过表达KyoT2对哮喘小鼠气道平滑肌细胞增殖和迁移的影响

Inhibitory effect of KyoT2 overexpression on proliferation and migration of airway smooth muscle cells in mice with asthma

目的研究KyoT2对哮喘小鼠气道平滑肌细胞(ASMC)增殖和迁移的影响。方法卵白蛋白(OVA)诱导BALB/c小鼠建立哮喘气道重塑模型后,行ASMC分离和培养,并以原代培养的正常小鼠ASMC作为对照组。检测对照组和哮喘组小鼠ASMC中Kyo T2的表达。哮喘小鼠ASMC转染p CMV-Myc(空载体组)和过表达KyoT2质粒p CMV-Myc-KyoT2(KyoT2过表达组)48h后,采用RT-PCR和Westernblot方法检测各组KyoT2m RNA和蛋白表达;采用MTT法和Brd U法检测ASMC增殖;采用Transwell法检测ASMC的迁移。Westernblot用于检测过表达KyoT2对RBP-Jκ、PTEN和AKT蛋白表达水平的影响。结果与对照组比较,哮喘组ASMC中KyoT2表达下调,KyoT2过表达组ASMC中KyoT2表达显著上调(P<0.05)。与空载体组比较,KyoT2过表达抑制细胞增殖和细胞迁移(P<0.05);且KyoT2过表达能下调RBP-Jκ和AKT的表达,上调PTEN的表达(P<0.05)。结论 KyoT2抑制哮喘ASMC增殖和迁移,其机制可能是通过负调控RBP-Jκ/PTEN/AKT信号通路来实现。

ObjectiveTo investigate the effect of KyoT2 on the proliferation and migration of airway smooth muscle cells （ASMCs） in mice with asthma.MethodsOvalbumin （OVA） was used to establish the asthmatic model of airway remodeling in BALB/c mice. ASMCs were isolated and cultured, and primarily cultured ASMCs were used as the control group. The expression of KyoT2 in ASMCs was measured in the control and asthma groups. After the ASMCs from asthmatic mice were transfected with pCMV-Myc （empty vector group） or pCMV-Myc-KyoT2 plasmid with overexpressed KyoT2 （KyoT2 expression group） for 48 hours, RT-PCR and Western blot were used to measure the mRNA and protein expression of KyoT2, the MTT assay and BrdU assay were used to measure the proliferation of ASMCs, and Transwell assay was used to measure the migration of ASMCs. Western blot was used to determine the effect of KyoT2 overexpression on the protein expression of RBP-Jκ, PTEN, and AKT.ResultsCompared with the control group, the asthma group had signiifcantly downregulated expression of KyoT2 in ASMCs, and the KyoT2 expression group had signiifcantly upregulated expression of KyoT2 in ASMCs （P〈0.05）. Compared with the empty vector group, overexpressed KyoT2 signiifcantly inhibited cell proliferation and migration, downregulated the expression of RBP-Jκ and AKT, and upregulated the expression of PTEN.ConclusionsOverexpressed KyoT2 can inhibit the proliferation and migration of ASMCs through the negative regulation of RBP-Jκ/PTEN/AKT signaling pathway.