CAJ | 학술논문

目的筛选矽肺大鼠肺组织与正常大鼠肺组织的差异表达基因,并对差异表达基因基质金属蛋白每-12(MMP-12)和组织蛋白酶E(Cathepsin E)进行鉴定,探讨其在矽肺发病中的作用.方法将健康SD大鼠随机分为模型组(24只)和对照组(6只),采用非气管暴露法建立雄性SD大鼠矽肺模型,HE染色和VG染色进行肺组织病理观察.对染尘60d的大鼠肺组织,应用基因芯片筛选大鼠肺组织中的差异表达基因.选择明显上调的MMP-12和Cathepsin E基因,用反转录聚合酶链式反应(RT-PCR)、免疫组织化学法及蛋白质印迹(Western blot)法进行鉴定.结果从26 962个基因中共筛选出模型组和对照组的差异表达基因338个,其中上调基因267个,下调基因71个.经RT-PCR验证,染尘后30、60、90d时,模型组大鼠肺组织中MMP-12 mRNA表达的吸光度值分别为对照组的4.306、5.338、6.713倍,Cathepsin E分别为1.434、2.974、3.889倍.经免疫组织化学法验证,染尘后30、60、90 d时,模型组大鼠肺组织中MMP-12蛋白表达的平均吸光度值分别为对照组的1.435、1.746、2.069倍,Cathepsin E分别为1.372、1.663、2.103倍.经Western blot法验证,染尘后30、60、90d时,模型组大鼠肺组织中MMP-12蛋白表达的平均吸光度值分别为对照组的1.214、1.531、1.959倍,Cathepsin E分别为1.262、1.828、1.907倍.与对照组相比,模型组大鼠肺组织中MMP-12和Cathepsin E mRNA和蛋白表达量明显上调,差异均有统计学意义(P＜0.05).结论应用基因芯片筛选出矽肺大鼠肺组织差异表达基因并得以验证,MMP-12和Cathepsin E可能通过降解肺泡壁基底膜及参与免疫应答等参与矽肺的发生发展过程.
목적 사선석폐대서폐조직여정상대서폐조직적차이표체기인,병대차이표체기인기질금속단백매-12(MMP-12)화조직단백매E(Cathepsin E)진행감정,탐토기재석폐발병중적작용.방법 장건강SD대서수궤분위모형조(24지)화대조조(6지),채용비기관폭로법건립웅성SD대서석폐모형,HE염색화VG염색진행폐조직병리관찰.대염진60d적대서폐조직,응용기인심편사선대서폐조직중적차이표체기인.선택명현상조적MMP-12화Cathepsin E기인,용반전록취합매련식반응(RT-PCR)、면역조직화학법급단백질인적(Western blot)법진행감정.결과 종26 962개기인중공사선출모형조화대조조적차이표체기인338개,기중상조기인267개,하조기인71개.경RT-PCR험증,염진후30、60、90d시,모형조대서폐조직중MMP-12 mRNA표체적흡광도치분별위대조조적4.306、5.338、6.713배,Cathepsin E분별위1.434、2.974、3.889배.경면역조직화학법험증,염진후30、60、90 d시,모형조대서폐조직중MMP-12단백표체적평균흡광도치분별위대조조적1.435、1.746、2.069배,Cathepsin E분별위1.372、1.663、2.103배.경Western blot법험증,염진후30、60、90d시,모형조대서폐조직중MMP-12단백표체적평균흡광도치분별위대조조적1.214、1.531、1.959배,Cathepsin E분별위1.262、1.828、1.907배.여대조조상비,모형조대서폐조직중MMP-12화Cathepsin E mRNA화단백표체량명현상조,차이균유통계학의의(P＜0.05).결론 응용기인심편사선출석폐대서폐조직차이표체기인병득이험증,MMP-12화Cathepsin E가능통과강해폐포벽기저막급삼여면역응답등삼여석폐적발생발전과정.
Objective To screen the differentially expressing genes between silicotic lung tissue and normal lung tissue,to identify the differentially expressing genes of matrix metalloproteinase-12(MMP-12)and Cathepsin E and to explore the roles of those genes in silicosis development.Methods Thirty male SD rats were divided randomly into two groups: control group(6 rats)and exposure group(24 rats)which was exposed to SiO2 by intra-tracheal perfusion.On the 30 th,60 th and 90 th days after exposure,8 rats in model group and 2 rats in control group were executed and the lung tissues were obtained.The morphologic changes of lung tissues were observed with HE staining and VG staining under a light microscope.The gene microarrays were used to identify differentially expressing genes of lung tissues in rats exposed to SiO2 for 60 days.Two significantly up-regulated genes,MMP-12 and Cathepsin E,were validated using RT-PCR,immunohistochemistry and Western Blot assay.Results A total of 338 differentially expressing genes were identified from the 26 962genes between silicotic rats and normal rats,including 267 up-regulated genes and 71 down-regulated genes.The results of RT-PCR showed that in the lung tissues of exposure group on the 30 th,60 th and 90 th days,the mRNA expression levels of MMP-12 were 4.306,5.338,6.713 times higher than those in the control group,the mRNA expression levels of Cathepsin E were 1.434,2.974,3.889 times higher than those in the control group,respectively.The results of immunohistochemical showed that in the lung tissues of exposure group on the 30 th,60 th and 90 th days,the mRNA expression levels of MMP-12 were 1.435,1.746,2.069 times higher than those in the control group,the mRNA expression levels of Cathepsin E were 1.372,1.663,2.103 times higher than those in the control group,respectively.The results of immunohistochemical showed that in the lung tissues of exposure group on the 30 th,60 th and 90 th days,the expression levels of MMP-12 protein were 1.214,1.531,1.959 times higher than those in the control group,the expression levels of Cathepsin E protein were 1.262,1.828,1.907 times higher than those in the control group,respectively.Compared with the control group,the mRNA and protein expression levels of MMP-12 and Cathepsin E in lung tissues of exposure group were significantly up-regulated(P＜0.05).Conclusion The differentially expressing genes in rat lung tissues screened by gene chip were validated,which suggested that a complex gene regulatory network may be contributed to occurrence of silicosis.MMP-12 and Cathepsin E genes may be involved in the development of silicotic pulmonary fibrosis by degrading the basement membrane of alveolar wall and participating in the immune response.