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

摘要:

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KyoT2的表达。哮喘小鼠ASMC转染pCMV-Myc （空载体组）和过表达KyoT2质粒pCMV-Myc-KyoT2 （KyoT2过表达组）48 h后，采用RT-PCR和Western blot方法检测各组KyoT2 mRNA和蛋白表达；采用MTT法和BrdU法检测ASMC增殖；采用Transwell法检测ASMC的迁移。Western blot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。

Abstract:

Objective To investigate the effect of KyoT2 on the proliferation and migration of airway smooth muscle cells (ASMCs) in mice with asthma. Methods Ovalbumin (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. Results Compared with the control group, the asthma group had significantly downregulated expression of KyoT2 in ASMCs, and the KyoT2 expression group had significantly upregulated expression of KyoT2 in ASMCs (P < 0.05). Compared with the empty vector group, overexpressed KyoT2 significantly inhibited cell proliferation and migration, downregulated the expression of RBP-Jκ and AKT, and upregulated the expression of PTEN. Conclusions Overexpressed KyoT2 can inhibit the proliferation and migration of ASMCs through the negative regulation of RBP-Jκ/PTEN/AKT signaling pathway.