MIRO1修饰的人脐带间充质干细胞通过线粒体自噬修复肺上皮细胞损伤的实验研究

刘雯丽; 赵玲萍; 孙亚楠; 刘金霞; 徐梦; 习佳飞; 梁志欣

doi:10.12435/j.issn.2095-5227.2024.078

MIRO1修饰的人脐带间充质干细胞通过线粒体自噬修复肺上皮细胞损伤的实验研究

MIRO1-modified MSC hUCMSCs in repairing lung epithelial cell injury through mitochondrial autophagy

摘要

摘要:
背景间充质干细胞(mesenchymal stem cells，MSCs)为急性肺损伤(acute lung injury，ALI)的治疗修复提供了新的策略，目前单独的MSCs治疗效果欠佳，需要探索增强 MSCs 疗效的方法。
目的探讨过表达MIRO1(mitochondrial Rho GTPase 1，Rhot1)的人脐带间充质干细胞(human umbilical cord mesenchymal stem cells，hUCMSCs)对脂多糖诱导的人肺支气管上皮细胞(bronchial epithelium transformed with Ad12-SV40 2B，BEAS-2B)损伤的治疗作用。
方法包装过表达MIRO1的慢病毒，通过慢病毒感染构建过表达MIRO1的hUCMSCs稳转细胞株；实验细胞随机分为4组：对照组(Control)、脂多糖组(LPS)、脂多糖 + 人脐带间充质干细胞组(Con-hUCMSCs)、脂多糖 + 过表达MRIO1的人脐带间充质干细胞组(MIRO1-hUCMSCs)。脂多糖刺激BEAS-2B细胞24 h，通过Transwell小室分别与Con-hUCMSCs和MIRO1-hUCMSCs细胞共培养24 h，收集BEAS-2B细胞并使用Western blot和RT-qPCR实验方法检测肿瘤坏死因子α(tumor necrosis factor-α，TNF-α)、白细胞介素1β(interleukin，IL-1β)、IL-6和线粒体自噬相关蛋白(PINK1、PARKIN、MIRO1、SQSTM1)表达水平。
结果成功筛选获得过表达MIRO1的hUCMSCs稳转细胞株。通过Transwell小室共培养，MIRO1-hUCMSCs组可抑制脂多糖组炎症因子TNF-α、IL-1β和IL-6的升高(P＜0.05)，恢复线粒体自噬蛋白PINK1、SQSTM1、MIRO1的低表达(P＜0.05)，降低PARKIN蛋白高表达(P＜0.05)。MIRO1-hUCMSCs组相比LPS组、Con-hUCMSCs组治疗有统计学差异(P＜0.05)。
结论 MIRO1修饰的hUCMSCs与单纯hUCMSCs相比，可通过调节线粒体自噬PINK1-Parkin通路中的PINK1、PARKIN、MIRO1和SQSTM1蛋白改善脂多糖引起的BEAS-2B细胞损伤。

Abstract:
Background Mesenchymal stem cells (MSCs) provide a new strategy for the treatment and repair of acute lung injury (ALI). Currently, MSCs alone have poor therapeutic effect, and methods to enhance the efficacy of MSCs need to be explored.
Objective To investigate the therapeutic effect of mitochondrial Rho GTPase 1(Rhot1) overexpression of human umbilical cord mesenchymal stem cells (hUCMSCs) on lipopolysaccharide-induced injury of human lung bronchial epithelial cells (BEAS-2B).
Methods Lentivirus overexpressing MIRO1 was packaged and hUCMSCs overexpressing MIRO1 were constructed by lentivirus infection. The experimental cells were randomly divided into control group, lipopolysaccharide group (LPS), lipopolysaccharide + human umbilical cord mesenchymal stem cells (Con-hUCMSCs) group and lipopolysaccharide + human umbilical cord mesenchymal stem cells overexpressing MRIO1 (MIRO1-hUCMSCs) group. Lipopolysaccharide stimulated BEAS-2B cells for 24h and co-cultured with Con-hUCMSCs and MIRO1-hUCMSCs cells through Transwell chamber for 24h. The BEAS-2B cells were collected, and tumor necrosis factor-α (TNF-α) and interleukin (1β) were detected by Western blot and RT-qPCR. The expression levels of IL-1β, interleukin (IL-6), and mitochondrial autophagy associated proteins (PINK1, PARKIN, MIRO1, SQSTM1) were leukin.
Results hUCMSCs stable cell lines overexpressing MIRO1 were successfully screened. Through Transwell cell co-culture, MIRO1-hUCMSCs group inhibited the increase of inflammatory factors TNF-α, IL-1β and IL-6 in LPS group (P<0.05), restored the low expression of mitochondrial autophagy proteins PINK1, SQSTM1 and MIRO1 (P<0.05), decreased the high expression of PARKIN protein (P<0.05). The effect in MIRO1-hUCMSCs group showed statistical significance compared with LPS group and Con-hUCMSCs group (P<0.05).
Conclusion Compared with hUCMSCs alone, MiRO1-modified hUCMSCs can ameliorate lipopolysaccharids-induced BEAS-2B cell damage by regulating PINK1, PARKIN, MIRO1 and SQSTM1 proteins in mitochondrial autophagy Pink1-Parkin pathway.

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