C8-神经酰胺诱导肺泡Ⅱ型上皮细胞凋亡的研究

Effect of C8-ceramide on apoptosis of alveolar type Ⅱ epithelial cells

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18dICR孕鼠，分离、培养胎鼠肺泡Ⅱ型上皮细胞（alveolartype1Iepithelialcell，AEC1I），分别加入0、20、40、80μmol／L的c8-神经酰胺溶液，继续培养24h，MTF法检测c8-神经酰胺对AECII增殖的影响，使用80μmol／LC8．神经酰胺溶液培养AECII3、6、12、24h，MTF法检测AECⅡ活力。使用0、20、40、80μmo]／LC8-神经酰胺溶液对AECII进行体外干预12和24h，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、80μmol／L时，12和24h细胞总凋亡率分别为（20．634-0．86）％、（22．67±1．72）％、（42．03±1．34）％、（50．404±1．30）％和（20．93±2．51）％、（28．934±3．19）％、（47．004±1．08）％、（57．774±3．04）％（除12h20pμmol／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、80μmol／L时，12和24h的凋亡率分别为（5．514±1．41）％、（13．24±2．62）％、（35．104±4．59）％、（75．07±4．37）％和（5．564±1．36）％、（21．344±2．09）％、（37．12±2．11）％、（91．334±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c8一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。

Objective To investigate the effects of exogenous C8-ceramide on primary alveolar type Ⅱ epithelial cells （AEC Ⅱ ）. Methods AEC 11 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 cells 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 txmol/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 μmol/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 ）. Conelusion The results demonstrate that C8-eeramide can decrease the viability of AEC Ⅱ andinduce apoptosis.