CAJ | 학술논문

目的探讨云锡矿粉诱导人肺上皮细胞转化及成纤维细胞活化过程中的相互作用.方法常规培养永生化人支气管上皮细胞(BEAS-2B)及人胚肺成纤维细胞(WI-38),每6天传代1次;100μg/ml的云锡矿粉隔代诱导BEAS-2B及WI-38至第9代,共诱导5次,自第11代分组共培养至第40代.刀豆凝集素A及锚着独立性生长实验检测细胞恶性转化,流式细胞术检测细胞周期变化,免疫细胞化学法检测成纤维细胞α-平滑肌肌动蛋白(α-SMA)表达.结果 (1)与正常BEAS-2B细胞相比,矿粉诱导的BEAS-2B细胞单独培养至第28代细胞形态开始出现改变;与WI-38细胞共培养后,细胞形态改变提早至第20代;与矿粉诱导的WI-38细胞共培养后,细胞形态改变进一步提早至第16代.矿粉诱导的BEAS-2B细胞培养至第26代时凝集实验出现阳性;与WI-38细胞及矿粉诱导的WI-38细胞共培养后,细胞凝集时间缩短.与WI-38细胞共培养的矿粉诱导BEAS-2B及与矿粉诱导WI-38细胞共培养的矿粉诱导的BEAS-2B细胞分别在第26及第36代锚着独立性生长实验出现阳性,第36代的克隆形成率分别为6.00‰±1.00‰和15.33‰±2.52‰,且差异有统计学意义(P<0.05).矿粉诱导的各组上皮细胞S期细胞所占比例随培养代数增加逐渐升高,细胞发生恶性转化.(2)100 μg/ml矿粉诱导的成纤维细胞培养至第26代出现α-SMA表达;与上皮细胞共培养后,成纤维细胞α-SMA表达增强,且随培养代数的增加,阳性表达细胞数明显增多,着色程度增强.结论个旧矿粉能诱导支气管上皮细胞恶性转化及成纤维细胞活化;肺上皮细胞是矿粉诱癌的主要靶细胞;肺上皮细胞的转化和成纤维细胞的活化相互影响,相互促进.
목적 탐토운석광분유도인폐상피세포전화급성섬유세포활화과정중적상호작용.방법 상규배양영생화인지기관상피세포(BEAS-2B)급인배폐성섬유세포(WI-38),매6천전대1차;100μg/ml적운석광분격대유도BEAS-2B급WI-38지제9대,공유도5차,자제11대분조공배양지제40대.도두응집소A급묘착독립성생장실험검측세포악성전화,류식세포술검측세포주기변화,면역세포화학법검측성섬유세포α-평활기기동단백(α-SMA)표체.결과 (1)여정상BEAS-2B세포상비,광분유도적BEAS-2B세포단독배양지제28대세포형태개시출현개변;여WI-38세포공배양후,세포형태개변제조지제20대;여광분유도적WI-38세포공배양후,세포형태개변진일보제조지제16대.광분유도적BEAS-2B세포배양지제26대시응집실험출현양성;여WI-38세포급광분유도적WI-38세포공배양후,세포응집시간축단.여WI-38세포공배양적광분유도BEAS-2B급여광분유도WI-38세포공배양적광분유도적BEAS-2B세포분별재제26급제36대묘착독립성생장실험출현양성,제36대적극륭형성솔분별위6.00‰±1.00‰화15.33‰±2.52‰,차차이유통계학의의(P<0.05).광분유도적각조상피세포S기세포소점비례수배양대수증가축점승고,세포발생악성전화.(2)100 μg/ml광분유도적성섬유세포배양지제26대출현α-SMA표체;여상피세포공배양후,성섬유세포α-SMA표체증강,차수배양대수적증가,양성표체세포수명현증다,착색정도증강.결론 개구광분능유도지기관상피세포악성전화급성섬유세포활화;폐상피세포시광분유암적주요파세포;폐상피세포적전화화성섬유세포적활화상호영향,상호촉진.
Objective To study the interaction between transformation of human pulmonary epithelial ceils and activation of fibroblasts induced by Yunnan tin mine dust. Methods (1) The immortalized human bronchial epithelial cell line BEAS-2B and human embryo lung fibroblast cell hne WI-38 were grown in MEM medium containing 5% and 10% FBS, respectively, at 37 ℃ and 5% CO2 with saturated humidity. The cells were subcultured every 6 days. BEAS-2B cells and WI-38 cells were induced with Yunnan tin mine dust on every other generation at the concentration of 100 μg/ml. From the 11th generation, the cells were co-cultured. Epithelial cell transformation was tested using concanavalin A (ConA)agglutination and anchorage-indepen-dent growth assays. The cell cycles were analyzed through flow cytometry. The expressions of α-SMA in fi-broblasts were determined with immunocytochemistry. Results (1) Cell morphology of mine dust-exposed epithelial cells began to transform at the 28th generation. Similar transformations were observed with mine dust-induced epithelial cells co-cultured with fibroblasts from the 20th generation and mine dust-induce ep-ithelial ceils co-cultured with mine dust-induced fibroblasts from the 16th generation. ConA agglutination assay and anchorage-independent growth assays were negative in normal BEAS-2B cells. At the 26 th generation, the agglutination test result of the mine dust-exposed epithelial cells was positive. Co-cultured with fibroblasts and mine dust-exposed fibroblasts, the agglutination time of the mine dust-exposed epithelial cells became short. Epithelial cell anchorage-independent growth assay was positive for mine dust-exposed epithelial cells co-cul-tured with fibroblasts at the 36th generations and for mine dust-exposed epithelial cells co-cultured with mine dust-exposed fibroblasts at the 26th generations. The clone formation rate of the 26th generation was 6.00‰±1.00‰ and 15.33‰±2.52‰ respectively, with the significant differences (P<0.05). With generation adding, the portion of S phase increased for mine dust-exposed epithelial cells. (2) At the 26th generations, fibroblasts expressed α-SMA. Co-cultured with epithelial cell, the α-SMA expression of fibroblasts increased. Especially, positive cell numbers and intensity of staining dramatically increased with generation adding. Conclusions (1) The tin mine dust can induce malignant transformation of human pulmonary epithelial cells BEAS-2B and activation of fibroblasts WI-38. (2) The epithelial cells are major target in carcinogenesis induced by Yunnan tin mine dust. (3) Transformation of epithelia and activation of fibroblasts co-evolve in the developing process of induced lung cancer by Yunnan tin mine dust.