CAJ | 학술논문

目的利用基因组芯片筛选Ⅱ型肺泡上皮细胞(AECⅡ)凋亡相关微小RNA (microRNA,miRNA),以期为临床防治高氧性急性肺损伤(HALI)提供新策略.方法原代培养雄性SD大鼠AECⅡ36 h,加入0.5 mmol/L H2O2诱导建立AECⅡ凋亡模型,透射电镜下鉴定AECⅡ并观察凋亡细胞形态；于制模前及制模后2.5、6、12、24 h收集细胞,流式细胞术检测细胞凋亡率；基因芯片技术筛选AECⅡ凋亡相关miRNA,并采用实时荧光聚合酶链反应(RT-PCR)对基因芯片结果进行验证.结果透射电镜下观察可见特征性的微绒毛及嗜锇性板层小体,鉴定为AECⅡ.透射电镜下可见细胞质回缩,染色质凝聚、边聚,细胞表面微绒毛稀少,嗜锇性板层小体排空.H2O2损伤细胞后,细胞凋亡率较空白对照组显著增加,且随损伤时间延长而逐渐上升[制模前及制模后2.5、6、12、24 h分别为(9.43±1.02)％、(18.38±2.91)％、(28.57±1.18)％、(35.83±2.66)％、(57.68±2.22)％,两两比较P＜ 0.05].与制模前相比,制模24h细胞伴有多种miRNAs的差异表达,其中rno-miR-449a-5p、rno-miR-34b/c-5p、rno-miR-200a/c-3p、rno-miR-146a-5p、rno-miR-141-3p、rno-miR-21-5p、rno-miR-375-3p、rno-miR-29b-3p、rno-miR-214-5p、rno-miR-210-5p及rno-miR-214-3p与凋亡密切相关.RT-PCR结果验证凋亡相关miRNAs结果与芯片结果一致.结论利用基因组芯片筛选AECⅡ凋亡相关miRNA,其中miR-34家族可能是AECⅡ凋亡调控的主要参与者.rno-miR-21-5p可能是AECⅡ内重要的抗凋亡基因.
목적 이용기인조심편사선Ⅱ형폐포상피세포(AECⅡ)조망상관미소RNA (microRNA,miRNA),이기위림상방치고양성급성폐손상(HALI)제공신책략.방법 원대배양웅성SD대서AECⅡ36 h,가입0.5 mmol/L H2O2유도건립AECⅡ조망모형,투사전경하감정AECⅡ병관찰조망세포형태；우제모전급제모후2.5、6、12、24 h수집세포,류식세포술검측세포조망솔；기인심편기술사선AECⅡ조망상관miRNA,병채용실시형광취합매련반응(RT-PCR)대기인심편결과진행험증.결과 투사전경하관찰가견특정성적미융모급기철성판층소체,감정위AECⅡ.투사전경하가견세포질회축,염색질응취、변취,세포표면미융모희소,기철성판층소체배공.H2O2손상세포후,세포조망솔교공백대조조현저증가,차수손상시간연장이축점상승[제모전급제모후2.5、6、12、24 h분별위(9.43±1.02)％、(18.38±2.91)％、(28.57±1.18)％、(35.83±2.66)％、(57.68±2.22)％,량량비교P＜ 0.05].여제모전상비,제모24h세포반유다충miRNAs적차이표체,기중rno-miR-449a-5p、rno-miR-34b/c-5p、rno-miR-200a/c-3p、rno-miR-146a-5p、rno-miR-141-3p、rno-miR-21-5p、rno-miR-375-3p、rno-miR-29b-3p、rno-miR-214-5p、rno-miR-210-5p급rno-miR-214-3p여조망밀절상관.RT-PCR결과험증조망상관miRNAs결과여심편결과일치.결론 이용기인조심편사선AECⅡ조망상관miRNA,기중miR-34가족가능시AECⅡ조망조공적주요삼여자.rno-miR-21-5p가능시AECⅡ내중요적항조망기인.
Objective To screen type Ⅱ alveolar epithelial cell (AEC Ⅱ) apoptosis related microRNA (miRNA) with gene chip technology,provide a new strategy for the prevention and treatment of hyperoxia-induced acute lung injury (HALI).Methods AEC Ⅱ of male Sprague-Dawley (SD) rats was primarily cultured for 36 hours,then exposed to 0.5 mmol/L H2O2 to establish apoptosis model.Transmission electron microscope (TEM) was used to identify AEC Ⅱ and to observe apoptosis cell morphology.Before and after H2O2 injury for 2.5,6,12 and 24 hours,fluorescence-activated cell sorting (FACS) was employed to detect apoptosis rate.Additionally,gene chip technology and real time polymerase chain reaction (RT-PCR) were used to screen and verify apoptosis related miRNA respectively.Results Microvilli and osmiophilic multilamellar body were found under TEM,which were the characteristic structure of AEC Ⅱ.This proved that the cells cultured were AEC Ⅱ.After H2O2 injury for 24 hours,cytoplamic retraction,chromation condensation and margination,microvilli and osmiophilic multilamellar body disappearance could be found under TEM.Compared with the blank control group,the apoptosis rate of AEC Ⅱ was significantly increased after exposed to 0.5 mmol/L H2O2,and gradually increased with time [the early apoptosis rate before and after H2O2 injury for 2.5,6,12 and 24 hours were (9.43 ± 1.02)％,(18.38 ± 2.91)％,(28.57 ± 1.18)％,(35.83 ± 2.66)％ and (57.68 ± 2.22)％,respectively,all P＜0.05].Compared with before H2O2 injury,cells at 24 hours accompanied a significantly changed miRNA expression profiling,in which apoptosis related miRNA had been screened,they were rno-miR-449a-5p,rno-miR-34b/c-5p,rno-miR-200a/c-3p,rno-miR-146a-5p,rno-miR-141-3p,rno-miR-21-5p,rno-miR-375-3p,rno-miR-29b-3p,rno-miR-214-5p,rno-miR-210-5p and rno-miR-214-3p,and the results of RT-PCR were consistent with the gene chip results.Conclusions Screened out AEC lⅡ apoptosis related miRNA,and miR-34 family may play a key role in AEC Ⅱ apoptosis regulation.Rno-miR-21-5p may be an important anti-apoptotic gene in AEC Ⅱ apoptosis regulation.