobus, the cortex and the rhizoma anemarrhenae, the mulberry bark, the lacquer of the mountain, the mushroom, the doodoldenlandia and the unerutin. (6) The results of correlation analysis: The most important drug combination of lung cancer is analyzed by association rules, such as Radix Glehniae, Radix Glehniae, Radix Ophiopogonis, Sagittarius, and Zea; and using the association rules to analyze the correlation between the common machine, the pathological factors and the drug, such as: The medicine which is associated with the phlegm-stasis and heat-toxic drug-blocking lung comprises the following components in parts by weight: mountain-type mushroom, cat claw grass, lacquer, Bombyx Batryticatus, Coicis Semen, Nidus Vespae, Herba Hedyotidis Diffusae, Herba Pileae Scriptae, Herba Rubri, and Prunellae Spica; and the drug associated with the sputum is prepared from Rhizoma Pinelliae, Bombyx Batryticatus, Nidus Vespae, and Radix Araliae, Arisaema arisaematis, and Zea, Such as the mushroom, the coix seed, the perilla, the white mustard seed, the fritillary bulb, and the like. (7) The results of the experiment: Yifei Jiedu Decoction can inhibit the proliferation of A549 cells, and this effect is time-and dose-dependent, which can reduce the synthesis of A549 cell DNA, block the cell in G0/ G1 phase, and present dose-dependence, which can promote the apoptosis and the death rate of (11.6-3.07%). 22.70% (2.33%, 26.67% 3.69% vs 8.07% 2.62%), the expression of clear-caspase3 and clear-caspase3 showed a concentration-dependent increase, and the high-dose group was significantly different (P0.05), and the p-jn-, p-p38MARK, p-erk protein expression was up-regulated and the high-dose group was significantly different (P0.05). The changes of the morphological changes of the cells can be induced by hypoxia. After the intervention of the Yifei Jiedu Decoction, the changes of the cell morphology induced by hypoxia can be inhibited, the invasion and the migration ability of the cells can be inhibited. Compared with the hypoxia group, the hypoxia + traditional Chinese medicine group can increase the expression of E-cadherin and P-gsk-3 (P0.05), and down-regulate the Vimentin, Snail, HCO3-catenin, P-Smad2/3 protein expression (P0.05). The research conclusion (1) is the basic pathogenesis of the disease, such as phlegm, blood stasis, heat and cancer, and the disease is in the lung, involving the spleen, the stomach, the liver, the kidney and the like. (2) Professor Zhou's treatment of lung cancer emphasizes the importance of eliminating pathogenic factors and strengthening body resistance, and has the functions of clearing away heat and toxic materials, eliminating phlegm and resolving hard mass, promoting blood circulation, removing blood stasis, invigorating qi and nourishing yin, and the The basic method comprises the following raw materials in part by weight:18 Chinese-medicinal materials such as Carapax Trionycis, Radix Glehniae, Radix Ophiopogonis, Radix Pseudostellariae, Herba et Gemma Agrimoniae, Rhizoma Arisaematis, and Pseudosciatherae, and Herba Euphorbiae Laevigatae, Radix Arachiae, Bombyx Batryticatus, Herba Hedyotidis Diffusae, and Herba Scutellariae Barbatae. The treatment is based on the basic prescription, along with the pathogenesis and symptoms of the clip. (3) Yifei Jiedu Decoction can inhibit the proliferation of lung cancer A549 cells, promote its apoptosis, and can inhibit the morphological changes, invasion, migration and epithelial-mesenchymal transition (EMT) of lung cancer A549 cells induced by hypoxia, and the mechanism may be related to the inhibition of Wnt/ I-catenin pathway.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R249;R273
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8 周琴;左明焕;李泉旺;姜敏;孙韬;肖俐;乔占兵;胡凯文;;基于中药寒热属性理论使用华蟾素治疗恶性胸腹水的临床研究[J];北京中医药大学学报(中医临床版);2013年04期
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相关博士学位论文 前2条
1 詹金彪;天花粉蛋白抗肿瘤作用的分子机理研究[D];浙江大学;2008年
2 程志强;中药消水Ⅱ号外敷治疗恶性胸水的临床与实验研究[D];北京中医药大学;2002年
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