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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 effect of free silicon dioxide (SiO 2) on epithelial-mesenchymal transition (EMT) induced by direct or indirect interaction with RLE-6TN in rat alveolar type Ⅱ epithelial cell line and its effect on the expression of RLE-6TN surface marker protein . Methods (1) Bronchoalveolar lavage was used to extract alveolar macrophages (AM) from SD rats without specific pathogen. AM and RLE-6TN were treated with 0-140 mg / L SiO 2 suspension and cultured for 24, 48 and 72 h. The cell viability was detected by CCK-8 assay, and the dose of dust was screened in subsequent experiments. (2) AM cells were seeded on Transwell and co-cultured with RLE-6TN. The direct control group (RLE-6TN alone, no dying treatment), the direct dyed dust group (RLE-6TN alone, the mass concentration of 100 mg / L SiO 2 suspension treatment), the indirect control group (AM and RLE- 6TN co-culture, no dust treatment) and indirect dusting group (AM and RLE-6TN co-cultured to give a mass concentration of 100 mg / L SiO 2 suspension treatment AM), respectively, in the dust 0,24,48 and After 72 h, cells were harvested and the relative expression levels of E-cadherin and α-SMA in RLE-6TN were determined by immunoblotting. Results (1) According to the result of CCK-8, the dose of SiO 2 dust in the follow-up experiment was set as 100 mg / L. (2) The relative expression levels of E-cad and a-SMA in RLE-6TN were statistically significant (P <0.01) in the interaction effect between dust treatment and dusting time. Among them, the relative expression levels of E-cad in RLE-6TN at both direct exposure and indirect exposure were lower at 48 and 72 hours than those at the same time point (P <0.05) (P <0.05). The relative expression levels of E-cadherin at the time point of 48 and 72 h were all significantly lower in the indirect infected dust group than in the same group at the time points of 0 and 24 h (P <0.05). At 48 and 72 h, the relative expression of α-SMA in the direct-dyed and indirect-dyed groups was higher than that in the corresponding control group at the same time point (P <0.05) ), And the relative expression level of α-SMA in RLE-6TN was higher than that in direct dust group (P <0.05). The relative expression level of α-SMA in RLE-6TN in two dust groups was significantly higher than that in direct dust group The time-effect relationship increased with the increase of dust time (P <0.05). CONCLUSIONS: Direct or indirect exposure to free SiO 2 can induce EMT in RLE-6TN, which leads to the down-regulation of E-cadherin and the up-regulation of α-SMA expression. Indirect dyestuff may induce EMT in RLE-6TN more easily.