周仲瑛教授從痰瘀熱毒辨治肺癌的臨床經(jīng)驗及益肺解毒湯的抗腫瘤實驗研究
發(fā)布時間：2019-06-12 06:52
【摘要】：研究目的從多層次、多角度對國醫(yī)大師周仲瑛教授治療的肺癌病案進行挖掘分析,基于病機辨證總結(jié)其治療肺癌的臨床經(jīng)驗和學(xué)術(shù)思想,形成周仲瑛教授病機辨治肺癌的基本方案;同時對周教授治療肺癌的基本方(益肺解毒湯)進行體外抗腫瘤實驗研究,探討其抗腫瘤效果及作用機制。研究方法(1)"人機結(jié)合,以人為本",即在跟隨周仲瑛教授門診抄方學(xué)習(xí)的基礎(chǔ)上,結(jié)合論著學(xué)習(xí)、病案研習(xí)、名醫(yī)訪談等,收集、整理周教授近10年來的肺癌門診病案,嚴格按照診斷標準、排除標準篩選出符合納入標準的病案共646例(共計2451診次),建立病案數(shù)據(jù)庫,運用頻數(shù)法、關(guān)聯(lián)規(guī)則和系統(tǒng)聚類等方法挖掘分析病案中證候、病機、方藥之間的規(guī)律,提煉總結(jié)周教授辨治肺癌的臨證經(jīng)驗、辨治方案和學(xué)術(shù)思想。(2)實驗部分以人肺腺癌A549細胞為研究對象,正常條件下培養(yǎng),再用低、中、高濃度的益肺解毒湯進行干預(yù),運用MTT法觀察其對A549細胞的增殖抑制作用,流式細胞術(shù)檢測細胞的凋亡情況及細胞周期,Westernblot方法觀察A549細胞凋亡相關(guān)蛋白的表達;使A549細胞在缺氧條件下培養(yǎng),再用益肺解毒湯干預(yù),在顯微鏡下觀察細胞形態(tài)學(xué)改變,運用Transwell實驗觀察細胞侵襲能力,細胞劃痕實驗檢測細胞遷移能力,Western blot方法觀察A549細胞發(fā)生上皮間充質(zhì)轉(zhuǎn)化(EMT)相關(guān)的蛋白表達。研究結(jié)果(1)一般情況:646例肺癌患者,共計2451診次,其中男性332例,共1557診次,女性患者314例,共計894診次,年齡最大的87歲,最小26歲,平均年齡61.75±10.05歲。(2)證候分布情況:涉及的臨床表現(xiàn)共324種,主要的臨床表現(xiàn)有咳嗽、少痰、口干、胸悶、胸痛等;涉及舌、脈象共61種,主要的舌脈有:質(zhì)暗紫、質(zhì)暗紅、有裂紋,苔黃薄膩、苔薄黃膩、苔淡黃膩,脈滑、細、弦。(3)病機分布情況:病案中病機描述共133種,主要的病機有氣陰兩傷、痰瘀熱毒、癌毒走注、痰瘀郁毒。主要的病理因素是痰、瘀、癌毒、熱、氣郁,最常見的病性因素為陰虛、氣虛,主要的病位因素有肺、胃、脾、腎、肝。(4)常用方藥分布:藥物總計涉及345種,主要的藥物有北沙參、南沙參、麥冬、山慈菇、澤漆、仙鶴草、太子參、貓爪草、白花蛇舌草、僵蠶、腫節(jié)風(fēng)、薏苡仁、制南星、鱉甲、半枝蓮、法半夏,使用頻次超過400次的藥物有46味,藥物分祛邪、扶正、調(diào)理3大類,祛邪以清熱解毒,化痰散結(jié),活血化瘀,祛風(fēng)通絡(luò)攻毒,清肺化痰類藥物為主,分別占10.93%,12.74%,9.45%,7.86%,5.09%;扶正以益氣養(yǎng)陰,補益氣血,滋補肝腎類藥物為主,分別占16.48%,6.54%,4.80%;調(diào)理以健脾助運藥物為主。(5)K-均值聚類法:對總體肺癌病案中涉及的345種藥物進行聚類分析,并結(jié)合周教授本人意見,得出肺癌基本方:炙鱉甲,南北沙參,天麥冬,太子參,仙鶴草,制南星,山慈菇,澤漆,貓爪草,炙僵蠶,白花蛇舌草,半枝蓮等18味(暫時公開14味)。采用聚類法進行分析,在較低距離尺度水平,發(fā)現(xiàn)若干藥物與藥物的相關(guān)性藥物組合,如黃芪+黨參+靈芝+雞血藤,地骨皮+知母+桑白皮,澤漆+山慈菇,狗舌草+漏蘆。(6)關(guān)聯(lián)分析結(jié)果:通過關(guān)聯(lián)規(guī)則分析肺癌最核心的藥物組合為南沙參、北沙參、麥冬、山慈菇、澤漆;運用關(guān)聯(lián)規(guī)則分析常見病機、病理因素與藥物的相關(guān)性,例如:與痰瘀熱毒阻肺相關(guān)聯(lián)的藥物包括:山慈菇、貓爪草、澤漆、僵蠶、薏苡仁、露蜂房、白花蛇舌草、腫節(jié)風(fēng)、漏蘆、白毛夏枯草等;與痰相關(guān)聯(lián)的藥物為:法半夏、僵蠶、露蜂房、貓爪草、制南星、澤漆、山慈菇、薏苡仁、蘇子、白芥子、貝母等。(7)實驗研究結(jié)果:益肺解毒湯能抑制A549細胞增殖,且這種作用呈時間和劑量依賴性,可減少A549細胞DNA的合成,使細胞阻滯于G0/G1期,并且呈現(xiàn)劑量依賴性,可促使其調(diào)亡,調(diào)亡率分別為(11.6±3.07%,22.70±2.33%,26.67±3.69%vs 8.07±2.62%),cleaved-caspase3、cleaved-caspase9蛋白表達均出現(xiàn)濃度依賴性上調(diào),高劑量組差異顯著(P0.05),p-jnκ、p-p38MARK、p-erk蛋白表達亦成濃度依賴性上調(diào),高劑量組差異顯著(P0.05)。缺氧可誘導(dǎo)細胞發(fā)生形態(tài)改變;益肺解毒湯干預(yù)后可抑制缺氧誘導(dǎo)的細胞形態(tài)改變、抑制細胞侵襲和遷移能力;與缺氧組相比,缺氧+中藥組可上調(diào)E-cadherin,P-gsk-3β蛋白表達(P0.05),下調(diào) Vimentin,Snail,β-catenin,P-Smad2/3 蛋白的表達(P0.05)。研究結(jié)論(1)痰瘀熱毒、氣陰兩傷為本病的基本病機;病理因素以痰、瘀、熱、癌毒為主;病位在肺,涉及脾、胃、肝、腎等臟腑。(2)周教授治療肺癌強調(diào)祛邪重于扶正,采用清熱解毒,化痰散結(jié),活血化瘀,益氣養(yǎng)陰等復(fù)法組方。基本方為:炙鱉甲,南北沙參,天麥冬,太子參,仙鶴草,制南星,山慈菇,澤漆,貓爪草,炙僵蠶,白花蛇舌草,半枝蓮等18味(暫時公開14味)。治療以基本方為主,隨兼夾病機、癥狀加減。(3)益肺解毒湯能抑制肺癌A549細胞增殖、促進其凋亡,并且能抑制缺氧誘導(dǎo)的肺癌A549細胞的形態(tài)改變、侵襲、遷移能力及上皮間充質(zhì)轉(zhuǎn)化(EMT),其機制可能與抑制Wnt/β-catenin 通路有關(guān)。
[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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R249;R273
本文編號：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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R249;R273
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