【摘要】：目的:研究不同年齡段(青年、老年)小鼠實驗性肺纖維化中上皮細胞-間充質細胞轉化(epithelial-mesenchymal transition,EMT)相關指標:上皮細胞標志物E-鈣粘蛋白(E-Cadherin,E-Cad),間質細胞標志物α平滑肌肌動蛋白(alpha smooth muscle Actin,α-SMA)、波形蛋白(Vimentin)及轉錄因子Snail隨病程進展的表達變化,初步探討增齡對小鼠肺纖維化病變程度及EMT的影響,為進一步探索增齡促進肺纖維化進展的可能機制提供依據(jù)。方法:20周齡雄性C57/BL6小鼠60只,隨機分為兩組:青年組(20周齡)、老年組(飼養(yǎng)至26周齡)。上述兩組每組再隨機分為5組,其中1組作為生理鹽水對照組(NS),給予氣管內注入0.9%氯化鈉注射液,于第28d處死;其余4組為肺纖維化模型組,按5mg/kg劑量,予氣管內注入博來霉素(bleomycin,BLM),分別于造模后第7d、14d、21d及28d處死,取左肺制備石蠟切片,右肺液氮凍存后行相關蛋白RT-PCR檢測。石蠟切片行HE染色及Masson染色,通過肺組織形態(tài)改變從而判斷肺纖維化病變進展程度;免疫組織化學染色檢測EMT相關指標E-Cadherin、Vimentin、α-SMA蛋白的表達水平,平均光灰度值使用美國Universal Imaging Porporation圖像分析系統(tǒng)測量,反映病程中EMT的動態(tài)變化;RT-PCR法檢測液氮凍存肺組織轉錄因子Snail1、Snail2及其調控蛋白E-cad的m RNA的表達水平。使用SPSS19.0對上述數(shù)據(jù)進行統(tǒng)計分析,所有數(shù)據(jù)均采取“均數(shù)標準差”表示,P0.05,差異有統(tǒng)計學意義。結果:HE染色及Masson染色結果示BLM誘導的小鼠肺纖維化模型組,隨病程進展,肺實質破壞、膠原生成量較對照組增加明顯,表明造模成功。將老年與青年小鼠同時間點的纖維化病變程度進行對比,前者更嚴重。免疫組化法結果顯示,E-Cadherin主要表達于肺泡上皮細胞、支氣管黏膜上皮細胞。青年、老年兩組小鼠對照組表達較多,各時間點模型組的表達較對照組減少,且隨病程進展,減少更為明顯,差異有統(tǒng)計學意義(P0.05);進一步將兩組模型小鼠的各個對應時間點表達量進行比較,老年較青年組表達較少(P0.05),而兩組對照組無明顯差異。Vimentin主要表達于支氣管黏膜上皮及肺間質細胞,α-SMA在對照組只表達于支氣管及血管平滑肌細胞,而在模型組肺間質肌成纖維細胞中還可觀測到其表達。上述兩個指標在各時間點模型組表達較對照組增加,隨病程進展,增多更顯著(P0.05);同時間點老年模型組較青年模型組表達較多(P0.05),兩組對照組無明顯差異。RT-PCR法結果示在青年、老年兩組小鼠中,隨時間推移,Snail1及Snail2的表達均呈現(xiàn)先升高后下降的趨勢(相較對照組,模型組14d表達量明顯增加,28d較14d表達明顯減少,P0.05)。對比兩組小鼠同時間點的表達情況,老年模型組較青年模型組于14d增加更為明顯,28d減少更為緩慢(P0.05),兩組對照組無明顯差異。E-Cadherin模型組14d、28d的表達較對照組減少,且隨病程進展,減少更為明顯(P0.05)。相較青年模型組,同時間點老年模型組表達較少(P0.05),兩組對照組無明顯差異,與免疫組化結果基本一致。結論:BLM氣管內注入誘導小鼠肺纖維化模型成功,同時期老年模型組相較青年模型組肺纖維化及EMT程度更嚴重,而對照組無明顯差異。老年模型組Snail1、Snail2、E-Cadherin較青年模型組變化更為明顯。增齡可能通過影響小鼠肺纖維化模型EMT進程中轉錄因子Snail1、Snail2及其下游調節(jié)蛋白E-Cadherin等表達從而加重肺纖維化病變程度。
[Abstract]:Objective: To study the related indexes of epithelial cell-mesenchymal transition (EMT) in experimental pulmonary fibrosis of mice with different age groups (young and old): E-cadherin (E-Cadiin, E-Cad). The effect of increasing age on the degree of pulmonary fibrosis and EMT in mice was studied. To further explore the possible mechanism of increasing age to promote the progression of pulmonary fibrosis. Methods: 60-week-old male C57/ BL6 mice were randomly divided into two groups: the young group (20 weeks old) and the old group (26 weeks old). The two groups were randomly divided into 5 groups, one group was used as the normal saline control group (NS), and 0.9% sodium chloride injection was injected into the trachea, and was sacrificed on day 28d; the remaining 4 groups were pulmonary fibrosis model group, and the bleomycin (BLM) was injected into the trachea according to the dose of 5mg/ kg. The left lung was used to prepare the paraffin section and the right lung liquid nitrogen was frozen and the related protein RT-PCR was detected. The paraffin sections were stained with HE and Masson, and the degree of progression of pulmonary fibrosis was determined by the change of the morphology of the lung. The expression levels of E-Cadherin, Vimentin, and S-SMA were measured by immunohistochemical staining. The average light gray values were measured using the American Universal Imaging Porptron image analysis system. The dynamic changes of EMT were reflected in the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the course of the disease. The above data were statistically analyzed using SPSS19.0, and all the data were expressed in 鈥渕ean standard deviation鈥，

本文編號：2345962