β-catenin过表达的间充质干细胞对改善海水吸入型肺损伤治疗效果的研究

β-catenin overexpression mesenchymal stem cells improve seawater inhalation induced acute lung injury

目的构建过表达β-catenin的慢病毒载体并建立大鼠MSCs稳转细胞株;研究过表达β-catenin的MSCs对损伤的肺泡上皮细胞的修复作用,以及对治疗海水吸入型肺损伤(seawater inhalation induced acute lung injury,SWI-ALI)大鼠模型的疗效。方法 MSCs转染:MSCs(2×105/孔)接种于六孔板过夜培养,慢病毒转染。转染慢病毒载体的MSCs表达稳定性和转染效率检测。雄性SD大鼠36只随机分为四组(每组9只),分别为空白对照组、SWI-ALI模型组、MSCs治疗组、MSCs-Ctnnb1治疗组。造模后4 h取各组肺组织标本进行苏木精-伊红染色(Hematoxylin and eosin staining,HE staining)行病理组织学检查评估肺损伤。NIR815染料标记对照组MSCs和过表达组MSCs-Ctnnb1干细胞,注入两组SWI-ALI大鼠模型气道,分别在干预后12、48 h行体外近红外光谱肺部成像对比干细胞定殖效果。最后进行数据统计分析。结果构建活化β-catenin的慢病毒稳转大鼠MSCs细胞株:培养至第10代,通过荧光显微镜观察检测转染有目的基因的MSCs表达感染效率仍大于90%。动物实验:病理损伤评估:海水吸入型肺损伤大鼠肺组织标本经苏木精-伊红染色镜下观察到肺泡壁增厚,肺泡间质炎性浸润,肺泡充血、水肿。这些组织病理学特征在MSCs治疗组和MSCs-Ctnnb1治疗组海水干预后4 h比SWI-ALI模型组明显缓解。MSCs-Ctnnb1治疗组比MSCs治疗组病理改善效果更好。MSCs在肺内定殖:MSCs治疗组和MSCs-Ctnnb1治疗组大鼠在海水干预后12 h和48 h肺组织进行体外近红外光谱成像追踪肺内MSCs,MSCs-Ctnnb1治疗组比MSCs治疗组海水干预后12 h和48 h荧光信号表达更强,定殖效果更好。结论通过β-catenin过表达激活经典Wnt/β-catenin通路提高间充质干细胞的增殖及向受损肺组织的迁移能力,增加MSCs在肺内的定殖、缓解病理损伤,并提高MSCs对SWI-ALI的治疗效果。

Objective To identify MSCs and buildβ-catenin overexpression MSCs lines,and confirm the effects ofβ-catenin overexpression on the repair of injured alveolar epithelia and its overall therapeutic effect in seawater inhalation induced-acute lung injury(SWI-ALI)in rats.Methods MSCs transfection:The EF1α-MCS-3FLAG-CMV-EGFP-T2A-Puromycin(over-expressing Ctnnb1)lentivector was constructed and packaged.MSCs transduced overexpression of the target genes,induced from the lentiviral vectors.The MSCs were cultured in normal culture medium for 10 passages after transduction to assay their long-term transfection efficiency.This was observed by fluorescence microscopy.The rats were randomly divided into four groups:the normal control,the seawater(SW)+PBS group,the SW+MSCs control group,and SW+MSCs-Ctnnb1 group.Samples were collected from each rat for hematoxylin and eosin staining.NIR815-labeled cells were directly instilled into the trachea of the SW+MSCs control rats and the SW+MSCs-Ctnnb1 rats.Ex vivo lungs from three subjects per group were imaged at two time points(12 h and 48 h post-instillation)using a Maestro in-vivo optical imaging system.Results Lentiviral vector transduction efficiency in MSCs:The effciency of the lentiviral vector transduction of MSCs-Ctnnb1(overexpression of Ctnnb1)after 10 passages in MSCs was detected via fluorescence microscopy.The analysis revealed transduction efficiencies above 90%.These results suggest that the lentivirus-mediated transduction was efficient and stable.Histopathological examination:Increased thickening of the alveolar wall,alveolar and interstitial inflammatory cell infiltration,hemorrhaging,alveolar exudates,and edema were observed in the lung tissue of rats after seawater-induced lung injury.However,these histopathological characteristics were alleviated at 4 h in the SW+MSCs control and SW+MSCs-Ctnnb1 groups compared to that of the SW+PBS group.The effect was higher in the SW+MSCs-Ctnnb1 group than in the SW+MSCs control group.Stem cells reservations:Color-coded fluorescence images indicated that the signals in the SW+MSCs-Ctnnb1 group were stronger compared to those in the SW+MSCs control group towards the end of 12 h and 48 h of exposure to seawater.Conclusions Activatedβ-catenin was used for improving stem cell proliferation,colonization,and differentiation in seawater inhalation induced acute lung injury via lentiviral vectors.It increased MSCs retention in the lung,further improved the lung epithelial permeability,thus contributing to an improved therapeutic effect of MSCs in ARDS.