【摘要】 目的 观察奥沙利铂对肝星状细胞（HSC）活化的影响，进一步探讨奥沙利铂与micro RNA-30a-5p及自噬的关系。方法 培养HSC-LX2并做如下分组：（1）对照组、PDGF处理组、奥沙利铂处理组、奥沙利铂+PDGF处理组；（2）对照组、micro RNA-30a-5p转染组，PDGF处理组、micro RNA-30a-5p转染+PDGF处理组；（3）对照组、3-MA组、micro RNA-30a-5p抑制剂组、micro RNA-30a-5p抑制剂+3-MA组。应用免疫蛋白印迹技术（WesternBlot）分析HSC活化相关蛋白Ⅰ型胶原（Collagen-Ⅰ）、抗α-平滑肌肌动蛋白（α-SMA）及HSC自噬相关蛋白Beclin 1、p62、LC3B的表达；溶酶体示踪剂和免疫荧光检测LC3B自噬小体的表达。实时荧光定量PCR(RT-PCR)检测microRNA-30a-5p表达水平。应用生物信息学技术预测microRNA-30a-5p在HSC中的潜在靶点。正态分布的计量资料两组间比较采用成组t检验；多组间比较采用单因素方差分析，进一步两两比较采用LSD-t检验。结果 应用奥沙利铂处理细胞时，RT-PCR结果显示奥沙利铂处理组micro RNA-30a-5p的表达水平高于对照组（P<0.01）;Western Blot结果显示奥沙利铂处理组的HSC活化相关蛋白α-SMA、Collagen-Ⅰ及自噬相关蛋白Beclin 1、LC3BⅡ/Ⅰ水平均降低（P值均<0.001）；免疫荧光结果显示奥沙利铂处理组自噬小体低于对照组（P<0.05）。进一步应用microRNA-30a-5p模拟类似物（mimic）转染HSC-LX2结果显示，与对照组相比，microRNA-30a-5p mimic组自噬相关蛋白Beclin 1和LC3BⅡ/Ⅰ的表达水平均降低（P值均<0.05）;HSC活化相关蛋白Collagen-Ⅰ的表达水平亦降低（P<0.001）；应用microRNA-30a-5p抑制剂转染HSC-LX2,Western Blot结果显示，与对照组比较，microRNA-30a-5p抑制剂组HSC活化相关蛋白Collagen-Ⅰ、α-SMA，自噬相关蛋白Beclin 1的表达水平均升高（t值分别为2.41、2.32、4.57,P值均<0.05）。Western Blot结果显示，与对照组相比，micro RNA-30a-5p抑制剂组HSC自噬相关蛋白Beclin 1及活化相关蛋白α-SMA表达水平均升高（P值均<0.05），应用自噬抑制剂3-MA处理后，两组间上述蛋白表达无显著差异（P>0.05）。通过TargetScan、PicTar和miRanda生物信息软件分析表明，自噬相关蛋白Beclin 1是microRNA-30a-5p的潜在靶标。结论 奥沙利铂可通过上调microRNA-30a-5p表达从而抑制HSC激活，为肝纤维化的治疗提供新的思路和作用靶点。更多还原

【Abstract】 Objective To investigate the effect of oxaliplatin on the activation of hepatic stellate cells (HSCs),as well as the association of oxaliplatin with microRNA-30a-5p and autophagy.Methods HSC-LX2 cells were cultured and divided into groups according to the following three protocols:control group,PDGF treatment group,oxaliplatin treatment group,oxaliplatin+PDGF treatment group;control group,microRNA-30a-5p transfection group,PDGF treatment group,microRNA-30a-5p transfection+PDGF treatment group;control group,3-MA group,microRNA-30a-5p inhibitor group,microRNA-30a-5p inhibitor+3-MA group.Western Blot was used to measure the expression of HSC activation-related proteins (Collagen-I and alpha-smooth muscle actin[α-SMA]) and HSC autophagy-related proteins (Beclin-1,P62,and LC3B);LysoTracker staining and immunofluorescence assay were used to measure the expression of LC3B autophagosomes;RT-PCR was used to measure the expression level of microRNA-30a-5p;bioinformatics techniques were used to predict the potential targets of microRNA-30a-5p in HSCs.The independentsamples t test was used for comparison of normally distributed continuous data between two groups;a one-way analysis of variance was used for comparison between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results After the cells were treated with oxaliplatin,RT-PCR results showed that the oxaliplatin treatment group had a significantly higher expression level of microRNA-30a-5p than the control group (P<0.01);Western Blot showed that the oxaliplatin treatment group had significant reductions in the expression levels of the HSC activation-related proteins α-SMA and Collagen-Ⅰand the autophagy-related proteins Beclin 1 and LC3BⅡ/Ⅰ（all P<0.001);immunofluorescence assay showed that the oxaliplatin treatment group had a significantly lower number of autophagosomes than the control group (P<0.05).After HSC-LX2 cells were transfected with microRNA-30a-5p mimic,compared with the control group,the microRNA-30a-5p mimic group had significant reductions in the expression levels of the autophagy-related proteins Beclin 1 and LC3BⅡ/Ⅰ（P<0.05) and the HSC activation-related protein Collagen-Ⅰ（P<0.001);after HSC-LX2 cells were transfected with microRNA-30a-5p inhibitor,Western Blot showed that compared with the control group,the microRNA-30a-5p inhibitor group had significant increases in the expression levels of the HSC activation-related proteins Collagen-Ⅰand α-SMA and the autophagy-related protein Beclin 1 (t=2.41,2.32,and 4.57,all P<0.05).Western Blot showed that compared with the control group,the microRNA-30a-5p inhibitor group had significant increases in the expression levels of the HSC autophagy-related protein Beclin 1 and the HSC activation-related proteinα-SMA (both P<0.05),and after the treatment with the autophagy inhibitor 3-MA,there were no significant differences in the expression of these proteins between the two groups (P>0.05).The bioinformatics analysis using TargetScan,PicTar,and miRanda databases showed that the autophagy-related protein Beclin-1 might be a potential target of miRNA-30a-5p.Conclusion Oxaliplatin can inhibit the activation of HSCs by upregulating the expression of microRNA-30a-5p,which provides new ideas and a new target for the treatment of liver fibrosis.更多还原