生理学报
生理學報
생이학보
ACTA PHYSIOLOGICA SINICA
2006年
3期
244-254
,共11页
香烟烟雾%肺泡上皮细胞%凋亡%Fas受体
香煙煙霧%肺泡上皮細胞%凋亡%Fas受體
향연연무%폐포상피세포%조망%Fas수체
cigarette smoke%alveolar epithelial cells%apoptosis%Fas receptor
香烟烟雾提取物(cigarette smoke extract,CSE)中含有丰富的氧化剂和自由基,由它所引起的氧化应激可导致肺泡壁的损伤进而发展为肺气肿.近年来,围绕CSE损伤肺泡壁作用机制的研究较为活跃,但其结果却一直存在着分歧.本实验的目的是观察CSE对肺泡Ⅱ型上皮细胞的损伤作用并探讨与其相关的分子机制.MTT比色法的结果显示,CSE以时间和剂量依赖性的方式降低细胞的增殖活力,流式细胞术的分析结果表明细胞增殖周期被阻滞在G1/S期.Hoechst 33258染色以及透射电镜观察从形态上确认CSE诱导细胞凋亡的发生,DNA梯的出现和Annexin V-FITC/碘化丙啶双染色的结果从分子水平得到进一步的证实.同时,运用流式细胞术检测到CSE诱导的凋亡伴随着Fas受体的高表达和caspase-3的显著活化.另外,使用H2DCFDA染色,经激光共聚焦显微镜术测得细胞内氧自由基在细胞受到CSE刺激以后大量快速积累.结果表明CSE能够抑制肺泡Ⅱ型上皮细胞来源的A549细胞的生长和增殖,并诱导细胞凋亡,由Fas受体所介导的死亡受体途径参与此凋亡过程,而CSE所引起的氧化应激则可能是阻止肺泡上皮细胞生长增殖并诱导其凋亡的始动因素.
香煙煙霧提取物(cigarette smoke extract,CSE)中含有豐富的氧化劑和自由基,由它所引起的氧化應激可導緻肺泡壁的損傷進而髮展為肺氣腫.近年來,圍繞CSE損傷肺泡壁作用機製的研究較為活躍,但其結果卻一直存在著分歧.本實驗的目的是觀察CSE對肺泡Ⅱ型上皮細胞的損傷作用併探討與其相關的分子機製.MTT比色法的結果顯示,CSE以時間和劑量依賴性的方式降低細胞的增殖活力,流式細胞術的分析結果錶明細胞增殖週期被阻滯在G1/S期.Hoechst 33258染色以及透射電鏡觀察從形態上確認CSE誘導細胞凋亡的髮生,DNA梯的齣現和Annexin V-FITC/碘化丙啶雙染色的結果從分子水平得到進一步的證實.同時,運用流式細胞術檢測到CSE誘導的凋亡伴隨著Fas受體的高錶達和caspase-3的顯著活化.另外,使用H2DCFDA染色,經激光共聚焦顯微鏡術測得細胞內氧自由基在細胞受到CSE刺激以後大量快速積纍.結果錶明CSE能夠抑製肺泡Ⅱ型上皮細胞來源的A549細胞的生長和增殖,併誘導細胞凋亡,由Fas受體所介導的死亡受體途徑參與此凋亡過程,而CSE所引起的氧化應激則可能是阻止肺泡上皮細胞生長增殖併誘導其凋亡的始動因素.
향연연무제취물(cigarette smoke extract,CSE)중함유봉부적양화제화자유기,유타소인기적양화응격가도치폐포벽적손상진이발전위폐기종.근년래,위요CSE손상폐포벽작용궤제적연구교위활약,단기결과각일직존재착분기.본실험적목적시관찰CSE대폐포Ⅱ형상피세포적손상작용병탐토여기상관적분자궤제.MTT비색법적결과현시,CSE이시간화제량의뢰성적방식강저세포적증식활력,류식세포술적분석결과표명세포증식주기피조체재G1/S기.Hoechst 33258염색이급투사전경관찰종형태상학인CSE유도세포조망적발생,DNA제적출현화Annexin V-FITC/전화병정쌍염색적결과종분자수평득도진일보적증실.동시,운용류식세포술검측도CSE유도적조망반수착Fas수체적고표체화caspase-3적현저활화.령외,사용H2DCFDA염색,경격광공취초현미경술측득세포내양자유기재세포수도CSE자격이후대량쾌속적루.결과표명CSE능구억제폐포Ⅱ형상피세포래원적A549세포적생장화증식,병유도세포조망,유Fas수체소개도적사망수체도경삼여차조망과정,이CSE소인기적양화응격칙가능시조지폐포상피세포생장증식병유도기조망적시동인소.
Cigarette smoke extract (CSE) contains abundant oxidants and free radicals. Oxidative stress caused by cigarette smoking results in the destruction of the alveolar cell walls and emphysema. However, there exists discrepancy about how CSE works in the process. In the present study, we observed the effect of CSE on the cell growth of type Ⅱ alveolar epithelial cell-derived A549 cell line,and provided molecular understanding of this effect. The MTT assay results showed that CSE decreased the cell viability of A549 cells in a dose- and time-dependent manner, and cell cycle was arrested in G1/S phase. Furthermore, CSE-induced apoptosis of A549 cells was verified by Hoechst 33258 staining, electron microscopy in morphology, and the appearance of DNA fragmentation and annexin V-FITC/propidium iodide (PI) staining assay at molecular level. It was found that CSE treatment resulted in the upregulation of Fas/APO-1 receptor and activation of caspase-3. CSE also initiated accumulation of intracellular reactive oxygen species, which was detected by laser confocal microscopy. Taken together, CSE could inhibit the cell growth and induce apoptosis of A549 cells through Fas receptor pathway.Oxidative stress caused by CSE may be the radical factor leading to apoptosis as well as cell growth inhibition in alveolar epithelial cells.
