阻遏矽肺纤维化的实验研究
조알석폐섬유화적실험연구
The experimental study of suppressing silicosis fibrosis
  • 万方数据
    中华劳动卫生职业病杂志 중화노동위생직업병잡지
    CHINESE JOURNAL OF INDUSTRIAL HYGIENE AND OCCUPATIONAL DISEASES
    2011年 10期 740-745 ,共6页

    翁泽平%张继君%刘薇薇%陈娟%刘移民%余卫%唐丽娟%陈嘉榆%方茅%张程%叶更新%陈玲珍%钟雪云

    옹택평%장계군%류미미%진연%류이민%여위%당려연%진가유%방모%장정%협경신%진령진%종설운

    肝细胞生长因子%髓样组细胞%肺纤维化%羟脯氨酸 肝細胞生長因子%髓樣組細胞%肺纖維化%羥脯氨痠
    간세포생장인자%수양조세포%폐섬유화%간포안산
    Hepatocyte growth factor%Myeloid progenitor cells%Pulmonary fibrosis%Hydroxyproline
    目的 比较骨髓间充质样干细胞( BM-MSCs)与pcDNA3.1肝细胞生长因子(HGF)转染的BM-MSCs对SiO2所致大鼠肺泡炎和早期肺纤维化的影响并探讨其机制.方法 收集培养雄性Wistar 幼鼠的原代BM-MSCs,并进行4,6-联脒-2-苯基吲哚(DAPI)标记.将50只雄性Wistar大鼠气管滴注40mg/ml SiO2混悬液1ml造模后,随机分为模型组、BM-MSCs组和pcDNA 3.1-HGF+BM-MSCs组.造模次日,模型组(10只):尾静脉注入PBS 1 ml; BM-MSCs组(20只):尾静脉注入106个/ml BM-MSCs 1 ml;pcD-NA3.1-HGF+BM-MSCs组(20只):尾静脉注入106个/ml pcDNA3.1-HGF转染的BM-MSCs 1 ml.14和28 d后,分别处死大鼠,取肺组织冰冻切片,荧光显微镜下检测体内DAPI标记细胞；HE和Masson染色观察肺炮炎和纤维化情况；免疫印迹( Western blot)法测各组大鼠肺组织的HGF表达量；ELISA法检测肺组织中肿瘤坏死因子(TNF)-α的含量,样本碱水解法检测肺组织中羟脯氨酸(HYP)的含量.结果14、28 d时,pcDNA3.1-HGF+BM-MSCs组肺组织肺泡炎评分(14 d:1.16±0.13,28 d:0.82±0.20)均低于同时期BM-MSCs组(14 d:1.68±0.17,28 d:1.58±0.31)和模型组(14 d:2.36±0.17,28 d:2.80±0.14),BM-MSCs组肺泡炎评分均低于同时期模型组,差异均有统计学意义(P＜0.05).14、28 d时pcDNA3.1-HGF+BM-MSCs组肺纤维化评分( 14 d:0.10±0.11,28 d:1.16±0.13)均明显低于同时期BM-MSCs( 14 d:0.54±0.15,28 d:1.36±0.13)和模型组(14 d:0.64±0.09,28 d:1.84±0.17),差异均有统计学意义(P＜0.05).14、28 d时pcDNA3.1-HGF+BM-MSCs组肺组织匀浆中TNF-α含量[14 d:(280.48±23.11) pg/mg,28 d:(249.78±22.33) pg/mg]均明显低于BM-MSCs组[14 d:(342.58±35.34) pg/mg,28 d:(319.51±17.84) pg/mg]和模型组[14 d:(442.29±36.76) pg/mg,28 d:(348.53±33.95) pg/mg],BM-MSCs组肺组织匀浆中TNF-α含量低于模型组,差异均有统计学意义(P＜0.05).14、28 d时pcDNA3.1-HGF+BM-MSCs组肺组织匀浆中HYP表达量[14 d:(0.46±0.04) μg/mg,28 d:(0.65±0.05)μg/mg]均明显低于同时期BM-MSCs组[14 d:(0.63±0.04) μg/mg,28 d:(1.04±0.07) μg/mg]和模型组[14 d:(0.72±0.06) μg/mg,28 d:(1.39±0.06) μg/mg],差异均有统计学意义(P＜0.05)；28 d时,BM-MSCs组肺组织匀浆中HYP表达量低于模型组,差异有统计学意义(P＜0.05).结论 应用pcDNA3.1-HGF转染后的BM-MSCs比单独应用BM-MSCs能更有效地对抗SiO2诱导的大鼠肺泡炎和早期肺纤维化,作用机制可能与其减轻肺组织的炎症有关.
    목적 비교골수간충질양간세포( BM-MSCs)여pcDNA3.1간세포생장인자(HGF)전염적BM-MSCs대SiO2소치대서폐포염화조기폐섬유화적영향병탐토기궤제.방법 수집배양웅성Wistar 유서적원대BM-MSCs,병진행4,6-련미-2-분기신타(DAPI)표기.장50지웅성Wistar대서기관적주40mg/ml SiO2혼현액1ml조모후,수궤분위모형조、BM-MSCs조화pcDNA 3.1-HGF+BM-MSCs조.조모차일,모형조(10지):미정맥주입PBS 1 ml; BM-MSCs조(20지):미정맥주입106개/ml BM-MSCs 1 ml;pcD-NA3.1-HGF+BM-MSCs조(20지):미정맥주입106개/ml pcDNA3.1-HGF전염적BM-MSCs 1 ml.14화28 d후,분별처사대서,취폐조직빙동절편,형광현미경하검측체내DAPI표기세포；HE화Masson염색관찰폐포염화섬유화정황；면역인적( Western blot)법측각조대서폐조직적HGF표체량；ELISA법검측폐조직중종류배사인자(TNF)-α적함량,양본감수해법검측폐조직중간포안산(HYP)적함량.결과14、28 d시,pcDNA3.1-HGF+BM-MSCs조폐조직폐포염평분(14 d:1.16±0.13,28 d:0.82±0.20)균저우동시기BM-MSCs조(14 d:1.68±0.17,28 d:1.58±0.31)화모형조(14 d:2.36±0.17,28 d:2.80±0.14),BM-MSCs조폐포염평분균저우동시기모형조,차이균유통계학의의(P＜0.05).14、28 d시pcDNA3.1-HGF+BM-MSCs조폐섬유화평분( 14 d:0.10±0.11,28 d:1.16±0.13)균명현저우동시기BM-MSCs( 14 d:0.54±0.15,28 d:1.36±0.13)화모형조(14 d:0.64±0.09,28 d:1.84±0.17),차이균유통계학의의(P＜0.05).14、28 d시pcDNA3.1-HGF+BM-MSCs조폐조직균장중TNF-α함량[14 d:(280.48±23.11) pg/mg,28 d:(249.78±22.33) pg/mg]균명현저우BM-MSCs조[14 d:(342.58±35.34) pg/mg,28 d:(319.51±17.84) pg/mg]화모형조[14 d:(442.29±36.76) pg/mg,28 d:(348.53±33.95) pg/mg],BM-MSCs조폐조직균장중TNF-α함량저우모형조,차이균유통계학의의(P＜0.05).14、28 d시pcDNA3.1-HGF+BM-MSCs조폐조직균장중HYP표체량[14 d:(0.46±0.04) μg/mg,28 d:(0.65±0.05)μg/mg]균명현저우동시기BM-MSCs조[14 d:(0.63±0.04) μg/mg,28 d:(1.04±0.07) μg/mg]화모형조[14 d:(0.72±0.06) μg/mg,28 d:(1.39±0.06) μg/mg],차이균유통계학의의(P＜0.05)；28 d시,BM-MSCs조폐조직균장중HYP표체량저우모형조,차이유통계학의의(P＜0.05).결론 응용pcDNA3.1-HGF전염후적BM-MSCs비단독응용BM-MSCs능경유효지대항SiO2유도적대서폐포염화조기폐섬유화,작용궤제가능여기감경폐조직적염증유관.
    Objective To compare the difference of effects on SiO2-induced alveolitis and early fibrosis between bone marrow-derived mesenchymal-like stem cells (BM-MSCs) and BM-MSCs transfected by pcD-NA3.1-HGF and to explore the mechanism of this effects.Methods The Primary BM-MSCs from Wistar male young rats were cultured and labeled by 4,6-diamidino-2-phenylindole (DAPI).Fifty Wistar rats were randomly divided into 3 groups:model group ( 10 rats),which was administered with SiO2 by the trache,the next day,injected PBS via the tail vein; BM-MSCs group (20 rats),which was administered with SiO2 by the trache,the next day,injected with 1 ml suspension of BM-MSCs via the tail vein; PcDNA3.1-HGF plus BM-MSC group (20 rats),which was administered with SiO2 by the trache,the next day,injected with 1 ml suspension of BM-MSCs transfected by pcDNA3.1-HGF via the tail vein.On the 14th and 28th days after treatment,half of the animals were sacrificed,respectively,and the lungs were harvested for frozen section to observe the cell marked by DAP1.HE staining under a fluorescent microscope,and to observe the pulmonary alveolitis and fibrosis by HE and Masson staining under a light microscope.Western blot assay was used to detect the expression of HGF in rat lungs.The expression levels of tumor necrosis factor-α (TNF-α) in pulmonary tissues were analyzed quantitatively by ELISA.The contents of HYP in pulmonary tissues were analyzed quantitatively by sample hydrolysis method.Results On the 14th and 28th days after treatment,the scores of pulmonary alveolitis and early fibrosis in pcDNA3.1-HGF plus BM-MSCs group were 2.36±0.17,2.8±0.14 and 0.14-0.11,1.16±0.13,which were significantly lower than those ( 1.68±0.17,1.58±0.31 and 0.54±0.15,1.36±0.13) in BM-MSCs group,also which were significantly lower those (2.36±0.17,2.80±0.14 and 0.64±0.09,1.84±0.17) in model group (P＜0.05); On the 14th and 28th days after treatment,the TNF-α contents of pulmonary tissues in pcDNA3.1-HGF plus BM-MSCs group were 280.4±23.11 and 249.78±22.33 pg/mg,which were significantly lower than those (341.58±35.34,442.29±36.76 pg/mg and 319.51±17.84,348.53±33.95 pg/mg) in BM-MSCs and model groups (P＜0.05 ); On the 14th and 28th days after treatment,the HYP contents of pulmonary tissues in pcDNA3.1-HGF plus BM-MSCs group were 0.46±0.04 and 0.65±0.05 μg/mg,which were significantly lower than those (0.63±0.04,1.04±0.07μg/mg and 0.72±0.60,1.39±0.60μg/mg) in BM-MSCs and model groups (P＜0.05).Conclusion The effects of BM-MSCs transfected by pcDNA3.1-HGF on suppressing pulmonary alveolitis and early fibrosis induced by SiO2 were better than those of BM-MSCs.The mechanism may be associated with the reduced pulmonary inflammation.
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