间充质干细胞通过重编程胸腺上皮细胞的DNA甲基化来逆转胸腺衰老。

Mesenchymal stem cells reverse thymus aging by reprogramming the DNA methylation of thymic epithelial cells.

作者信息

Yang Zailing, Tian Chuan, He Zhixu, Zhu Xiangqing, He Jie, Pan Hang, Li Ye, Ruan Guangping, Wu XiJun, Pan Xinghua

机构信息

The Basic Medical Laboratory of the 920th Hospital of Joint Logistics Support Force of PLA, The Transfer Medicine Key Laboratory of Cell Therapy Technology of Yunan Province, The Integrated Engineering Laboratory of Cell Biological Medicine of State and Regions, Kunming 650032, Yunnan Province, China.

The Second Peoples Hospital of Guiyang, Medical Laboratory, Guiyang 550023, Guizhou Province, China.

出版信息

Regen Ther. 2024 Mar 26;27:126-169. doi: 10.1016/j.reth.2024.03.008. eCollection 2024 Dec.

BACKGROUND

A decrease in the number and activity of thymic epithelial cells (TECs) is an important factor in thymic degeneration. Mesenchymal stem cells (MSCs) treating thymic ageing is a promising strategy, but the DNA methylation modification mechanism in TECs remains unclear.

METHODS

Aged rhesus monkeys were treated with MSCs to establish a thymic senescence model, and hematoxylin-eosin (HE) staining, immunofluorescence staining, and ELISA were performed to observe the structure and function of the thymus. TEC aging model and MSCs co-culture system were established to detect DNA methylation modification and transcriptomic changes, correlation analysis between transcription factor methylation and mRNA expression, and q-PCR, immunofluorescence staining, and Western blot were used to identified key genes.

RESULTS

MSCs improved the structure and function of thymus in elderly macaque monkeys; reduced the expression levels of β-Gal, P16, and P21; and increased the activity of aging TECs. There were 501 genes with increased methylation in the promoter region in the treated group compared with the untreated group, among which 23 genes were involved in the negative regulation of cell growth, proliferation and apoptosis, while 591 genes had decreased methylation, among which 37 genes were associated with promoting cell growth and proliferation and inhibiting apoptosis. Furthermore, 66 genes showed a negative correlation between promoter methylation levels and gene transcription; specifically, PDE5A, DUOX2, LAMP1 and SVIL were downregulated with increased methylation, inhibiting growth and development, while POLR3G, PGF, CHTF18, KRT17, FOXJ1, NGF, DYRK3, LRP8, CDT1, PRELID1, F2R, KNTC1 and TRIM3 were upregulated with decreased methylation, promoting cell growth.

CONCLUSION

MSCs improve the structure and function of aged thymus, which involves the regulation of DNA methylation profiles and a decrease in the methylation level of the transcription factor NGF to specifically upregulate KRT17 and FOXJ1 to promote the proliferation of TECs.

背景

胸腺上皮细胞（TECs）数量和活性的减少是胸腺退化的一个重要因素。间充质干细胞（MSCs）治疗胸腺衰老不失为一种很有前景的策略，但TECs中的DNA甲基化修饰机制仍不清楚。

方法

用MSCs处理老年恒河猴以建立胸腺衰老模型，采用苏木精-伊红（HE）染色、免疫荧光染色和酶联免疫吸附测定（ELISA）来观察胸腺的结构和功能。建立TEC衰老模型与MSCs共培养体系，以检测DNA甲基化修饰和转录组变化、转录因子甲基化与mRNA表达之间的相关性分析，并运用定量聚合酶链反应（q-PCR）、免疫荧光染色和蛋白质免疫印迹法来鉴定关键基因。

结果

MSCs改善了老年猕猴胸腺的结构和功能；降低了β-半乳糖苷酶（β-Gal）、P16和P21的表达水平；并提高了衰老TECs的活性。与未处理组相比，处理组启动子区域甲基化增加的基因有501个，其中23个基因参与细胞生长、增殖和凋亡的负调控，而甲基化减少的基因有591个，其中37个基因与促进细胞生长和增殖以及抑制凋亡相关。此外，66个基因的启动子甲基化水平与基因转录呈负相关；具体而言，磷酸二酯酶5A（PDE5A）、双氧化酶2（DUOX2）、溶酶体相关膜蛋白1（LAMP1）和 supervillin（SVIL）随着甲基化增加而下调，抑制生长发育，而聚合酶Ⅲ亚基G（POLR3G）、胎盘生长因子（PGF）、染色体传递保真因子18（CHTF18）、角蛋白17（KRT17）、叉头框蛋白J1（FOXJ1）、神经生长因子（NGF）、双重特异性酪氨酸磷酸化调节激酶3（DYRK3）、低密度脂蛋白受体相关蛋白8（LRP8）、细胞分裂周期蛋白1（CDT1）、线粒体内膜蛋白1（PRELID1）、凝血因子Ⅱ受体（F2R）、核仁磷酸蛋白1（KNTC1）和三聚体结构域蛋白3（TRIM3）随着甲基化减少而上调，促进细胞生长。