论文部分内容阅读
目的:研究亚砷酸钠（NaAsOn 2）对人肝星状细胞（LX-2细胞）自噬蛋白微管相关蛋白轻链3（LC3）、Beclin-1的影响。n 方法:体外培养LX-2细胞，使用红色荧光蛋白-绿色荧光蛋白-LC3（RFP-GFP-LC3）慢病毒稳定感染LX-2细胞，流式细胞术进行筛选和感染率测定。采用成组设计，用不同浓度NaAsOn 2[μmol/L：5.00（感染+高砷剂量组）、0.50（感染+中砷剂量组）、0.05（感染+低砷剂量组）、0.00（感染组）]孵育稳定感染LX-2细胞，构建体外肝纤维化模型，同时设立空白组。采用CCK-8法检测NaAsOn 2对LX-2细胞活性的影响；采用实时荧光定量PCR和蛋白免疫印迹法检测LC3、Beclin-1、α-平滑肌肌动蛋白（α-SMA）mRNA和蛋白表达水平。n 结果:RFP-GFP-LC3慢病毒稳定感染LX-2细胞后，流式细胞术测定RFP、GFP荧光，感染率在70%左右，荧光显微镜下观测RFP和GFP的荧光强度无显著性差异，稳定感染细胞株建立成功。NaAsOn 2处理24、48、72 h，与空白组比较，感染组细胞活性差异无统计学意义（n P均> 0.05），其余各剂量组细胞活性均下降（n P均< 0.05）。各组间LC3、Beclin-1、α-SMA mRNA和蛋白表达水平比较差异有统计学意义（n F = 17.450、11.084，11.294、11.745，31.635、12.130，n P均< 0.05）。LC3 mRNA水平，感染组、感染+高砷剂量组、感染+低砷剂量组（20.09 ± 6.50、36.57 ± 9.68、14.19 ± 6.17）高于空白组（1.25 ± 0.21，n P均< 0.05），感染+高砷剂量组高于感染组（n P < 0.05）；Beclin-1 mRNA水平，各组（22.46 ± 0.66、13.38 ± 2.27、20.80 ± 6.95、24.31 ± 7.09）高于空白组（1.10 ± 0.53， n P均< 0.05）；α-SMA mRNA水平，感染+高砷剂量组、感染+中砷剂量组、感染+低砷剂量组（1.07 ± 0.27、1.65 ± 0.17、1.73 ± 0.26）高于空白组（0.60 ± 0.11）、感染组（0.31 ± 0.09，n P均< 0.05）。LC3蛋白表达，感染组、感染+中砷剂量组、感染+低砷剂量组（0.20 ± 0.06、0.15 ± 0.00、0.16 ± 0.01）高于空白组（0.04 ± 0.01，n P均< 0.05）；Beclin-1蛋白表达，感染+中砷剂量组、感染+低砷剂量组（0.83 ± 0.03、1.20 ± 0.02）高于空白组（0.25 ± 0.01，n P均< 0.05），感染+低砷剂量组高于感染组（0.53 ± 0.03，n P < 0.05）；α-SMA蛋白表达，感染+中砷剂量组、感染+低砷剂量组（0.78 ± 0.10、0.68 ± 0.06）高于空白组（0.40 ± 0.07）、感染组（0.48 ± 0.04， n P均 0.05), and the cell viability of other dose groups decreased ( n P < 0.05). There were significant differences in the expression levels of LC3, Beclin-1, α-SMA mRNA and protein in blank, infection, infection + high arsenic dose, infection + medium arsenic dose, and infection + low arsenic dose groups ( n F = 17.450, 11.084, 11.294, 11.745, 31.635, 12.130, n P < 0.05). In infection, infection + high arsenic dose, and infection + low arsenic dose groups, the levels of LC3 mRNA (20.09 ± 6.50, 36.57 ± 9.68, 14.19 ± 6.17) were higher than that of the blank group (1.25 ± 0.21, n P < 0.05), and the level of LC3 mRNA in infection + high arsenic dose group was higher than that of the infection group ( n P < 0.05); in infection, infection + high arsenic dose, infection + medium arsenic dose, and infection + low arsenic dose groups, the levels of Beclin-1 mRNA (22.46 ± 0.66, 13.38 ± 2.27, 20.80 ± 6.95, 24.31 ± 7.09) were higher than that of the blank group (1.10 ± 0.53, n P < 0.05); in infection + high arsenic dose, infection + medium arsenic dose, and infection + low arsenic dose groups, the levels of α-SMA mRNA (1.07 ± 0.27, 1.65 ± 0.17, 1.73 ± 0.26) were higher than that of the blank and infection groups (0.60 ± 0.11, 0.31 ± 0.09, n P < 0.05). In infection, infection + medium arsenic dose, and infection + low arsenic dose groups, the LC3 protein expressions (0.20 ± 0.06, 0.15 ± 0.00, 0.16 ± 0.01) were significantly increased compared to that of the blank group (0.04 ± 0.01, n P < 0.05); in infection + medium arsenic dose, and infection + low arsenic dose groups (0.83 ± 0.03, 1.20 ± 0.02), the Beclin-1 protein expressions were significantly increased compared to that of the blank group (0.25 ± 0.01, n P < 0.05), and the Beclin-1 protein expression in infection + low arsenic dose group was increased compared to that of the infection group (0.53 ± 0.03, n P < 0.05); in infection + medium arsenic dose, and infection + low arsenic dose groups (0.78 ± 0.10, 0.68 ± 0.06), the α-SMA protein expressions were significantly increased compared to that of the blank and infection groups (0.40 ± 0.07, 0.48 ± 0.04, n P < 0.05).n Conclusion:NaAsOn 2 may affect the process of arsenic-induced liver fibrosis by promoting the autophagy level of LX-2 cells.n