模拟微重力下传代培养对间充质干细胞生物学特性影响的转录组学研究

Transcriptomic study on the effects of subculture under simulated microgravity on the biological characteristics of mesenchymal stem cells

摘要

目的 基于转录组数据和不同功能典型标志物表达分析, 探究模拟微重力下传代培养对间充质干细胞(MSCs)干性、衰老、分化、增殖和凋亡等关键生物学特性的影响。方法 在2D回转器模拟的微重力环境中连续传代培养小鼠骨髓间充质干细胞, 分别提取模拟微重力组和对照组的第3代(P3)、第6代(P6)、第9代(P9)细胞的总RNA进行转录组测序和生物信息学分析, 并采用qRT-PCR验证MSCs关键功能标志物的表达。结果 与对照组相比, 模拟微重力培养的P3、P6、P9细胞差异表达基因显著富集在免疫应答、信号传导、衰老、凋亡、细胞周期等相关通路中。其中, P3、P6细胞增殖功能受抑制, P9代细胞增殖功能上调; P3、P6、P9细胞干性维持及成脂、成软骨、成神经相关分子表达上调, 衰老和凋亡相关分子也呈上调趋势(P<0.05)。结论 长时程模拟微重力能够重塑MSCs的生物学功能, 在提高干性、特定方向分化潜能和晚期增殖能力的同时激活衰老和凋亡过程, 为优化MSCs体外扩增体系提供了新视角。

Abstract

Objective To investigate the effects of prolonged passaging under simulated microgravity on critical biological properties of mesenchymal stem cells (MSCs), specifically their stemness, senescence, differentiation potential, proliferative capacity, and apoptotic regulation. Methods Mouse bone marrow-derived mesenchymal stem cells were continuously passaged in a simulated microgravity environment created by a 2D clinostat. Total RNA was extracted from cells at the 3rd (P3), 6th (P6), and 9th (P9) passages in both simulated microgravity group and control group for transcriptomic sequencing and bioinformatic analysis. The expression of key functional markers in MSCs was further validated using qRT-PCR. Results Compared with the control group, the differentially expressed genes of P3, P6, and P9 cells cultured under simulated microgravity were significantly enriched in pathways related to immune response, signal transduction, senescence and apoptosis, and cell cycle. Specifically, the proliferative capacity of P3 and P6 cells was suppressed, while that of P9 cells was upregulated. Moreover, the expression levels of molecules associated with stemness maintenance, adipogenic differentiation, chondrogenic differentiation, and neurogenic differentiation were elevated across P3, P6, and P9 cells, accompanied by the increased expression of senescence- and apoptosis-related molecules (P < 0.05). Conclusion Long-term simulated microgravity can reshape the biological functions of MSCs, enhancing stemness, specific differentiation potential, and late-stage proliferation ability while activating senescence and apoptosis processes, thereby offering a novel perspective for optimizing the in vitro MSCs expansion system.

关键词

间充质干细胞 / 模拟微重力 / 转录组学 / 干性维持 / 再生医学 / 衰老 / 凋亡 / 分化 / 增殖

Key words

mesenchymal stem cells / simulated microgravity / transcriptomics / stemness maintenance / regenerative medicine / aging / apoptosis / differentiation / proliferation

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基金资助

国家自然科学基金面上项目(32271289)

空军军医大学人才助推“砺剑行动”揭榜挂帅项目(2023RCJB05)