肺纤维化中医证候观察及肺痹汤干预肺纤维化小鼠模型机制研究

发布时间：2018-05-14 00:43

本文选题：肺痹汤 + 肺纤维化　；参考：《北京中医药大学》2016年博士论文

【摘要】：肺间质纤维化是一种以进行性呼吸困难、干咳、气短、喘憋为临床特征的慢性进行性肺间质疾病。该疾病可由多种肺系疾病、多种系统性疾病发展而来,包括无原发疾病的特发性肺间质纤维化。其主要病理特征是成纤维细胞、肌成纤维细胞的增殖,细胞外基质的过度沉积。随着高分辨率CT的普及,肺间质纤维化检出率逐年升高。其发病率随年龄增长而增高,确诊后生存期一般为3-8年,中位生存期为3年。该病预后差,除肺移植外尚无有效的治疗方法,被世界卫生组织(WHO)列为难治性疾病。而近几十年来,中医凭借其成熟的辨证论治体系,在治疗肺纤维化一病的认识和治疗上积累了丰富的临床经验,取得了一定的较好疗效,值得肯定,但需要进一步循证医学的积累和实验研究数据支撑。因此,探索中医药治疗本病的有效性具有十分重大的意义。研究目的观察肺间质纤维化一病的临床中医证候特征,为深化中医对该病的认识提供一定的循证医学依据；通过动物模型实验和细胞学实验,观察导师经验方肺痹汤干预肺纤维化的疗效及其干预的可能机制。研究方法1肺纤维化中医临床证候特征研究的方法按照特发性肺间质纤维化与继发性肺间质纤维的诊断标准,制定临床信息采集表,观察2006年～2015年北京中医药大学第二临床医学院东方医院门诊收集的116例肺纤维化病人资料,对其症状进行整理分析,利用SPSS软件进行统计学上的聚类分析法,研究肺纤维化病人的中医证候学特征。2肺痹汤干预肺纤维化疗效机制研究的方法2.1实验一：观察肺痹汤在博莱霉素诱导的肺纤维化小鼠模型造模过程中的影响,设置假手术对照组、正常模型组、激素干预组和肺痹汤干预组,从小鼠一般情况、死亡情况、支气管肺泡灌洗液中白细胞计数、肺组织内胶原总含量等几个方面,探讨肺痹汤治疗肺纤维化的可能机制和疗效。2.2实验二：观察人甲壳酶蛋白(YKL-40)和干扰素-γ(INF-γ)对人肺胚成纤维细胞(MRC5)在小鼠脱细胞肺组织片(1nouse lung scaffold)上的生长情况及蛋白表达,从细胞水平探讨研究热点的细胞因子YKL-40对成纤维细胞增殖和蛋白表达的影响及其在肺纤维化过程中的可能作用。2.3实验三：观察不同浓度的中药单体黄芪甲苷、丹参酮Ⅱ A粉,中药注射液黄芪注射液、丹参酮ⅡA注射液,中药肺痹汤对MRC5的生长影响,从细胞水平探讨中药肺痹汤及其主要成份干预成纤维细胞增殖的影响。研究结果1肺间质纤维化的中医证候学研究1.1症状：肺间质纤维化患者常见的症状共计60项,出现频率超过25%的症状共有22项,依次为：咳嗽,气短,喘息,咳痰,乏力,口干,汗出,咽干,咳嗽无力,畏寒,失眠,心慌,易感冒,痰粘,恶风,痰难咯,大便干结,耳聋耳鸣,唇甲紫暗,头晕,腰膝酸软,盗汗。1.2证候要素：在中医证候要素方面,主要提示了“痰”、“气虚”、“热”、“阴虚”、“血瘀”是肺间质纤维化患者的主要证素特点。1.3证候分类：1.3.1气虚血瘀型。咳嗽,咳嗽无力,咳痰,白痰,乏力,淡暗舌。本组中“咳嗽”、“咳痰”为肺间质纤维化患者常见症状,“咳嗽无力”、“乏力”提示气虚的表现,“淡暗舌”提示气虚血瘀。此组症状提示气虚血瘀证的表现。1.3.2痰热阻肺型。痰粘,痰难咯,大便干结,脉滑。本组中痰粘、痰难咯提示痰热的表现,肺与大肠相表里,肺热移于大肠故可见“大便干结”,脉清提示痰证。本组症状为痰热阻肺证的表现。1.3.3气阴两虚型。喘息,气短,畏寒,汗出,心慌,易感冒,失眠,恶风,口干,咽干。此组中症状较多,提示为肺气阴两虚证的表现。1.3.4肾阴亏虚型。盗汗,头晕,耳聋耳鸣,腰膝酸软,脉沉,脉细。此组症状提示为肾阴虚证的表现。1.3.5痰瘀阻肺型。唇甲紫暗,暗红舌。“唇甲紫暗”与“暗红舌”主要为血瘀的表现。结合临床经验,提示此组为痰瘀阻肺证的表现。2肺痹汤干预肺纤维化机制的实验研究2.1肺痹汤对博莱霉素诱导的小鼠肺纤维化模型有缓解作用,可改善小鼠一般情况,降低小鼠肺内胶原蛋白总含量,减少肺泡支气管灌洗液内炎症细胞数量。2.2研究热点细胞因子YKL-40可以促进MRC5增殖,并能促使其向肌成纤维细胞转化；其免疫荧光染色结果显示YKL-40刺激的MRC5内,蛋白Ki67、I型胶原和a-肌动蛋白合成较对照组显著增多,使其更易在小鼠脱细胞肺组织片(mouse lung scaffold)上粘附生长增殖,从而促进肺纤维化的进程。2.3肺痹汤及其主要成份对MRC5增殖的抑制作用中药单体黄芪甲苷对MRC5的抑制作用随浓度的增加而增强,其中从0.078ug/ml开始出现抑制,其抑制率为3.06±6.52%,直至200ug/ml时抑制率为12.55±15.22%。中药单体丹参酮ⅡA对MRC5的抑制作用随浓度的增加而增强,其中从10ug/ml开始出现抑制,其抑制率为2.17±5.39%,直至160ug/ml时抑制率为72.62±12.29%.黄芪注射液对MRC5的抑制作用随浓度的增加而增强,其中从6.25×103ug/ml开始出现抑制,其抑制率为8.89±5.88%,直至10Sug/ml浓度时抑制率为20.58±9.5%。丹参酮Ⅱ A注射液对MRC5的抑制作用随浓度的增加而增强,其中从0.625ug/ml时开始出现抑制,其抑制率为11.32±10.35%,直至320ug/ml时抑制率为45.06±37.72%。肺痹汤对MRC5的抑制作用随浓度的增加而增强,其中从0.156ul/ml开始出现抑制,其抑制率为2.89±14.7%,直至2.5ul/ml及更高的浓度,抑制作用尤其明显,均在60%以上,其中2.5u1/ml的抑制率为66.2±34.75%。结论从116例肺纤维化病人资料临床观察分析结果,认为肺纤维化一病常见证候可能包括：气虚血瘀、痰热阻肺、气阴两虚、肾阴亏虚、痰瘀阻肺；其中医证候特征以气虚血瘀较为常见,气虚为其发病之本,血瘀为久病之实。肺痹汤能够缓解博莱霉素诱导的小鼠肺纤维化模型的肺内情况,该疗效机制可能是从抑制炎症反应和抗胶原合成两个方面实现的。细胞因子YKL-40能够促进MRC5增殖,合成分泌胶原,并向肌成纤维细胞转化,是一种促肺纤维化的因子；而肺痹汤及其主要成份能够抑制MRC5的增殖,其中肺痹汤抑制效果最佳且多次重复实验的结果稳定,其主要成分丹参抑制效果亦可,且较黄芪的抑制效果好。在以肺纤维化常见证候气虚血瘀证基础上拟定的经验方肺痹汤,对肺纤维化有一定缓解作用,其作用机制可能是通过抑制成纤维细胞增殖,抑制肺内炎症反应程度和抑制胶原合成等几个方面。
[Abstract]:Pulmonary fibrosis is a chronic progressive interstitial lung disease characterized by progressive dyspnea, dry cough, shortness of breath, and asthma. This disease can be developed by a variety of lung diseases and multiple systemic diseases, including idiopathic pulmonary fibrosis without primary disease. Its main pathological feature is fibroblasts and myofibroblasts. Proliferation, excessive deposition of extracellular matrix. With the popularization of high resolution CT, the detection rate of pulmonary fibrosis is increasing year by year. The incidence of the pulmonary fibrosis increases with age. The survival period is generally 3-8 years and the median survival period is 3 years. The prognosis is poor, except for lung transplantation, there is no effective treatment, and the WHO (WHO) is a difficult one. In recent decades, with its mature syndrome differentiation and treatment system, Chinese medicine has accumulated rich clinical experience in the treatment of the disease of pulmonary fibrosis, and has achieved good curative effect. It is worth affirming, but it needs further evidence-based medicine to accumulate and verify the data support. Therefore, to explore the treatment of this disease by traditional Chinese medicine. The purpose of this study is to observe the characteristics of TCM Syndromes of pulmonary fibrosis (PF), and to provide a evidence-based medical basis for deepening the understanding of the disease. Through animal model experiments and cytological experiments, the effect and intervention of tutor experience Fang Fei Bi Decoction in the intervention of pulmonary fibrosis are observed. The method of study method 1 the clinical syndrome characteristics of TCM syndrome of pulmonary fibrosis, according to the diagnostic criteria of idiopathic pulmonary fibrosis and secondary pulmonary interstitial fiber, set up a clinical information collection table, and observe 116 cases of pulmonary fibrosis patients in the outpatient department of the second clinical medicine hospital of Beijing University of Chinese Medicine from 2006 to 2015. In order to investigate the effect of lung Bi Decoction on pulmonary fibrosis, the effect of lung Bi Decoction (.2) on the effect mechanism of pulmonary fibrosis was studied by SPSS software. The effect of lung Bi Decoction on the model of pulmonary fibrosis induced by bleomycin was observed. The possible mechanism and efficacy of lung Bi Decoction in the treatment of pulmonary fibrosis in the control group, the normal model group, the hormone intervention group and the lung Bi Decoction intervention group, from the mice general condition, the death situation, the white blood cell count in the bronchoalveolar lavage fluid and the total collagen content in the lung tissue, the possible mechanism and efficacy of lung Bi Decoction in the treatment of pulmonary fibrosis were discussed in.2.2 two: YKL-4 0) the growth and protein expression of human lung fibroblast cells (MRC5) on mouse 1nouse lung scaffold (1nouse lung scaffold), and the effect of cytokine YKL-40 on the proliferation and protein expression of fibroblasts and the possible role of.2.3 in the process of pulmonary fibrosis in the process of pulmonary fibrosis. Test three: To observe the effects of different concentrations of Radix Astragali, tanshinone II A, Radix Astragali, tanshinone II A, tanshinone II A, and lung Bi Decoction of traditional Chinese medicine on the growth of MRC5, and the effect of the traditional Chinese medicine lung Bi Decoction and its main components on the proliferation of fibroblast from the cell level. Results 1 TCM syndrome of pulmonary fibrosis Symptomatic study 1.1 symptoms: the common symptoms of pulmonary fibrosis were 60, and there were 22 symptoms of more than 25% of the symptoms. The symptoms were: cough, short breath, wheezing, expectoration, fatigue, dry mouth, sweat, dry throat, cough weakness, fear of cold, insomnia, palpitation, phlegm, phlegm, phlegm, deafness and tinnitus, labia dark, dizziness, .1.2 syndrome factors: "phlegm", "Qi Deficiency", "heat", "Yin Deficiency" and "blood stasis" are the main syndromes and characteristics of.1.3 syndrome in the patients with pulmonary interstitial fibrosis: 1.3.1 Qi deficiency and blood stasis type. Cough, cough weakness, expectoration, white phlegm, fatigue and dim tongue. "Cough" in this group. "Expectoration" is a common symptom of pulmonary fibrosis in the patients. "Cough is weak" and "fatigue" suggests Qi deficiency. "Light dark tongue" suggests Qi deficiency and blood stasis. The symptoms suggest that qi deficiency and blood stasis syndrome shows.1.3.2 phlegm heat resistance lung type. Phlegm sticky, phlegm difficult, stool dry and vein slippery. In intestinal form, the lung heat shifted to the large intestine. The symptoms of phlegm and heat resistance syndrome were.1.3.3 Qi and yin deficiency syndrome. The symptoms of this group were asthmatic, short, cold, perspiration, panic, cold, insomnia, evil wind, dry mouth, and dry pharynx. The symptoms of this group were more, suggesting the manifestation of the deficiency of.1.3.4 kidney yin deficiency, the two deficiency of Lung Qi Yin, night sweats, Dizziness, deafness and tinnitus, waist and knee soft, pulse sinks, pulse fine. The symptoms of this group are.1.3.5 phlegm and blood stasis syndrome. Lip a dark red tongue, dark red tongue. "Lip a dark" and "dark red tongue" are the main manifestations of blood stasis. Combined with clinical experience, it is suggested that this group is the performance of phlegm stasis syndrome in.2 lung Bi Decoction in the mechanism of pulmonary fibrosis. The study of 2.1 lung Bi decoction can relieve the pulmonary fibrosis model induced by bleomycin in mice. It can improve the general situation of mice, reduce the total content of collagen in the lung of mice and reduce the number of inflammatory cells in the alveolar lavage fluid.2.2 research hot spot factor YKL-40 can promote the proliferation of MRC5 and promote its conversion to myofibroblast. The results of immunofluorescence staining showed that the synthesis of protein Ki67, type I collagen and a- actin increased significantly in the MRC5 stimulated by YKL-40, making it easier to adhere and proliferate on the mouse cell lung tissue slices (mouse lung scaffold), and thus promote the progression of pulmonary fibrosis in the process of.2.3 lung Bi Decoction and its main components on the proliferation of MRC5. The inhibitory effect of Astragaloside on MRC5 was enhanced with the increase of concentration, in which the inhibitory rate was 3.06 + 6.52% from 0.078ug/ml, and the inhibition rate was 12.55 + 15.22%., and the inhibitory effect of tanshinone II A on MRC5 was enhanced with the increase of concentration, which began to appear from 10ug/ml. The inhibition rate was 2.17 + 5.39%, and the inhibition rate was 72.62 + 12.29%. at 160ug/ml. The inhibitory effect of Astragalus Membranaceus Injection on MRC5 increased with the increase of concentration, in which the inhibition rate was 8.89 + 5.88% from 6.25 x 103ug/ml, and the inhibition rate was 20.58 + 9.5%. tanshinone II A injection to MRC5 under 10Sug/ml concentration. With the increase of concentration, the inhibitory rate was 11.32 + 10.35%, the inhibition rate was 11.32 + 10.35%, and the inhibition rate was 45.06 + 37.72%., and the inhibitory effect of lung Bi Decoction on MRC5 increased with the increase of concentration, and the inhibitory rate was 2.89 + 14.7% from 0.156ul/ml, until 2.5ul/ml and higher concentration. The inhibitory effect was more than 60%, and the inhibitory rate of 2.5u1/ml was 66.2 + 34.75%.. Conclusion from 116 cases of pulmonary fibrosis, the common syndromes of pulmonary fibrosis may include Qi deficiency and blood stasis, phlegm heat resistance, deficiency of Qi and Yin, deficiency of kidney yin, phlegm and stasis of lung, and Qi deficiency syndrome characteristics of TCM syndrome. Blood stasis is more common, Qi deficiency is the origin of the disease, and blood stasis is a long-term disease. Lung Bi decoction can relieve the pulmonary fibrosis induced by bleomycin in the pulmonary fibrosis model of mice. This mechanism may be realized from two aspects: inhibition of inflammatory reaction and synthesis of anti collagen. Cytokine YKL-40 can promote the proliferation of MRC5, synthesize and secrete collagen. The transformation of myofibroblast is a factor for promoting pulmonary fibrosis, and the lung Bi Decoction and its main components can inhibit the proliferation of MRC5, in which the inhibitory effect of the lung Bi decoction is the best and the results of repeated repeated experiments are stable. The main ingredient of the main ingredient of Salvia miltiorrhiza is also better than the Astragalus. On the basis of stasis syndrome, empirical Fang Fei Bi decoction has some relieving effect on pulmonary fibrosis. Its mechanism may be by inhibiting the proliferation of fibroblasts, inhibiting the degree of inflammatory reaction in the lung and inhibiting the synthesis of collagen.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259

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