Objective To investigate the effects of exogenous C8-ceramide on primary alveolar type Ⅱ epithelial cells (AEC Ⅱ).Methods AEC Ⅱ were isolated from 18 day gestational age ICR mice,and cultured in Dulbecco' s Modified Eagle Medium with 95％ air and 5％ CO2 at 37 ℃.The cells were treated with different concentrations (0,20,40 and 80 μmol/L) of C8-ceramide for 24 h and treated with 80 μmol/L of C8-ceramide for different time (3,6,12,and 24 h) to observe the viability of cells by MTT assay.Then,the annexin V externalization assay and TUNEL assay were performed to determine apoptosis after the cells were treated with different concentrations (0,20,40,and 80 μmol/L) of C8-ceramide for different time (12 and 24 h).Results The results of MTT assay showed that cell viability decreased in a dose-and time-dependent manner.In Annexin V externalization assay,the ceils showed a distribution of (20.63±0.86)％,(22.67±1.72)％,(42.03±1.34)％,(50.40±1.30)％ and (20.93±2.51)％,(28.93 ± 3.19) ％,(47.00 ± 1.08) ％,(57.77 ± 3.04) ％ cells in apoptosis after treated with 0,20,40,80 μmol/L of C8-ceramide for 12 and 24 h (It is significant difference from untreated cells except for 20 μmol/L at 12 h point,all P ＜ 0.05).In TUNEL assay,the cells showed a distribution of (5.51 ± 1.41)％,(13.24±2.62)％,(35.10±4.59)％,(75.07±4.37)％ and (5.56±1.36)％,(21.34± 2.09)％,(37.12 ±2.11)％,(91.33 ±0.72)％ cells in apoptosis after treated with 0,20,40,80 μ mol/L of C8-ceramide for 12 and 24 h (It is significant difference from untreated cells at the same time point,all P ＜ 0.05).Conclusion The results demonstrate that C8-ceramide can decrease the viability of AEC Ⅱ and induce apoptosis.%目的 探讨外源性C8-神经酰胺诱导肺泡Ⅱ型上皮细胞凋亡的作用.方法 取妊娠18 d ICR孕鼠,分离、培养胎鼠肺泡Ⅱ型上皮细胞(alveolar typeⅡepithelial cell,AECⅡ),分别加入0、20、40、80 μmol/L的C8-神经酰胺溶液,继续培养24 h,MTr法检测C8-神经酰胺对AECⅡ增殖的影响,使用80 μmol/L C8-神经酰胺溶液培养AECⅡ3、6、12、24 h,MTT法检测AECⅡ活力.使用0、20、40、80 μmol/L C8-神经酰胺溶液对AECⅡ进行体外干预12和24 h,分别使用流式细胞术和TUNEL法检测细胞凋亡率.结果 C8-神经酰胺对AECⅡ增殖具有明显抑制作用,并且具有浓度效应和时间效应.C8-神经酰胺溶液浓度为0、20、40、80 μmol/L时,12和24 h细胞总凋亡率分别为(20.63±0.86)％、(22.67±1.72)％、(42.03±1.34)％、(50.40±1.30)％和(20.93±2.51)％、(28.93±3.19)％、(47.00±1.08)％、(57.77±3.04)％(除12 h 20 μmol/L组外,相同时间点,其他各组与对照组相比,差异均有统计学意义,均P＜ 0.05).C8-神经酰胺溶液浓度为0、20、40、80 μmol/L时,12和24 h的凋亡率分别为(5.51±1.41)％、(13.24±2.62)％、(35.10±4.59)％、(75.07±4.37)％和(5.56±1.36)％、(21.34±2.09)％、(37.12±2.11)％、(91.33±0.72)％(相同时间点,各浓度组与对照组相比,差异有统计学意义,均P ＜0.05).结论 C8-神经酰胺可降低肺泡上皮细胞生存率,并且具有明显的诱导细胞凋亡作用.
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