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.%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.
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