游离二氧化硅诱导肺泡Ⅱ型上皮细胞转化机制

Mechanism of epithelial-mesenchymal transition induced by free silicon dioxide in type Ⅱ alveolar epithelial cells

目的观察游离二氧化硅(SiO_2)直接或间接作用于大鼠肺泡Ⅱ型上皮细胞株RLE-6TN而诱导发生上皮间质转化(EMT)的情况,及其对RLE-6TN表面标志蛋白表达影响情况。方法 (1)采用支气管肺泡灌洗术提取无特定病原体级SD大鼠肺泡巨噬细胞(AM)。以质量浓度为0~140 mg/L的SiO_2混悬液处理AM和RLE-6TN并培养24、48和72 h后,采用CCK-8实验检测细胞存活率,筛选后续实验染尘剂量。(2)取AM接种于Transwell的小室层与RLE-6TN共培养。设直接对照组(单独RLE-6TN,不作染尘处理)、直接染尘组(单独RLE-6TN,予质量浓度为100 mg/L的SiO_2混悬液处理)、间接对照组(AM与RLE-6TN共培养,不作染尘处理)和间接染尘组(AM与RLE-6TN共培养,予质量浓度为100 mg/L的SiO_2混悬液处理AM),分别于染尘0、24、48和72 h后收获细胞,用免疫印迹法测定RLE-6TN中的E-黏钙蛋白(E-cad)和α-平滑肌肌动蛋白(α-SMA)的相对表达水平。结果 (1)根据CCK-8结果,后续实验的SiO_2染尘剂量设为100 mg/L。(2)RLE-6TN中E-cad和α-SMA相对表达水平在染尘处理和染尘时间的交互效应上均有统计学意义(P<0.01)。其中,直接染尘组和间接染尘组RLE-6TN在48和72 h的E-cad相对表达水平均低于同时间点直接对照组(P<0.05),直接染尘组RLE-6TN在72 h时间点的E-cad相对表达水平分别低于同组0和24 h时间点(P<0.05),间接染尘组RLE-6TN在48和72 h时间点的E-cad相对表达水平均低于同组0 h时间点(P<0.05);在48和72 h时间点,直接染尘组和间接染尘组α-SMA相对表达水平分别高于同时间点的相应对照组(P<0.05),且间接染尘组RLE-6TN中α-SMA相对表达水平均高于直接染尘组(P<0.05);2个染尘组RLE-6TN中α-SMA相对表达水平均呈随着染尘时间的增加而增加的时间-效应关系(P<0.05)。结论游离SiO_2直接或间接染尘均可诱导RLE-6TN发生EMT,导致E-cad表达下调,α-SMA表达呈现时间-效应性上调;间接染尘可能更易于诱导RLE-6TN发生EMT。

Objective To investigate the epithelial-mesenchymal transition （EMT） induced by direct or indirect exposure to free silicon dioxide （SiO2） and the expression of surface protein marker in rat typeⅡalveolar epithelial cell RLE-6TN.Methods i） The alveolar macrophages （AM） were isolated from specific pathogen-free SD rat by bronchoalveolar lavage.AM and RLE-6TN were treated with 0-140 mg/L （final concentration） of SiO2 suspension and were cultured conventionally for 24,48 and 72 hours.The cell viability was detected by CCK-8 assay.The result of CCK-8 essay was used to choose the SiO2 concentration for the following study.ii） To establish models of RLE-6TN co-cultured with AM that were seeded in Transwell.The cells were divided into 4 groups： the direct control group （RLE-6TN,no SiO2 exposed）,the direct exposure group （RLE-6TN,treated with 100 mg/L SiO2）,the indirect control group （RLE-6TN and AM were cocultured,no SiO2 exposed） and the indirect exposure group （RLE-6TN and AM were co-cultured,AM was treated with 100 mg/L SiO2 directly）.Western blotting was used to detect the expression of E-cadherin （E-cad） and α-smooth muscle protein （α-SMA） after cells were cultured for 0,24,48 and 72 hours.Results i） According to the CCK-8 assay,the final concentration of 100 mg/L SiO2 was chosen for the following study.ii） The difference of relative expression of E-cad and α-SMA in RLE-6TN was statistically significant in different treatment combination and time （P〈0.01）.The E-cad expression of RLE-6TN at 48 and 72 hours in the direct exposure group and the indirect exposure group was lower than that in direct control group at the same time point （P〈0.05）.The E-cad expression in RLE-6TN at 72 hours in the direct exposure group was lower than that in the 0 and 24 hours （P〈0.05）.The E-cad expression in RLE-6TN at 48 and 72 hours in the indirect exposure group was lower than that in the 0 hour （P〈0.05）.At 48 and 72 hours,the α-SMA expression in the indirect exposure group and the direct exposure group was higher than that in their control groups at the same time point （P〈0.05）.The expression of α-SMA in the indirect exposure group was higher than that in the direct exposure group （P〈0.05）.The expression of α-SMA in both exposure groups increased in a time-effect relationship （P〈0.05）.Conclusion Direct or indirect exposure to free SiO2 can induce EMT in RLE-6TN,and decrease the expression of E-cad and increase the expression of α-SMA in a time-effect relationship.Indirect exposure group is more susceptible to EMT.