[摘要] 目的 觀察氟伐他汀對放射性肺損傷的防治作用，為放射性肺炎和肺間質(zhì)纖維化的治療探索一種新的方法。方法 雌性SD大鼠50只隨機(jī)分為正常對照組(C組)，單純照射組(R組)和氟伐他汀防治組(F組)。治療組于照射前7天以氟伐他汀20 mg/(kg·d)灌胃，另兩組灌服等量生理鹽水直至實(shí)驗(yàn)結(jié)束。照射組和治療組給予直線加速器照射，照射劑量單次20 Gy/min，于照射后5、15、30、60天取肺組織作HE，Masson，甲苯胺藍(lán)染色及透射電鏡，免疫組化法測定TGF-β1的表達(dá)，HE及免疫組化染色以計(jì)算機(jī)圖像分析系統(tǒng)測定，數(shù)據(jù)用重復(fù)測量多因素方差分析和T檢驗(yàn)。 結(jié)果 治療組大鼠肺炎和肺水腫比照射組明顯減輕。照射組大鼠肺組織膠原纖維量比治療組明顯增多。照射組15、30、60天肥大細(xì)胞數(shù)量逐漸增多(P<0.01)，而治療組減少(P<0.01)，電鏡顯示Ⅱ型肺泡細(xì)胞和毛細(xì)血管內(nèi)皮細(xì)胞30天時(shí)照射組較治療組明顯損傷，照射組30、60天肺間質(zhì)增厚(P<0.01)，治療組減輕，照射組TGF-β1的表達(dá)在30、60天較治療組明顯增高(P<0.01)。結(jié)論 氟伐他汀能抑制放射性炎癥和組織損傷，可用于預(yù)防和治療放射性肺損傷。
[關(guān)鍵詞] 氟伐他汀；放射性肺損傷；形態(tài)學(xué)；TGF-β1
Preventive effects of fluvastatin on radiationinduced lung injuries in rats
YE Jiangfeng,QI Haowen,MENG Yulin,et al.Department of Respiratory Diseases,PLA General Hospital,Lanzhou Military Command,Urumqi 830000,China
[Abstract] Objective To assess the efficacy of fluvastatin for prevention and treatment of radiationinduced lung injuries in rat.To evaluate the possibility of using fluvastatin as a new therapy for pneumonia and pulmonary fibrosis induced by radiation. Methods 50 SD female rats were randomly divided into control,radiation group and treatment groups .Fluvastatin was given to rats in treatment groups at a dose of 20 mg/(kg·day) by gastrogavaging from seven days before radiation until the end of the experiment.The rats in other groups were gastrogavaged with the same amount of saline. Rats in the radiation group and treatment group were exposed to thoracic radiation at a dose of 20 Gy by linear accelerator. Lung tissue from each rat was obtained,fixed and examined by HE staining,masson staining,toluidine blue staining and transmission electron microscopy. Expression of TGFβ1 were measured by immunohistochemistry staining. The field of observation of HE staining and immunohistochemistry statining were measured by a computerized image analysis system. The data were analyzed by ANOVA and students t test. Results Pneumonia and pulmonary edema in rats of treatment group were much less severe than in radiation group.The amount of collagen fibers in lung tissue from rats of radiation group increased significantly compared to treatment group.The number of mast cells was increased gradually in radiation group on days 15,30,60(P<0.01),but decreased in treatment group(P<0.01).Electron microscopy showed that type Ⅱ pneumonocytes and lung capillary endothelial cells were much more severely injured in radiation group on day 30 than in treatment group.The area of mesenchyma was increased in radiation group on day 30 and day 60(P<0.01),and decreased in the treatment group(P<0.01). The expression of TGFβ1 was high in radiation group on day 30 and day 60 (P<0.01) than in treatment group on days 15,30 and 60(P<0.01). Conclusion Fluvastatin might inhibit radiationinduced inflammation and tissue damage and could be used to prevent and treat radiationinduced lung injuries.
[Key words] fluvastatin;radiationinduced lung injury;pathomorphology;TGFβ1
放射性肺損傷是核輻射事故,骨髓移植預(yù)處理及胸部腫瘤放療后常見的并發(fā)癥。由于放射性肺損傷早期的放射性肺炎,后期肺間質(zhì)纖維化的存在,很大程度上限制了胸部腫瘤放療及骨髓移植的發(fā)展,也使得平時(shí)、戰(zhàn)時(shí)核輻射損傷的防治更加困難。然而目前缺少有效的防治措施，如何解決這個難題是放射生物學(xué)研究的熱點(diǎn)之一。有報(bào)道氟伐他汀能有效地防治肺間質(zhì)纖維化[1]。為此,我們探索氟伐他汀對放射性肺損傷的防治作用，為放射性肺損傷的早期治療提供實(shí)驗(yàn)依據(jù)。
1 材料與方法
1.1 材料 直線加速器(英國公司的8MV-X),氟伐他汀(瑞士諾華公司提供)。圖像分析系統(tǒng)(德國LEICA公司的Q500MC),TGF-β1免疫組化試劑盒(武漢博士德生物工程有限公司提供)。
1.2 動物分組及給藥 雌性SD大鼠50只由第四軍醫(yī)大學(xué)實(shí)驗(yàn)動物中心提供，體重(180±20)g,隨機(jī)分為正常對照組(C組)、單純照射組(R組)、氟伐他汀防治組(F組)。F組于照射前7天以氟伐他汀20 mg/(kg·d)灌胃，另兩組灌服等量生理鹽水。R組、F組腹腔內(nèi)注射硫噴妥鈉40 mg/kg，動物麻醉后行直線加速器單次照射，劑量為20 Gy，面積4.5 cm× 4.5 cm(全胸)，劑量率為3 Gy/min，距離1 m。
1.3 病理學(xué)觀察及定量測定 于照射后5、15、30、60天以硫噴妥鈉40 mg/kg腹腔內(nèi)注射，麻醉后剪開胸腔，取大鼠右中下肺浸入10%福爾馬林溶液中固定，常規(guī)石蠟包埋，制作石蠟切片(5 μm)，行HE染色，光學(xué)顯微鏡下觀察，用 LEICAQ500MC圖像分析系統(tǒng)(德國)測定肺間質(zhì)面密度；Masson染色觀察基質(zhì)膠原纖維變化；甲苯胺藍(lán)染色觀察肥大細(xì)胞數(shù)密度變化。取左下肺組織1 mm3大小肺組織2~5塊置入3%戊二醛溶液固定，備作透射電鏡，觀察Ⅱ型肺泡上皮細(xì)胞，血管內(nèi)皮細(xì)胞超微結(jié)構(gòu)的變化。TGF-β1的表達(dá)選石蠟切片用免疫組化SABC法，按照試劑盒中說明書進(jìn)行操作，光學(xué)顯微鏡觀察結(jié)果。以PBS緩沖液替代一抗作陰性對照，試劑盒中提供陽性對照片。免疫組化陽性結(jié)果為細(xì)胞質(zhì)/膜染成棕黃色或黃色，采用圖像分析儀隨機(jī)測定細(xì)支氣管TGF-β1表達(dá)的灰度值，取其平均值。TGF-β1表達(dá)越弱，灰度值越大; TGF-β1表達(dá)越強(qiáng)，灰度值越小。
1.4 統(tǒng)計(jì)學(xué)方法 采用SPSS10.0版統(tǒng)計(jì)軟件包，以實(shí)驗(yàn)設(shè)計(jì)方差分析進(jìn)行顯著性檢驗(yàn)，所有數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示。
2 結(jié)果
2.1 大體肺組織標(biāo)本觀察 肺組織脆性增加,可呈肝樣實(shí)變,肺表面有白色灶狀病灶。
2.2 HE染色 C組：光學(xué)顯微鏡下觀察顯示正常肺組織。R組：照射后5天間質(zhì)細(xì)胞增多，主要為中性白細(xì)胞，15天出現(xiàn)肺泡腔減小，肺泡腔內(nèi)有泡沫細(xì)胞，炎癥細(xì)胞可見多形核細(xì)胞、巨噬細(xì)胞。30、60天除上述改變外，肺泡腔內(nèi)纖維素樣滲出和水腫液，肺間質(zhì)嚴(yán)重增厚，局部肺實(shí)變，有的出現(xiàn)肺膿腫，纖維母細(xì)胞不同程度增生，有較多中性白細(xì)胞、淋巴細(xì)胞、漿細(xì)胞、巨噬細(xì)胞浸潤，泡沫細(xì)胞成堆，紅細(xì)胞漏出。60天時(shí)局部有肉芽組織增生，成纖維細(xì)胞數(shù)量增多。F組：照射后5天炎癥細(xì)胞浸潤和肺水腫與R組比較無明顯變化。15天肺間質(zhì)增厚與R組比較變化不明顯，泡沫細(xì)胞、多形核細(xì)胞、巨噬細(xì)胞滲出減少。30、60天肺水腫明顯減輕，無肺實(shí)變發(fā)生，炎癥細(xì)胞滲出明顯減少。無肉芽組織增生。
[關(guān)鍵詞] 氟伐他汀；放射性肺損傷；形態(tài)學(xué)；TGF-β1
Preventive effects of fluvastatin on radiationinduced lung injuries in rats
YE Jiangfeng,QI Haowen,MENG Yulin,et al.Department of Respiratory Diseases,PLA General Hospital,Lanzhou Military Command,Urumqi 830000,China
[Abstract] Objective To assess the efficacy of fluvastatin for prevention and treatment of radiationinduced lung injuries in rat.To evaluate the possibility of using fluvastatin as a new therapy for pneumonia and pulmonary fibrosis induced by radiation. Methods 50 SD female rats were randomly divided into control,radiation group and treatment groups .Fluvastatin was given to rats in treatment groups at a dose of 20 mg/(kg·day) by gastrogavaging from seven days before radiation until the end of the experiment.The rats in other groups were gastrogavaged with the same amount of saline. Rats in the radiation group and treatment group were exposed to thoracic radiation at a dose of 20 Gy by linear accelerator. Lung tissue from each rat was obtained,fixed and examined by HE staining,masson staining,toluidine blue staining and transmission electron microscopy. Expression of TGFβ1 were measured by immunohistochemistry staining. The field of observation of HE staining and immunohistochemistry statining were measured by a computerized image analysis system. The data were analyzed by ANOVA and students t test. Results Pneumonia and pulmonary edema in rats of treatment group were much less severe than in radiation group.The amount of collagen fibers in lung tissue from rats of radiation group increased significantly compared to treatment group.The number of mast cells was increased gradually in radiation group on days 15,30,60(P<0.01),but decreased in treatment group(P<0.01).Electron microscopy showed that type Ⅱ pneumonocytes and lung capillary endothelial cells were much more severely injured in radiation group on day 30 than in treatment group.The area of mesenchyma was increased in radiation group on day 30 and day 60(P<0.01),and decreased in the treatment group(P<0.01). The expression of TGFβ1 was high in radiation group on day 30 and day 60 (P<0.01) than in treatment group on days 15,30 and 60(P<0.01). Conclusion Fluvastatin might inhibit radiationinduced inflammation and tissue damage and could be used to prevent and treat radiationinduced lung injuries.
[Key words] fluvastatin;radiationinduced lung injury;pathomorphology;TGFβ1
放射性肺損傷是核輻射事故,骨髓移植預(yù)處理及胸部腫瘤放療后常見的并發(fā)癥。由于放射性肺損傷早期的放射性肺炎,后期肺間質(zhì)纖維化的存在,很大程度上限制了胸部腫瘤放療及骨髓移植的發(fā)展,也使得平時(shí)、戰(zhàn)時(shí)核輻射損傷的防治更加困難。然而目前缺少有效的防治措施，如何解決這個難題是放射生物學(xué)研究的熱點(diǎn)之一。有報(bào)道氟伐他汀能有效地防治肺間質(zhì)纖維化[1]。為此,我們探索氟伐他汀對放射性肺損傷的防治作用，為放射性肺損傷的早期治療提供實(shí)驗(yàn)依據(jù)。
1 材料與方法
1.1 材料 直線加速器(英國公司的8MV-X),氟伐他汀(瑞士諾華公司提供)。圖像分析系統(tǒng)(德國LEICA公司的Q500MC),TGF-β1免疫組化試劑盒(武漢博士德生物工程有限公司提供)。
1.2 動物分組及給藥 雌性SD大鼠50只由第四軍醫(yī)大學(xué)實(shí)驗(yàn)動物中心提供，體重(180±20)g,隨機(jī)分為正常對照組(C組)、單純照射組(R組)、氟伐他汀防治組(F組)。F組于照射前7天以氟伐他汀20 mg/(kg·d)灌胃，另兩組灌服等量生理鹽水。R組、F組腹腔內(nèi)注射硫噴妥鈉40 mg/kg，動物麻醉后行直線加速器單次照射，劑量為20 Gy，面積4.5 cm× 4.5 cm(全胸)，劑量率為3 Gy/min，距離1 m。
1.3 病理學(xué)觀察及定量測定 于照射后5、15、30、60天以硫噴妥鈉40 mg/kg腹腔內(nèi)注射，麻醉后剪開胸腔，取大鼠右中下肺浸入10%福爾馬林溶液中固定，常規(guī)石蠟包埋，制作石蠟切片(5 μm)，行HE染色，光學(xué)顯微鏡下觀察，用 LEICAQ500MC圖像分析系統(tǒng)(德國)測定肺間質(zhì)面密度；Masson染色觀察基質(zhì)膠原纖維變化；甲苯胺藍(lán)染色觀察肥大細(xì)胞數(shù)密度變化。取左下肺組織1 mm3大小肺組織2~5塊置入3%戊二醛溶液固定，備作透射電鏡，觀察Ⅱ型肺泡上皮細(xì)胞，血管內(nèi)皮細(xì)胞超微結(jié)構(gòu)的變化。TGF-β1的表達(dá)選石蠟切片用免疫組化SABC法，按照試劑盒中說明書進(jìn)行操作，光學(xué)顯微鏡觀察結(jié)果。以PBS緩沖液替代一抗作陰性對照，試劑盒中提供陽性對照片。免疫組化陽性結(jié)果為細(xì)胞質(zhì)/膜染成棕黃色或黃色，采用圖像分析儀隨機(jī)測定細(xì)支氣管TGF-β1表達(dá)的灰度值，取其平均值。TGF-β1表達(dá)越弱，灰度值越大; TGF-β1表達(dá)越強(qiáng)，灰度值越小。
1.4 統(tǒng)計(jì)學(xué)方法 采用SPSS10.0版統(tǒng)計(jì)軟件包，以實(shí)驗(yàn)設(shè)計(jì)方差分析進(jìn)行顯著性檢驗(yàn)，所有數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示。
2 結(jié)果
2.1 大體肺組織標(biāo)本觀察 肺組織脆性增加,可呈肝樣實(shí)變,肺表面有白色灶狀病灶。
2.2 HE染色 C組：光學(xué)顯微鏡下觀察顯示正常肺組織。R組：照射后5天間質(zhì)細(xì)胞增多，主要為中性白細(xì)胞，15天出現(xiàn)肺泡腔減小，肺泡腔內(nèi)有泡沫細(xì)胞，炎癥細(xì)胞可見多形核細(xì)胞、巨噬細(xì)胞。30、60天除上述改變外，肺泡腔內(nèi)纖維素樣滲出和水腫液，肺間質(zhì)嚴(yán)重增厚，局部肺實(shí)變，有的出現(xiàn)肺膿腫，纖維母細(xì)胞不同程度增生，有較多中性白細(xì)胞、淋巴細(xì)胞、漿細(xì)胞、巨噬細(xì)胞浸潤，泡沫細(xì)胞成堆，紅細(xì)胞漏出。60天時(shí)局部有肉芽組織增生，成纖維細(xì)胞數(shù)量增多。F組：照射后5天炎癥細(xì)胞浸潤和肺水腫與R組比較無明顯變化。15天肺間質(zhì)增厚與R組比較變化不明顯，泡沫細(xì)胞、多形核細(xì)胞、巨噬細(xì)胞滲出減少。30、60天肺水腫明顯減輕，無肺實(shí)變發(fā)生，炎癥細(xì)胞滲出明顯減少。無肉芽組織增生。