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目的 针对传统方法分离大鼠胸主动脉中膜间充质干细胞（MSCs）时存在效率与纯度难以兼顾的局限，旨在建立并验证一种优化的双相收获法分离培养体系。方法 采用3～4周SD大鼠，无菌分离胸主动脉中膜并剪碎。实验设立3组，单纯酶消化法、预贴壁-酶消化序贯法及双相收获法组。系统比较3组在细胞首次迁出时间、原代融合时间、细胞获得率等生长动力学指标的差异。取第4代细胞，通过流式细胞术检测表面标志物（CD73、CD44、CD29、CD45、CD34、CD11b/c），并进行成骨、成脂及成软骨三系诱导分化实验以评估其干性。结果 双相收获法组原代融合时间（8.5±1.0）d短于单纯酶消化组（11.2±1.2）d和预贴壁-酶消化序贯组（12.8±1.5）d。流式鉴定显示，所获细胞稳定高表达CD73(87.2%±1.5%)、CD44(87.1%±0.8%)及CD29(96.8%±1.3%)，低表达CD45(3.22%±0.20%)、CD34(2.46%±0.5%)及CD11b/c(2.53%±2.1%)。经定向诱导，细胞可形成茜素红阳性钙结节、油红O阳性脂滴及阿利新蓝阳性糖胺聚糖，证实具备完整的三系分化潜能。细胞可稳定扩增至第8代，形态保持均一长梭形。结论 双相收获法是一种高效、稳定的大鼠胸主动脉中膜MSCs分离优化方案。该法通过时序性双步设计协同提升了细胞产量、纯度与干性，可为血管生物学与再生医学研究提供了优质的原位细胞来源。
Abstract：Objective To address the challenge of balancing efficiency and purity in isolating MSCs from rat tho‑racic aorta using traditional methods and to establish and validate an optimized isolation and culture system based on the dual-phase harvesting method.Methods Thoracic aortas were aseptically isolated from 3 to 4-week-old SD rats and minced. Three experimental groups were included: simple enzymatic digestion, pre-explant-enzymatic di‑gestion sequential method, and the dual-phase harvesting method. Growth kinetic parameters, including time to initial cell migration, time to primary confluence, and cell yield, were systematically compared among the three groups. Passage‑4 cells were examined for surface marker expression(CD73, CD44, CD29, CD45, CD34, and CD11b/c) by flow cytometry. Their stemness was evaluated through osteogenic, adipogenic, and chondrogenic tri‑lineage differentiation assays.Results The time to primary confluence in the dual-phase harvesting method group(8. 5 ± 1. 0) d was significantly shorter than that of the simple enzymatic digestion group(11. 2±1. 2) d and the pre‑explant enzymatic digestion sequential group(12. 8±1. 5) d. Flow cytometric analysis revealed that the har‑vested cells consistently showed high expression of CD73(87. 2±1. 5)%, CD44(87. 1±0. 8)%, and CD29(96. 8±1. 3)%, with low expression of CD45(3. 22±0. 2)%, CD34(2. 46±0. 5)%, and CD11b/c(2. 53±2. 1)%. Upon directed induction, the cells formed Alizarin Red‑positive calcium nodules, Oil Red O‑positive lipid droplets, and Alcian Blue-positive glycosaminoglycans, confirming complete trilineage differentiation potential. The cells could be stably expanded to passage 8 while maintaining a uniform elongated spindle-shaped morphology.Conclusion The dual‑phase harvesting method established in this study is an efficient and stable optimized protocol for isolating MSCs from the rat thoracic aorta media. This approach synergistically enhances cell yield, purity, and stemness through its temporally designed two‑step process, thereby providing a high-quality in situ cell source for vascular bi‑ology research and regenerative medicine.
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基本信息:
DOI：10.19405/j.cnki.issn1000-1492.2026.09.015
中图分类号:R318
引用信息:
[1]赖素玉,吴晶,池茗,等.基于双相收获法优化大鼠胸主动脉中膜来源间充质干细胞的分离培养体系[J].安徽医科大学学报,2026(09):1626-1633.DOI:10.19405/j.cnki.issn1000-1492.2026.09.015.
基金信息:
中国中医科学院科技创新工程重大攻关项目(No.CI2021A02109); 中央高水平中医医院临床研究和成果转化能力提升项目(No.HLCMHPP2023126)
2026-08-11
2026-08-11
2026-08-11