中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2015年
7期
574-578
,共5页
覃松%陈淼%戢慧%刘国跃%陈涛%李康%梅鸿
覃鬆%陳淼%戢慧%劉國躍%陳濤%李康%梅鴻
담송%진묘%집혜%류국약%진도%리강%매홍
肺泡上皮细胞,Ⅱ型%凋亡%微小RNA%miR-21-5p%分子机制
肺泡上皮細胞,Ⅱ型%凋亡%微小RNA%miR-21-5p%分子機製
폐포상피세포,Ⅱ형%조망%미소RNA%miR-21-5p%분자궤제
TypeⅡ alveolar epithelial cell%Apoptosis%MicroRNA%miR-21-5p%Molecule mechanism
目的：通过腺病毒过表达微小RNA-21-5p（miR-21-5p）后转染Ⅱ型肺泡上皮细胞（AECⅡ），观察其对过氧化氢（H2O2）诱导的AECⅡ细胞凋亡的影响，并探索其抗凋亡的分子机制。方法将传代培养的大鼠AECⅡ随机均分为4组：正常对照组（生理盐水）、H2O2损伤组（0.5 mmol/L H2O2）、miR-21-5p过表达组（miR-21-5p腺病毒+0.5 mmol/L H2O2）、 miR-21-5p阴性转染组（空腺病毒+0.5 mmol/L H2O2）。采用透射电镜及流式细胞仪分别检测细胞凋亡形态及早期凋亡率；取转染效率最高的时间点（6、12、24、48 h），采用实时荧光定量反转录-聚合酶链反应（RT-PCR）检测AECⅡ内miR-21-5p表达水平，蛋白质免疫印迹试验（Western Blot）检测细胞内Bcl-2、Bax、天冬氨酸特异性半胱氨酸蛋白酶3（caspase-3）的蛋白表达。结果① AECⅡ鉴定：荧光显微镜下可见AECⅡ的特征性结构（微绒毛及嗜锇性板层小体）。②细胞凋亡形态：透射电镜下可见AECⅡ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。③ AECⅡ内miR-21-5p表达：48 h miR-21-5p腺病毒转染效率最高。miR-21-5p过表达组AECⅡ细胞中miR-21-5p表达量明显高于H2O2损伤组和miR-21-5p阴性转染组（A值：1.54±0.02比1.02±0.02、0.56±0.03、0.58±0.02，均P＜0.05）。④细胞凋亡率：与正常对照组比较，H2O2损伤组、miR-21-5p阴性转染组和miR-21-5p过表达组细胞凋亡率均随损伤时间的延长逐渐升高。除损伤6 h外，miR-21-5p过表达组细胞凋亡率明显低于H2O2损伤组和miR-21-5p阴性转染组〔12 h：（10.73±2.80）%比（16.26±0.59）%、（16.04±0.70）%，24 h：（16.00±3.44）%比（23.29±2.78）%、（23.58±2.31）%，48 h：（31.30±3.55）%比（50.53±2.17）%、（49.41±1.97）%，均P＜0.05〕；miR-21-5p阴性转染组与H2O2损伤组各时间点比较差异均无统计学意义。⑤蛋白表达：miR-21-5p过表达组AECⅡ细胞Bax、caspase-3蛋白表达明显低于H2O2损伤组和miR-21-5p阴性转染组〔Bax（A值）：0.07±0.01比0.18±0.01、0.13±0.01，caspase-3（A值）：0.07±0.01比0.23±0.01、0.12±0.01，均P＜0.05〕，Bcl-2蛋白表达水平明显高于H2O2损伤组和miR-21-5p阴性转染组（A值：0.26±0.01比0.06±0.01、0.10±0.01，均P＜0.05）。结论① miR-21-5p具有抗AECⅡ凋亡的作用。②腺病毒载体是转染AECⅡ的一种比较成功的基因转导载体。③ miR-21-5p抗AECⅡ凋亡可能与下调Bax、caspase-3蛋白表达水平及上调Bcl-2蛋白表达水平有关。
目的：通過腺病毒過錶達微小RNA-21-5p（miR-21-5p）後轉染Ⅱ型肺泡上皮細胞（AECⅡ），觀察其對過氧化氫（H2O2）誘導的AECⅡ細胞凋亡的影響，併探索其抗凋亡的分子機製。方法將傳代培養的大鼠AECⅡ隨機均分為4組：正常對照組（生理鹽水）、H2O2損傷組（0.5 mmol/L H2O2）、miR-21-5p過錶達組（miR-21-5p腺病毒+0.5 mmol/L H2O2）、 miR-21-5p陰性轉染組（空腺病毒+0.5 mmol/L H2O2）。採用透射電鏡及流式細胞儀分彆檢測細胞凋亡形態及早期凋亡率；取轉染效率最高的時間點（6、12、24、48 h），採用實時熒光定量反轉錄-聚閤酶鏈反應（RT-PCR）檢測AECⅡ內miR-21-5p錶達水平，蛋白質免疫印跡試驗（Western Blot）檢測細胞內Bcl-2、Bax、天鼕氨痠特異性半胱氨痠蛋白酶3（caspase-3）的蛋白錶達。結果① AECⅡ鑒定：熒光顯微鏡下可見AECⅡ的特徵性結構（微絨毛及嗜鋨性闆層小體）。②細胞凋亡形態：透射電鏡下可見AECⅡ胞質迴縮，染色質凝聚、邊聚，細胞錶麵微絨毛稀少，嗜鋨性闆層小體排空。③ AECⅡ內miR-21-5p錶達：48 h miR-21-5p腺病毒轉染效率最高。miR-21-5p過錶達組AECⅡ細胞中miR-21-5p錶達量明顯高于H2O2損傷組和miR-21-5p陰性轉染組（A值：1.54±0.02比1.02±0.02、0.56±0.03、0.58±0.02，均P＜0.05）。④細胞凋亡率：與正常對照組比較，H2O2損傷組、miR-21-5p陰性轉染組和miR-21-5p過錶達組細胞凋亡率均隨損傷時間的延長逐漸升高。除損傷6 h外，miR-21-5p過錶達組細胞凋亡率明顯低于H2O2損傷組和miR-21-5p陰性轉染組〔12 h：（10.73±2.80）%比（16.26±0.59）%、（16.04±0.70）%，24 h：（16.00±3.44）%比（23.29±2.78）%、（23.58±2.31）%，48 h：（31.30±3.55）%比（50.53±2.17）%、（49.41±1.97）%，均P＜0.05〕；miR-21-5p陰性轉染組與H2O2損傷組各時間點比較差異均無統計學意義。⑤蛋白錶達：miR-21-5p過錶達組AECⅡ細胞Bax、caspase-3蛋白錶達明顯低于H2O2損傷組和miR-21-5p陰性轉染組〔Bax（A值）：0.07±0.01比0.18±0.01、0.13±0.01，caspase-3（A值）：0.07±0.01比0.23±0.01、0.12±0.01，均P＜0.05〕，Bcl-2蛋白錶達水平明顯高于H2O2損傷組和miR-21-5p陰性轉染組（A值：0.26±0.01比0.06±0.01、0.10±0.01，均P＜0.05）。結論① miR-21-5p具有抗AECⅡ凋亡的作用。②腺病毒載體是轉染AECⅡ的一種比較成功的基因轉導載體。③ miR-21-5p抗AECⅡ凋亡可能與下調Bax、caspase-3蛋白錶達水平及上調Bcl-2蛋白錶達水平有關。
목적：통과선병독과표체미소RNA-21-5p（miR-21-5p）후전염Ⅱ형폐포상피세포（AECⅡ），관찰기대과양화경（H2O2）유도적AECⅡ세포조망적영향，병탐색기항조망적분자궤제。방법장전대배양적대서AECⅡ수궤균분위4조：정상대조조（생리염수）、H2O2손상조（0.5 mmol/L H2O2）、miR-21-5p과표체조（miR-21-5p선병독+0.5 mmol/L H2O2）、 miR-21-5p음성전염조（공선병독+0.5 mmol/L H2O2）。채용투사전경급류식세포의분별검측세포조망형태급조기조망솔；취전염효솔최고적시간점（6、12、24、48 h），채용실시형광정량반전록-취합매련반응（RT-PCR）검측AECⅡ내miR-21-5p표체수평，단백질면역인적시험（Western Blot）검측세포내Bcl-2、Bax、천동안산특이성반광안산단백매3（caspase-3）적단백표체。결과① AECⅡ감정：형광현미경하가견AECⅡ적특정성결구（미융모급기철성판층소체）。②세포조망형태：투사전경하가견AECⅡ포질회축，염색질응취、변취，세포표면미융모희소，기철성판층소체배공。③ AECⅡ내miR-21-5p표체：48 h miR-21-5p선병독전염효솔최고。miR-21-5p과표체조AECⅡ세포중miR-21-5p표체량명현고우H2O2손상조화miR-21-5p음성전염조（A치：1.54±0.02비1.02±0.02、0.56±0.03、0.58±0.02，균P＜0.05）。④세포조망솔：여정상대조조비교，H2O2손상조、miR-21-5p음성전염조화miR-21-5p과표체조세포조망솔균수손상시간적연장축점승고。제손상6 h외，miR-21-5p과표체조세포조망솔명현저우H2O2손상조화miR-21-5p음성전염조〔12 h：（10.73±2.80）%비（16.26±0.59）%、（16.04±0.70）%，24 h：（16.00±3.44）%비（23.29±2.78）%、（23.58±2.31）%，48 h：（31.30±3.55）%비（50.53±2.17）%、（49.41±1.97）%，균P＜0.05〕；miR-21-5p음성전염조여H2O2손상조각시간점비교차이균무통계학의의。⑤단백표체：miR-21-5p과표체조AECⅡ세포Bax、caspase-3단백표체명현저우H2O2손상조화miR-21-5p음성전염조〔Bax（A치）：0.07±0.01비0.18±0.01、0.13±0.01，caspase-3（A치）：0.07±0.01비0.23±0.01、0.12±0.01，균P＜0.05〕，Bcl-2단백표체수평명현고우H2O2손상조화miR-21-5p음성전염조（A치：0.26±0.01비0.06±0.01、0.10±0.01，균P＜0.05）。결론① miR-21-5p구유항AECⅡ조망적작용。②선병독재체시전염AECⅡ적일충비교성공적기인전도재체。③ miR-21-5p항AECⅡ조망가능여하조Bax、caspase-3단백표체수평급상조Bcl-2단백표체수평유관。
ObjectiveTo study the effect of hydrogen peroxide (H2O2) in inducing apoptosis of typeⅡalveolar epithelial cell (AECⅡ) after overexpression by adenoviral transfection of micro RNA-21-5p (miR-21-5p), and to explore the mechanism of its anti-apoptosis.Methods Subculture AECⅡ were randomly divided into four groups: normal control group (normal saline), H2O2 challenge group ( 0.5 mmol/L H2O2), miR-21-5p overexpression group (miR-21-5p adenovirus+ 0.5 mmol/L H2O2), miR-21-5p negative transfection group (adenovirus void+0.5 mmol/L H2O2). Transmission electron microscopy and flow cytometry were used to detect apoptotic morphology and early apoptotic rate. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-21-5p in AECⅡ, and Western Blot was used to detect the protein expressions of Bcl-2, Bax, and caspase-3 at the highest transfection efficiency at different time points (6, 12, 24, 48 hours).Results ① AECⅡ identification: fluorescence microscopy showed the presence of characteristic structure of AECⅡ, i.e. microvilli and osmiophilic lamellar bodies.② Apoptotic morphology: transmission electron microscopy showed cytoplasmic retraction, chromatin condensation, margination, lack of cell surface microvilli, and emptying of osmiophilic lamellar bodies in AECⅡ.③ The expression of miR-21-5p in AECⅡ: the highest transfection efficiency was found at 48 hours. The expression of miR-21-5p in miR-21-5p overexpression group was significantly higher than that of the normal control group, H2O2 challenge group and miR-21-5p negative transfection group (A value: 1.54±0.02 vs. 1.02±0.02, 0.56±0.03, 0.58±0.02, allP< 0.05).④ The rate of early apoptosis: compared with normal control group, the early apoptotic rates in H2O2 challenge group, miR-21-5p negative transfection group and miR-21-5p overexpression group were gradually elevated with the prolongation of injury time. The early apoptotic rate in miR-21-5p overexpression group was significantly lower than that of the H2O2 challenge group and miR-21-5p negative transfection group at all time points except 6 hours [12 hours: (10.73±2.80)% vs. (16.26±0.59)%, (16.04±0.70)%; 24 hours:(16.00±3.44)% vs. (23.29±2.78)%, (23.58±2.31)%; 48 hours: (31.30±3.55)% vs. (50.53±2.17)%, (49.41±1.97)%, allP< 0.05]. There was no significant difference in early apoptotic rate between miR-21-5p negative transfection group and H2O2 challenge group at each time point.⑤ Protein expression: the expressions of Bax and caspase-3 in miR-21-5p overexpression group were significantly lower than those of the H2O2 challenge group and miR-21-5p negative transfection group [Bax (A value): 0.07±0.01 vs. 0.18±0.01, 0.13±0.01; caspase-3 (A value): 0.07±0.01 vs. 0.23±0.01, 0.12±0.01, allP< 0.05], and Bcl-2 protein expression was significantly higher than that of the H2O2 challenge group and miR-21-5p negative transfection group (A value: 0.26±0.01 vs. 0.06±0.01, 0.10±0.01, both P< 0.05).Conclusions① miR-21-5p has the function of anti-apoptosis of AECⅡ.② Adenoviral vector is a successful gene transfer vector when transfected with AECⅡ.③ The anti-apoptosis of AECⅡ by miR-21-5p may be associated with reduced Bax and caspase-3 protein levels and raised expression levels of Bcl-2 protein.
