摘要
目的探究配对盒基因6(Pax6)对血小板衍生因子(PDGF-BB)诱导下人肝星状细胞迁移能力的影响。方法体外培养人肝星状细胞系(LX-2),慢病毒感染LX-2敲减Pax6,使用PDGF-BB诱导其活化,实验分为4组:空白组(shNC+PBS)、实验组(shPax6+PBS)、PDGF-BB处理的空白组(shNC+PDGF-BB)、PDGF-BB处理的实验组(shPax6+PDGF-BB);采用划痕实验、Transwell小室细胞迁移实验,分析细胞迁移能力;细胞免疫荧光及蛋白质印迹技术检测细胞迁移相关蛋白的表达水平。结果PDGF-BB诱导下的LX-2活化,可促进其迁移能力增强;敲降Pax6后,能够抑制活化后细胞增强的迁移能力;蛋白质印迹技术实验结果表明,PDGF-BB刺激下能明显上调LX-2中N-cad表达、抑制E-cad表达;敲降Pax6后能抑制PDGF-BB诱导的N-cad表达、增加E-cad表达;细胞免疫荧光实验结果显示,PDGF-BB诱导下能明显上调Vimentin表达,敲降Pax6后,PDGF-BB对Vimentin的上调作用被抑制,E-cad与N-cad趋势同上述蛋白质印迹技术检测结果一致。结论敲降Pax6能抑制PDGF-BB诱导下LX-2增强的迁移能力,为临床抗肝纤维化药物开发提供了可能靶点,为治疗肝纤维化提供了新的策略。
Objective To explore the effect of paired box gene 6(pax6)on the migration of human hepatic stellate cells(HSCs)induced by platelet derived growth factor BB(PDGF-BB).Methods HSC line(LX-2)was cultured in vitro.LX-2 cells were infected with lentivirus to knock down Pax6.And PDGF-BB was added to stimulate the activation.The cells were divided into four groups:blank group[shNC+phosphate buffer saline(PBS)],experimental group(shPax6+PBS),blank group treated with PDGF-BB(shNC+PDGF-BB),and experimental group treated with PDGF-BB(shPax6+PDGF-BB).Scratch assay and Transwell assay were used to analyze cells migration ability.Cell immunofluorescence and Western blot(WB)were used to detect the expression levels of migration related proteins.Results PDGF-BB-induced activation of LX-2 cells promoted the enhancement of their migration ability.Knockdown of Pax6 inhibited the enhanced migration ability of activated cells.WB results show that PDGF-BB stimulation significantly up-regulated the expression of N-cadherin(N-cad)and inhibited the expression of E-cadherin(E-cad)in LX-2 cells.Knockdown of Pax6 inhibited the expression of N-cad and increased the expression of E-cad induced by PDGF-BB.The results of cell immunofluorescence experiments showed that the expression of Vimentin was significantly up-regulated under the induction of PDGF-BB.After knocking down Pax6,the up-regulation effect of PDGF-BB on Vimentin was inhibited.The trend of E-cad and N-cad was consistent with the above WB detection results.Conclusion Knockdown of Pax6 can inhibit the enhanced migration ability of LX-2 cells induced by PDGF-BB,which provides a possible target for the development of clinical anti-hepatic fibrosis drugs,and provides choice and application of new strategies for the treatment of liver fibrosis.
作者
张迪
谭悦浩
刘漪沦
李灿
刘蓉
胡雪
唐斌
邓峰美
Zhang Di;Tan Yuehao;Liu Yilun;Li Can;Liu Rong;Hu Xue;Tang Bin;Deng Fengmei(School of Basic Medical Sciences,Chengdu Medical College,Chengdu 610500,China;The First Affiliated Hospital of Chengdu Medical College,Chengdu 610500,China;Ya′an Mingshan District People′s Hospital,Ya′an 625000,China)
出处
《成都医学院学报》
CAS
2022年第4期421-425,共5页
Journal of Chengdu Medical College
基金
四川省中医药管理局基金项目(No:2021MS508)
雅安市科技局项目(No:2020yyjskf05)
四川养老与老年健康协同创新中心项目(No:YLZBZ2010)。
