中华实用儿科临床杂志
中華實用兒科臨床雜誌
중화실용인과림상잡지
Journal of Applied Clinical Pediatrics
2014年
14期
1060-1065
,共6页
王靓%刘伟%李文斌%程婷婷%高春芳%莫露霞%常立文
王靚%劉偉%李文斌%程婷婷%高春芳%莫露霞%常立文
왕정%류위%리문빈%정정정%고춘방%막로하%상립문
高体积分数氧肺损伤%氧化应激%角化上皮生长因子%肺泡Ⅱ型上皮细胞
高體積分數氧肺損傷%氧化應激%角化上皮生長因子%肺泡Ⅱ型上皮細胞
고체적분수양폐손상%양화응격%각화상피생장인자%폐포Ⅱ형상피세포
Hyperoxia-induced lung injury%Oxidative stress%Keratinocyte growth factor%Alveolar epithelial type Ⅱ cells
目的 探讨高体积分数氧(高氧)环境下,角化上皮生长因子(KGF)对Sprague-Dawley(SD)大鼠胎鼠肺泡Ⅱ型上皮细胞(ATⅡCs)增殖/存活、凋亡及死亡水平的影响.方法 提取SD大鼠胎鼠ATⅡCs进行原代培养,在常氧环境下[210 mL/L氧气(O2)]、高氧环境(950 mL/L O2)下分别培养细胞,并给予不同质量浓度的KGF(15 μg/L、25 μg/L、50 μg/L、75 μg/L、100 μg/L),将细胞按随机数字表法分为单纯常氧组、常氧+KGF组、单纯高氧组、高氧+KGF组,采用流式细胞仪检测细胞内活性氧(ROS)水平,采用细胞增殖检测法(MTT比色法)、乳酸脱氢酶释放试验(LDH法)检测细胞存活、死亡情况,Western Blot检测细胞凋亡指标活化的含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspase-3)蛋白的表达.结果 常氧环境下培养细胞,常氧+KGF组KGF 15～ 100 μg/L质量浓度范围内,各质量浓度组MTT水平显著高于单纯常氧组,25～ 100 μg/L质量浓度范围内各浓度组LDH显著低于单纯常氧组.与单纯常氧组相比,高氧环境暴露下细胞内ROS水平明显升高,呈现时间依赖性,高氧暴露4h以上,细胞活力显著下降、细胞死亡水平明显升高.高氧暴露0.5～1.0 h,高氧+KGF组15～75 μg/L质量浓度范围内,各质量浓度组MTT水平显著高于单纯高氧组,15 ～75 μg/L KGF内各质量浓度组LDH显著低于单纯高氧组.高氧暴露4h后,15 μg/L及25 μg/L高氧+KGF组细胞MTT水平高于单纯高氧组,25 ～ 100 μg/L范围内LDH释放水平低于单纯高氧组.延长高氧暴露时间达8h,仅高氧+50 μg/LKGF组细胞存活水平高于单纯高氧组,高氧+75 μg/L KGF组细胞死亡水平低于单纯高氧组.高氧暴露达12 h时,与单纯高氧组相比,高氯+KGF各质量浓度组细胞存活、死亡水平均无显著差异.高氧暴露4～8 h时,与单纯常氧组相比,细胞凋亡指标活化的Caspase-3水平显著升高;与此同时,在上述时间点分别需要25 μg/L和75 μg/L KGF,才能降低活化的Caspase-3表达水平.结论 KGF能够在常氧、短时间高氧情况下促进ATⅡCs的增殖/存活、抑制凋亡及死亡,发挥细胞保护作用;但长时间高氧暴露会降低KGF敏感性,抑制KGF保护效应.
目的 探討高體積分數氧(高氧)環境下,角化上皮生長因子(KGF)對Sprague-Dawley(SD)大鼠胎鼠肺泡Ⅱ型上皮細胞(ATⅡCs)增殖/存活、凋亡及死亡水平的影響.方法 提取SD大鼠胎鼠ATⅡCs進行原代培養,在常氧環境下[210 mL/L氧氣(O2)]、高氧環境(950 mL/L O2)下分彆培養細胞,併給予不同質量濃度的KGF(15 μg/L、25 μg/L、50 μg/L、75 μg/L、100 μg/L),將細胞按隨機數字錶法分為單純常氧組、常氧+KGF組、單純高氧組、高氧+KGF組,採用流式細胞儀檢測細胞內活性氧(ROS)水平,採用細胞增殖檢測法(MTT比色法)、乳痠脫氫酶釋放試驗(LDH法)檢測細胞存活、死亡情況,Western Blot檢測細胞凋亡指標活化的含半胱氨痠的天鼕氨痠蛋白水解酶-3(Caspase-3)蛋白的錶達.結果 常氧環境下培養細胞,常氧+KGF組KGF 15～ 100 μg/L質量濃度範圍內,各質量濃度組MTT水平顯著高于單純常氧組,25～ 100 μg/L質量濃度範圍內各濃度組LDH顯著低于單純常氧組.與單純常氧組相比,高氧環境暴露下細胞內ROS水平明顯升高,呈現時間依賴性,高氧暴露4h以上,細胞活力顯著下降、細胞死亡水平明顯升高.高氧暴露0.5～1.0 h,高氧+KGF組15～75 μg/L質量濃度範圍內,各質量濃度組MTT水平顯著高于單純高氧組,15 ～75 μg/L KGF內各質量濃度組LDH顯著低于單純高氧組.高氧暴露4h後,15 μg/L及25 μg/L高氧+KGF組細胞MTT水平高于單純高氧組,25 ～ 100 μg/L範圍內LDH釋放水平低于單純高氧組.延長高氧暴露時間達8h,僅高氧+50 μg/LKGF組細胞存活水平高于單純高氧組,高氧+75 μg/L KGF組細胞死亡水平低于單純高氧組.高氧暴露達12 h時,與單純高氧組相比,高氯+KGF各質量濃度組細胞存活、死亡水平均無顯著差異.高氧暴露4～8 h時,與單純常氧組相比,細胞凋亡指標活化的Caspase-3水平顯著升高;與此同時,在上述時間點分彆需要25 μg/L和75 μg/L KGF,纔能降低活化的Caspase-3錶達水平.結論 KGF能夠在常氧、短時間高氧情況下促進ATⅡCs的增殖/存活、抑製凋亡及死亡,髮揮細胞保護作用;但長時間高氧暴露會降低KGF敏感性,抑製KGF保護效應.
목적 탐토고체적분수양(고양)배경하,각화상피생장인자(KGF)대Sprague-Dawley(SD)대서태서폐포Ⅱ형상피세포(ATⅡCs)증식/존활、조망급사망수평적영향.방법 제취SD대서태서ATⅡCs진행원대배양,재상양배경하[210 mL/L양기(O2)]、고양배경(950 mL/L O2)하분별배양세포,병급여불동질량농도적KGF(15 μg/L、25 μg/L、50 μg/L、75 μg/L、100 μg/L),장세포안수궤수자표법분위단순상양조、상양+KGF조、단순고양조、고양+KGF조,채용류식세포의검측세포내활성양(ROS)수평,채용세포증식검측법(MTT비색법)、유산탈경매석방시험(LDH법)검측세포존활、사망정황,Western Blot검측세포조망지표활화적함반광안산적천동안산단백수해매-3(Caspase-3)단백적표체.결과 상양배경하배양세포,상양+KGF조KGF 15～ 100 μg/L질량농도범위내,각질량농도조MTT수평현저고우단순상양조,25～ 100 μg/L질량농도범위내각농도조LDH현저저우단순상양조.여단순상양조상비,고양배경폭로하세포내ROS수평명현승고,정현시간의뢰성,고양폭로4h이상,세포활력현저하강、세포사망수평명현승고.고양폭로0.5～1.0 h,고양+KGF조15～75 μg/L질량농도범위내,각질량농도조MTT수평현저고우단순고양조,15 ～75 μg/L KGF내각질량농도조LDH현저저우단순고양조.고양폭로4h후,15 μg/L급25 μg/L고양+KGF조세포MTT수평고우단순고양조,25 ～ 100 μg/L범위내LDH석방수평저우단순고양조.연장고양폭로시간체8h,부고양+50 μg/LKGF조세포존활수평고우단순고양조,고양+75 μg/L KGF조세포사망수평저우단순고양조.고양폭로체12 h시,여단순고양조상비,고록+KGF각질량농도조세포존활、사망수평균무현저차이.고양폭로4～8 h시,여단순상양조상비,세포조망지표활화적Caspase-3수평현저승고;여차동시,재상술시간점분별수요25 μg/L화75 μg/L KGF,재능강저활화적Caspase-3표체수평.결론 KGF능구재상양、단시간고양정황하촉진ATⅡCs적증식/존활、억제조망급사망,발휘세포보호작용;단장시간고양폭로회강저KGF민감성,억제KGF보호효응.
Objective To explore the survival/proliferation,apoptotic and death effects of keratinocyte growth factor (KGF) in alveolar epithelial type Ⅱ cells (AT Ⅱ Cs) exposed to hyperoxia.Methods Primary culture of AT Ⅱ Cs from the Sprague-Dawley rat fetuses was studied under room air condition (210 mL/L O2) and hyperoxic condition (950 mL/L O2) for 0.5-12.0 h.Various concentrations of KGF (15 μg/L,25 μg/L,50 μg/L,75 μg/L,100 μg/L)were added into the cell cultures.Cells were randomly divided into room-air group,room-air-KGF group,hyperoxic-exposure group and hyperoxic-exposure-KGF group.The levels of intracellular reactive oxygen species (ROS),cleaved cysteinyl aspartate specific proteinase-3 (Caspase-3),cell death and proliferation of AT Ⅱ Cs were measured by flow cytometer,Western Blot,release of lactate dehydrogenase assays (LDH assays) and 3-(4,5-Dimethyhhiazol-2-yl)-2,5-diphenyhetrazolium bromide assays (MTT assays),respectively.Results Under room air condition,KGF could significantly increase AT Ⅱ Cs proliferation with 15-100 μg/L in a dose-dependent manner and significantly decrease LDH production at concentrations of 25-100 μg/L.Exposure to hyperoxia resulted in a significant increase in intracellular ROS production in AT Ⅱ Cs in a time-dependent manner compared with that of the room air group.Cell viability decreased and LDH release increased significantly in a time-dependent manner when AT Ⅱ Cs were exposed to 950 mL/L O2 for more than 4 h.After exposure to hyperoxia for 0.5 h and 1 h,KGF could significantly increase AT Ⅱ Cs proliferation in 15-75 μ g/L and significantly decrease LDH production at concentrations of 25-75 μg/L.After exposure to hyperoxia up to 4 h,higher viability was observed in 15 μg/L and 25 μg/L KGF group,and lower death rate presented in 25-100 μg/L KGF group.Further,prolnged hyperoxic exposure for 8 h,high viabilitv was shown only in 50 μg/L KGF group,and less death rate was observed only in 75 μg/L KGF group.In addition,no significant difference in viability and mortality was found between hyperoxic group and hyperoxic-KGF group after hyperoxic exposure for 12 h.Expression of cleaved Caspase-3 was significant higher after 4 h and 8 h hyperoxic exposure than that in room-air group ;at the same time,by adding 25 μg/L and 75.μg/L KGF led to decreased expression of Caspase-3 was detected,compared to hyperoxic group.Conclusions KGF may promote survival/proliferation,inhibited apoptosis and death of rat fetal AT Ⅱ Cs in room air condition or under temporary exposure to hyperoxia in vitro.However,prolonged exposure to hyperoxia may decrease the sensitivity of AEC Ⅱ Cs to KGF and limit its protective effects on lung injury.
