培土生金法对COPD大鼠膈肌细胞增殖与凋亡的影响及临床应用

发布时间：2018-04-14 06:35

本文选题：培土生金 + 慢性阻塞性肺疾病　；参考：《广州中医药大学》2016年博士论文

【摘要】：目的：1.介绍导师治疗慢性阻塞性肺疾病的学术思想和临证经验,总结其用药规律及临证化裁。2.观察不同浓度培土生金法中药对COPD模型大鼠体重,肺与膈肌组织形态学、动脉血气(PH、PO2、PCO2)、膈肌细胞凋亡率、增殖率与凋亡率的比值、细胞凋亡途径(Fas、FasL、Bax、Bcl-2)、核转录因子-κB (NP-κB P65)的影响,试从膈肌细胞增殖与凋亡角度探讨培土生金法对COPD膈肌结构的保护作用,并从细胞凋亡途径及NP-κB通路探讨该治法在调节膈肌细胞增殖/凋亡平衡方面的作用及机制,为临床辩证应用培土生金法防治COPD提供实验依据。方法：1.通过仔细阅读导师多年来发表论文、学术讲稿,以及收集学生跟诊过程中导师对慢阻肺的辨证论治、遣方用药,整理总结孙志佳教授治疗慢阻肺的学术思想、辨证思路和处方规律。2.取Wistar大鼠为研究对象,采用气管内注入脂多糖(LPS)联合香烟烟熏法建立COPD大鼠模型,观察不同浓度培土生金法方药对大鼠一般情况,体重的影响。通过HE染色镜下观察大鼠肺和膈肌的组织形态学改变,结合动脉血气分析(PH、PO2、 PCO2)共同评估COPD大鼠模型建立情况和培土生金法对COPD大鼠整体保护作用。3.通过免疫组化法检测PCNA在膈肌中的表达,并计算增殖指数(PI),采用TUNEL方法检测膈肌细胞凋亡情况,并计算凋亡指数(AI)及PI/AI值,评估COPD大鼠膈肌细胞增殖/凋亡平衡情况和培土生金法对其影响。4.通过免疫组化法检测细胞凋亡途径相关蛋白Fas、FasL、Bax、Bcl-2及NF-κB通路相关蛋白P65在膈肌中的表达,并采用RT-PCR技术检测NF-κB P65 mRNA的表达,拟从细胞增殖与凋亡角度探讨培土生金法对COPD的治疗疗效和对膈肌保护作用的作用机制。结果：1.导师孙志佳教授治疗慢阻肺的学术思想可概括为3点：辨别虚实,治病求本,尤重脾土；审明疾病阶段及病在何脏,重视脏腑相关；注重体质,防病传变。临证导师应用培土生金法代表方六君子汤的基础上多有发挥：做到分期辨治,急则治标,以祛邪为主,重清热行气,兼顾痰、瘀,不禁猛药；稳定治本,补虚为主,尤重培土生金；同时注意生活饮食调摄,增强肺康复锻炼。导师临证应用六君子汤基础上多有发挥,临床疗效显著,进一步总结出导师培土生金基础方进行下一步实验验证。2. COPD大鼠模型复制成功,肉眼观察COPD模型组出现肺脏色暗红偏紫,无光泽,有些夹苍白色,肺体积增大；HE染色观察肺组织有明显气道炎症,肺泡结构破坏,肺疱腔扩大等表现；膈肌组织出现不同程度的膈肌纤维排列紊乱、间隙增宽,部分出现膈肌纤维断裂、溶解等。培土生金法能改善上述COPD肺和膈肌组织结构的破坏。3. COPD模型组大鼠体重及动脉PO：明显低于空白组,PCO2明显高于空白组；而培土生金法高剂量中药能明显升高COPD大鼠体重,同时增加动脉血PO：,降低PCO2。4. COPD模型组大鼠膈肌细胞凋亡指数(AI)明显高于空白组,增殖指数与凋亡指数的比值(PI/AI)明显低于空白组；培土生金法中药高剂量方药能降低大鼠膈肌AI,同时能增高PI/AI。5. COPD模型组大鼠膈肌组织Fas、FasL、Bax、及NF-κ B P65蛋白表达升高,Bcl-2蛋白表达降低；培土生金法能明显降低Fas、FasL、Bax、及NF-κ B P65蛋白的表达,同时能升高Bc卜2蛋白表达。NF-κB P65 mRNA基因在空白组、模型组、中药组中的表达未见明显差异。结论：1.导师孙志佳教授治疗COPD注重辨别虚实及疾病所处阶段,尤重脾土对COPD发病及治疗的关键作用,同时兼顾各脏腑之间的整体联系,关注个体体质对COPD传变的重要意义。基于以上辨证思想,导师提出COPD的治疗急性期以祛邪为主,重清热行气,兼顾痰瘀,用药应效专力宏,不能过于拘泥于老年体虚而妄用滋补；稳定期以补虚为主,尤重培土生金。注意情志疏导与饮食调控相结合,加强气息锻炼、传统功法等肺康复训练。2.培土生金法能减轻COPD大鼠肺和膈肌组织的损伤,改善大鼠体重、精神、活动度,呼吸道症状等整体状态。3.培土生金法能有效改善COPD大鼠低氧血症和高碳酸血症发展趋势,提高肺通气和换气功能,改善呼吸功能。4.培土生金法能通过抑制Fas/FasL死亡受体途径和线粒体途径相关蛋白在膈肌的表达,减少膈肌细胞的凋亡,从而起到对COPD肌的保护和呼吸功能的改善作用。5.培土生金法能通过抑制NF-κB通路关键蛋白P65的表达减少膈肌细胞的凋亡,维持膈肌细胞增殖与凋亡的平衡,此过程可能通过对NF-κB P65蛋白抑制减少,而使NF-κB P65蛋白的相对增多而产生,并不是通过增加NF-κB P65 mRNA转录表达而引起相应效应。
[Abstract]:Objective : 1 . To observe the effects of different concentrations of Chinese traditional medicine on the control of chronic obstructive pulmonary disease ( COPD ) in rats with chronic obstructive pulmonary disease ( COPD ) . To distinguish the deficiency , cure the disease and seek the book , especially the spleen soil ;
To examine the stage of disease and how the disease is in the viscera , and attach importance to the viscera - related ;
The present invention focuses on the constitution and the disease prevention and transmission , and the application of the Linzheng Shengjin method on the basis of the present invention has the advantages that the treatment of stages can be realized , the treatment is urgent , the symptoms are eliminated , the pathogenic factors are the main , the heat and heat circulation is heavy , the phlegm , the blood stasis , and the like are taken into account ;
stabilizing the constitution and tonifying deficiency , especially the cultivation of gold ;
At the same time , attention should be paid to life diet adjustment , to enhance the exercise of pulmonary rehabilitation . The clinical curative effect is remarkable , and the clinical curative effect is remarkable , and the next experimental verification is carried out on the basis of the foundation of the tutor cultivation . The model of COPD rats was successfully replicated , the visual observation of COPD model group showed that the lungs were dark red and purple , dull , some were white , the volume of lung increased ;
HE staining was used to observe the manifestations of airway inflammation , alveolar structure destruction and blister cavity enlargement .
The diaphragm muscle fibers were disordered in different degrees , the gaps were widened , and the rupture and dissolution of diaphragm muscle fibers appeared in some parts . The results showed that the structure of the above COPD lung and diaphragm muscle tissue could be improved . Body weight and arterial PO in COPD model group were significantly lower than those in blank group , and PCO2 was significantly higher than that in blank group .
However , the high - dose Chinese traditional medicine could obviously increase the weight of COPD rats and increase the PO of arterial blood , and reduce PCO2 . 4 . The apoptosis index ( AI ) was significantly higher in COPD model group than in blank group , and the ratio of proliferation index to apoptosis index ( PI / AI ) was significantly lower than that in blank group .
Traditional Chinese medicine high - dose prescription can reduce the AI of rat diaphragm while increasing PI / AI . 5 . The expression of Fas , FasL , Bax and NF - 魏B P65 protein in the diaphragm muscle tissue of COPD model group increased , and the expression of Bcl - 2 protein decreased .
The expression of Fas , FasL , Bax and NF - 魏B P65 protein can be significantly reduced by means of the cultivation method . The expression of NF - 魏B P65 mRNA in the blank group , the model group and the traditional Chinese medicine group is not obvious . Conclusion : 1 . Professor Sun Zhijia ' s treatment of COPD is a key role in the pathogenesis and treatment of COPD .
3 . The method can effectively improve the development trend of lung and diaphragm muscle in COPD rats , improve the lung ventilation and ventilation function , and improve the respiratory function .

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

【相似文献】