IPF患者中医证候分布规律研究及参七虫草胶囊对肺纤维化大鼠EMT过程的影响

发布时间：2018-01-14 07:42

  本文关键词：IPF患者中医证候分布规律研究及参七虫草胶囊对肺纤维化大鼠EMT过程的影响　出处：《安徽中医药大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 特发性肺纤维化 中医证候 参七虫草胶囊 细胞间质转化 Wnt/β-catenin信号通络

【摘要】：目的1探讨特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)患者中医证候分布规律。2探讨参七虫草胶囊对肺泡上皮细胞间质转化(epithelial mesenchymal transition,EMT)过程的影响。3探讨Wnt/β-catenin通路在EMT发生过程中的可能作用。方法1临床观察:查阅相关文献,制定IPF患者中医证候调查表,通过临床问卷调查的方式,搜集76例IPF患者一般情况(包括性别、年龄、病程、吸烟情况、合并疾病等)和中医四诊信息等,通过分析得出IPF患者中医证候分布规律。2动物实验:将128只雄性SD大鼠随机分为四组:空白组(C组)、模型组(M组)、参七虫草组(S组)和泼尼松组(P组),每组32只。适应性饲养7天后,3组大鼠均采用气管内注入博来霉素(bleomycin,BLM)的方法复制肺纤维化模型,空白组气管内注入等量生理盐水。造模后第2天开始灌胃给药,空白组灌入等体积生理盐水。灌胃后第7、14、21和28天取材,苏木精-伊红染色法(Hematoxylin Eosin staining,HE)观察肺组织病理结构,Masson染色法观察肺组织胶原沉积情况,免疫组化(immunohistochemical IHC)法检测肺组织中上皮细胞钙粘素(E-Cadherin,E-Cad)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和波形蛋白(Vimentin)的蛋白表达及反转录聚合酶链反应(reverse transcriptionpolymerase chain reaction,RT-PCR)检测肺组织中Wnt1、Wnt2和β-catenin mRNA表达的动态变化。结果1临床观察:76例IPF患者证候前五:气阴两虚夹瘀(13.16%)、痰热壅肺(10.53%)、痰瘀阻肺(9.21%)、肺肾气虚(7.89%)和肺气阴两虚(7.89%)。2动物实验:2.1 HE染色:空白组大鼠肺泡结构基本清晰完整。模型组大鼠第7天可见肺泡结构紊乱,大量肺泡腔塌陷,肺泡腔内炎性细胞浸润,肺泡间隔开始增宽;第14天见肺泡腔内大量炎性细胞浸润,肺泡间隔继续增宽,成纤维细胞增生,纤维组织形成,胶原沉积;第21天肺泡腔内炎性细胞较第14天减少,肺泡间隔进一步增宽;第28天基本无炎性细胞浸润,以大量成纤维细胞增生、纤维组织形成为主。参七虫草组及泼尼松组大鼠肺组织分别与模型组相应时间点比较,上述病理改变均有不同程度的减轻,参七虫草组与泼尼松组病理变化程度相近。2.2Masson染色:空白组大鼠肺泡间隔有少量蓝染胶原纤维分布。模型组大鼠肺组织第7天可见染成蓝色的胶原纤维沉积,主要集中于终末支气管壁、血管壁及肺泡间隔;第14天各部位蓝色染区颜色加深,胶原纤维进一步沉积;第21和28天,蓝色染区进一步增多,颜色进一步加深,表明大量胶原沉积,弥漫性纤维组织形成。参七虫草组及泼尼松组大鼠肺组织分别与模型组相应时间点比较,各组肺泡间隔宽度较模型组减小,蓝染胶原沉积量减少,纤维化程度减轻。参七虫草组与泼尼松组胶原沉积程度相近。2.3E-cad、α-SMA和Vimentin蛋白:第7、14、21和28天,E-cad蛋白表达模型组低于空白组,α-SMA和Vimentin蛋白表达模型组高于空白组,组间比较差异均有统计学意义(P0.05),参七虫草组和泼尼松组E-cad蛋白表达高于模型组,α-SMA和Vimentin蛋白表达低于模型组,组间比较差异均有统计学意义(P0.05),参七虫草组和泼尼松组E-cad和Vimentin蛋白表达相近,组间比较差异无统计学意义(P(29)0.05),第28天,参七虫草组α-SMA蛋白表达低于泼尼松组,差异有统计学意义(P(27)0.05);空白组4个时间点肺组织中E-cad、α-SMA和Vimentin蛋白表达无明显差异(P(29)0.05),模型组、参七虫草组和泼尼松组第14、21和28天肺组织中E-cad蛋白表达逐渐降低,α-SMA和Vimentin蛋白表达逐渐升高,组内比较差异有统计学意义(P0.05)。2.4 Wnt1、Wnt2和β-catenin mRNA:第7、14、21和28天,Wnt1、Wnt2和β-catenin mRNA表达模型组高于空白组,组间比较差异均有统计学意义(P0.05),参七虫草组和泼尼松组Wnt1、Wnt2和β-catenin mRNA表达低于模型组,组间比较差异均有统计学意义(P0.05),参七虫草组和泼尼松组组间比较,差异无统计学意义(P(29)0.05);空白组4个时间点肺组织中Wnt1、Wnt2和β-catenin mRNA的表达无明显差异(P(29)0.05),模型组第14和21天,肺组织中Wnt1、Wnt2和β-catenin mRNA表达逐渐增加,第21天最高,第28天有所下降,组内比较差异有统计学意义(P(27)0.05)。参七虫草组和泼尼松组第14和21天,肺组织中Wnt1和Wnt2 mRNA表达逐渐增加,第21天最高,第28天有所下降,组内比较差异有统计学意义(P(27)0.05),而β-catenin mRNA表达组内比较差异无统计学意义(P(29)0.05)。结论1气阴两虚夹瘀型为IPF患者常见证候类型。2参七虫草胶囊能够抑制肺纤维化大鼠肺组织的炎症及胶原沉积,抑制E-cad蛋白表达的降低,抑制α-SMA和Vimentin蛋白表达的升高,从而抑制EMT过程的发生,一定程度上抑制肺纤维化。3参七虫草胶囊能够抑制肺纤维化大鼠肺组织中Wnt1、Wnt2和β-catenin mRNA的表达升高,推测其抑制肺纤维化可能与抑制Wnt/β-catenin信号通路有关。
[Abstract]:Objective to investigate 1 idiopathic pulmonary fibrosis (idiopathic pulmonary, fibrosis, IPF) with the distribution of TCM Syndromes of ginseng.2 seven Cordyceps capsule on alveolar epithelial mesenchymal transition (epithelial mesenchymal transition, EMT.3) affect the process of Wnt/ beta -catenin pathway in EMT possible effect during clinical observation. Methods: 1 access to relevant literature, making IPF patients with TCM syndromes questionnaire, through clinical questionnaire, collected 76 cases of IPF patients in general (including gender, age, disease duration, smoking status, disease etc.) and TCM four diagnostic information, through the analysis of TCM syndrome in patients with IPF syndrome distribution.2 animal experiment: 128 male SD rats were randomly divided into four groups: control group (C group), model group (M group), seven ginseng Cordyceps group (S group) and prednisone group (P group), 32 rats in each group. After 7 days of adaptation, 3 groups of rats were intratracheal Injection of bleomycin (bleomycin, BLM) method to induce pulmonary fibrosis, blank group received intratracheal instillation of saline. Rats second days after intragastric administration, control group injected into saline. After intragastric administration at 7,14,21 and 28 days, hematoxylin eosin staining (Hematoxylin Eosin staining, HE) to observe the pathological structure of lung tissue, collagen deposition in lung tissues were observed by Masson staining, immunohistochemistry (immunohistochemical IHC) epithelial cadherin was detected in lung tissue (E-Cadherin, E-Cad), alpha smooth muscle actin (-smooth muscle alpha actin, alpha -SMA) and vimentin (Vimentin) protein expression and reverse transcription polymerase chain reaction (reverse transcriptionpolymerase chain reaction, RT-PCR Wnt1) in lung tissues was detected, the dynamic changes of the expression of Wnt2 and beta -catenin mRNA. Results of 1 clinical observation: 76 patients with IPF syndrome before five: Qi and Yin Two (13.16%), deficiency of blood stasis phlegm heat obstructing the lung (10.53%), the pbsl (9.21%), lung and kidney qi deficiency (7.89%) and two (7.89%) lung yin deficiency.2 animal experiment: 2.1 HE staining: the alveolar structure of the rats in the blank group basically clear and complete. The model seventh group rats alveolar days structure disorder, a large number of alveolar collapse, alveolar inflammatory cell infiltration, alveolar septum widened to fourteenth days; alveolar inflammatory cells infiltration, alveolar septum widened further, fibroblast proliferation, fibrous tissue formation and collagen deposition; alveolar inflammatory cells twenty-first days compared to fourteenth days reduced alveolar the interval further widened; twenty-eighth days no infiltration of inflammatory cells, with a large number of fibroblasts, mainly fibrous tissue formation. Ginseng seven Cordyceps group and prednisone group, the lung tissue of rats were compared with the corresponding time points in the model group, the pathological changes were reduced in different degrees, seven ginseng Cordyceps group With the prednisone group, the pathological changes similar to.2.2Masson staining: the blank group rats have a small amount of alveolar interval seventh days. Blue collagen fiber distribution in lung tissue of rats in the model group were found in collagen deposition blue, mainly concentrated in the end of the bronchial wall, vascular wall and alveolar septum; fourteenth days of each part of the blue stained area color deepens, further deposition of collagen fibers; twenty-first and 28 days, the blue stained area further increased, the color deepened, shows that a large number of collagen deposition, diffuse fibrous tissue formation. Ginseng seven Cordyceps group and prednisone group, the lung tissue of rats were compared with the model group at the same time, the width of alveolar septa compared with model group decreased, blue collagen deposition to reduce the amount of fibrosis severity. Ginseng seven Cordyceps group and prednisone group. The degree of collagen deposition on.2.3E-cad, alpha -SMA and Vimentin protein: 7,14,21 and 28 days, the expression of E-cad protein in model group. In the blank group, alpha -SMA and Vimentin protein expression in the model group than control group, there were significant differences among the groups (P0.05), seven Cordyceps group and prednisone group E-cad expression was higher than that in model group, alpha -SMA and Vimentin protein expression was lower than that of model group, there were significant differences among the groups (P0.05), ginseng seven Cordyceps group and prednisone group, E-cad and Vimentin protein expression were similar, there was no significant difference between groups (P (29) 0.05), the twenty-eighth day, the expression of -SMA protein alpha seven ginseng Cordyceps group than the prednisone group, the difference was statistically significant (P (27) 0.05); control group E-cad 4 time points lung tissue, alpha -SMA and Vimentin protein expression had no significant difference (P (29) 0.05), the model group, decreased expression of E-cad protein in seven ginseng Cordyceps group and prednisone group at 14,21 and 28 days in the lung tissue, alpha -SMA and Vimentin protein expression increased gradually within the group there was statistically significant difference 涔，

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