【摘要】：目的:研究不同年齡段(青年、老年)小鼠實(shí)驗(yàn)性肺纖維化中上皮細(xì)胞-間充質(zhì)細(xì)胞轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)相關(guān)指標(biāo):上皮細(xì)胞標(biāo)志物E-鈣粘蛋白(E-Cadherin,E-Cad),間質(zhì)細(xì)胞標(biāo)志物α平滑肌肌動(dòng)蛋白(alpha smooth muscle Actin,α-SMA)、波形蛋白(Vimentin)及轉(zhuǎn)錄因子Snail隨病程進(jìn)展的表達(dá)變化,初步探討增齡對(duì)小鼠肺纖維化病變程度及EMT的影響,為進(jìn)一步探索增齡促進(jìn)肺纖維化進(jìn)展的可能機(jī)制提供依據(jù)。方法:20周齡雄性C57/BL6小鼠60只,隨機(jī)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(jī)分為5組,其中1組作為生理鹽水對(duì)照組(NS),給予氣管內(nèi)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(nèi)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(guān)蛋白R(shí)T-PCR檢測(cè)。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(jìn)展程度;免疫組織化學(xué)染色檢測(cè)EMT相關(guān)指標(biāo)E-Cadherin、Vimentin、α-SMA蛋白的表達(dá)水平,平均光灰度值使用美國(guó)Universal Imaging Porporation圖像分析系統(tǒng)測(cè)量,反映病程中EMT的動(dòng)態(tài)變化;RT-PCR法檢測(cè)液氮凍存肺組織轉(zhuǎn)錄因子Snail1、Snail2及其調(diào)控蛋白E-cad的m RNA的表達(dá)水平。使用SPSS19.0對(duì)上述數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,所有數(shù)據(jù)均采取“均數(shù)標(biāo)準(zhǔn)差”表示,P0.05,差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:HE染色及Masson染色結(jié)果示BLM誘導(dǎo)的小鼠肺纖維化模型組,隨病程進(jìn)展,肺實(shí)質(zhì)破壞、膠原生成量較對(duì)照組增加明顯,表明造模成功。將老年與青年小鼠同時(shí)間點(diǎn)的纖維化病變程度進(jìn)行對(duì)比,前者更嚴(yán)重。免疫組化法結(jié)果顯示,E-Cadherin主要表達(dá)于肺泡上皮細(xì)胞、支氣管黏膜上皮細(xì)胞。青年、老年兩組小鼠對(duì)照組表達(dá)較多,各時(shí)間點(diǎn)模型組的表達(dá)較對(duì)照組減少,且隨病程進(jìn)展,減少更為明顯,差異有統(tǒng)計(jì)學(xué)意義(P0.05);進(jìn)一步將兩組模型小鼠的各個(gè)對(duì)應(yīng)時(shí)間點(diǎn)表達(dá)量進(jìn)行比較,老年較青年組表達(dá)較少(P0.05),而兩組對(duì)照組無明顯差異。Vimentin主要表達(dá)于支氣管黏膜上皮及肺間質(zhì)細(xì)胞,α-SMA在對(duì)照組只表達(dá)于支氣管及血管平滑肌細(xì)胞,而在模型組肺間質(zhì)肌成纖維細(xì)胞中還可觀測(cè)到其表達(dá)。上述兩個(gè)指標(biāo)在各時(shí)間點(diǎn)模型組表達(dá)較對(duì)照組增加,隨病程進(jìn)展,增多更顯著(P0.05);同時(shí)間點(diǎn)老年模型組較青年模型組表達(dá)較多(P0.05),兩組對(duì)照組無明顯差異。RT-PCR法結(jié)果示在青年、老年兩組小鼠中,隨時(shí)間推移,Snail1及Snail2的表達(dá)均呈現(xiàn)先升高后下降的趨勢(shì)(相較對(duì)照組,模型組14d表達(dá)量明顯增加,28d較14d表達(dá)明顯減少,P0.05)。對(duì)比兩組小鼠同時(shí)間點(diǎn)的表達(dá)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(duì)照組無明顯差異。E-Cadherin模型組14d、28d的表達(dá)較對(duì)照組減少,且隨病程進(jìn)展,減少更為明顯(P0.05)。相較青年模型組,同時(shí)間點(diǎn)老年模型組表達(dá)較少(P0.05),兩組對(duì)照組無明顯差異,與免疫組化結(jié)果基本一致。結(jié)論:BLM氣管內(nèi)注入誘導(dǎo)小鼠肺纖維化模型成功,同時(shí)期老年模型組相較青年模型組肺纖維化及EMT程度更嚴(yán)重,而對(duì)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(jìn)程中轉(zhuǎn)錄因子Snail1、Snail2及其下游調(diào)節(jié)蛋白E-Cadherin等表達(dá)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(hào)：2345962