本文關(guān)鍵詞：IPF患者中醫(yī)證候分布規(guī)律研究及參七蟲草膠囊對肺纖維化大鼠EMT過程的影響　出處：《安徽中醫(yī)藥大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 特發(fā)性肺纖維化 中醫(yī)證候 參七蟲草膠囊 細(xì)胞間質(zhì)轉(zhuǎn)化 Wnt/β-catenin信號通絡(luò)

【摘要】：目的1探討特發(fā)性肺纖維化(idiopathic pulmonary fibrosis,IPF)患者中醫(yī)證候分布規(guī)律。2探討參七蟲草膠囊對肺泡上皮細(xì)胞間質(zhì)轉(zhuǎn)化(epithelial mesenchymal transition,EMT)過程的影響。3探討Wnt/β-catenin通路在EMT發(fā)生過程中的可能作用。方法1臨床觀察:查閱相關(guān)文獻(xiàn),制定IPF患者中醫(yī)證候調(diào)查表,通過臨床問卷調(diào)查的方式,搜集76例IPF患者一般情況(包括性別、年齡、病程、吸煙情況、合并疾病等)和中醫(yī)四診信息等,通過分析得出IPF患者中醫(yī)證候分布規(guī)律。2動物實驗:將128只雄性SD大鼠隨機分為四組:空白組(C組)、模型組(M組)、參七蟲草組(S組)和潑尼松組(P組),每組32只。適應(yīng)性飼養(yǎng)7天后,3組大鼠均采用氣管內(nèi)注入博來霉素(bleomycin,BLM)的方法復(fù)制肺纖維化模型,空白組氣管內(nèi)注入等量生理鹽水。造模后第2天開始灌胃給藥,空白組灌入等體積生理鹽水。灌胃后第7、14、21和28天取材,蘇木精-伊紅染色法(Hematoxylin Eosin staining,HE)觀察肺組織病理結(jié)構(gòu),Masson染色法觀察肺組織膠原沉積情況,免疫組化(immunohistochemical IHC)法檢測肺組織中上皮細(xì)胞鈣粘素(E-Cadherin,E-Cad)、α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)和波形蛋白(Vimentin)的蛋白表達(dá)及反轉(zhuǎn)錄聚合酶鏈反應(yīng)(reverse transcriptionpolymerase chain reaction,RT-PCR)檢測肺組織中Wnt1、Wnt2和β-catenin mRNA表達(dá)的動態(tài)變化。結(jié)果1臨床觀察:76例IPF患者證候前五:氣陰兩虛夾瘀(13.16%)、痰熱壅肺(10.53%)、痰瘀阻肺(9.21%)、肺腎氣虛(7.89%)和肺氣陰兩虛(7.89%)。2動物實驗:2.1 HE染色:空白組大鼠肺泡結(jié)構(gòu)基本清晰完整。模型組大鼠第7天可見肺泡結(jié)構(gòu)紊亂,大量肺泡腔塌陷,肺泡腔內(nèi)炎性細(xì)胞浸潤,肺泡間隔開始增寬;第14天見肺泡腔內(nèi)大量炎性細(xì)胞浸潤,肺泡間隔繼續(xù)增寬,成纖維細(xì)胞增生,纖維組織形成,膠原沉積;第21天肺泡腔內(nèi)炎性細(xì)胞較第14天減少,肺泡間隔進一步增寬;第28天基本無炎性細(xì)胞浸潤,以大量成纖維細(xì)胞增生、纖維組織形成為主。參七蟲草組及潑尼松組大鼠肺組織分別與模型組相應(yīng)時間點比較,上述病理改變均有不同程度的減輕,參七蟲草組與潑尼松組病理變化程度相近。2.2Masson染色:空白組大鼠肺泡間隔有少量藍(lán)染膠原纖維分布。模型組大鼠肺組織第7天可見染成藍(lán)色的膠原纖維沉積,主要集中于終末支氣管壁、血管壁及肺泡間隔;第14天各部位藍(lán)色染區(qū)顏色加深,膠原纖維進一步沉積;第21和28天,藍(lán)色染區(qū)進一步增多,顏色進一步加深,表明大量膠原沉積,彌漫性纖維組織形成。參七蟲草組及潑尼松組大鼠肺組織分別與模型組相應(yīng)時間點比較,各組肺泡間隔寬度較模型組減小,藍(lán)染膠原沉積量減少,纖維化程度減輕。參七蟲草組與潑尼松組膠原沉積程度相近。2.3E-cad、α-SMA和Vimentin蛋白:第7、14、21和28天,E-cad蛋白表達(dá)模型組低于空白組,α-SMA和Vimentin蛋白表達(dá)模型組高于空白組,組間比較差異均有統(tǒng)計學(xué)意義(P0.05),參七蟲草組和潑尼松組E-cad蛋白表達(dá)高于模型組,α-SMA和Vimentin蛋白表達(dá)低于模型組,組間比較差異均有統(tǒng)計學(xué)意義(P0.05),參七蟲草組和潑尼松組E-cad和Vimentin蛋白表達(dá)相近,組間比較差異無統(tǒng)計學(xué)意義(P(29)0.05),第28天,參七蟲草組α-SMA蛋白表達(dá)低于潑尼松組,差異有統(tǒng)計學(xué)意義(P(27)0.05);空白組4個時間點肺組織中E-cad、α-SMA和Vimentin蛋白表達(dá)無明顯差異(P(29)0.05),模型組、參七蟲草組和潑尼松組第14、21和28天肺組織中E-cad蛋白表達(dá)逐漸降低,α-SMA和Vimentin蛋白表達(dá)逐漸升高,組內(nèi)比較差異有統(tǒng)計學(xué)意義(P0.05)。2.4 Wnt1、Wnt2和β-catenin mRNA:第7、14、21和28天,Wnt1、Wnt2和β-catenin mRNA表達(dá)模型組高于空白組,組間比較差異均有統(tǒng)計學(xué)意義(P0.05),參七蟲草組和潑尼松組Wnt1、Wnt2和β-catenin mRNA表達(dá)低于模型組,組間比較差異均有統(tǒng)計學(xué)意義(P0.05),參七蟲草組和潑尼松組組間比較,差異無統(tǒng)計學(xué)意義(P(29)0.05);空白組4個時間點肺組織中Wnt1、Wnt2和β-catenin mRNA的表達(dá)無明顯差異(P(29)0.05),模型組第14和21天,肺組織中Wnt1、Wnt2和β-catenin mRNA表達(dá)逐漸增加,第21天最高,第28天有所下降,組內(nèi)比較差異有統(tǒng)計學(xué)意義(P(27)0.05)。參七蟲草組和潑尼松組第14和21天,肺組織中Wnt1和Wnt2 mRNA表達(dá)逐漸增加,第21天最高,第28天有所下降,組內(nèi)比較差異有統(tǒng)計學(xué)意義(P(27)0.05),而β-catenin mRNA表達(dá)組內(nèi)比較差異無統(tǒng)計學(xué)意義(P(29)0.05)。結(jié)論1氣陰兩虛夾瘀型為IPF患者常見證候類型。2參七蟲草膠囊能夠抑制肺纖維化大鼠肺組織的炎癥及膠原沉積,抑制E-cad蛋白表達(dá)的降低,抑制α-SMA和Vimentin蛋白表達(dá)的升高,從而抑制EMT過程的發(fā)生,一定程度上抑制肺纖維化。3參七蟲草膠囊能夠抑制肺纖維化大鼠肺組織中Wnt1、Wnt2和β-catenin mRNA的表達(dá)升高,推測其抑制肺纖維化可能與抑制Wnt/β-catenin信號通路有關(guān)。
[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，

本文編號：1422666