ObjectiveTo address the challenge of balancing efficiency and purity in isolating MSCs from rat thoracic aorta using traditional methods and to establish and validate an optimized isolation and culture system based on the dual-phase harvesting method. MethodsThoracic 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 digestion 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 trilineage differentiation assays. ResultsThe 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 harvested 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. ConclusionThe 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 biology research and regenerative medicine.

Chiropractic therapy is a therapeutic approach for regulating the spine and is widely employed in clinical practice for multiple disorders, including constipation. The theory of "bone strengthening and tendon softening" originated from Huang Di Nei Jing, holding that bones maintain normal anatomical forms and relative positional relationships, and tendons are supple and free from damage. Only when the structures and functions of both are normal can the human body sustain normal physiological activities. In accordance with this theory, clinical treatment mainly aims at adjusting the relationship between tendons and bones and restoring the balance between them to address diseases. Based on the theory of "bone strengthening and tendon softening" and taking the essence of "tendons" as the starting point, this article explored the pathogenic mechanism of outlet obstructive constipation and the clinical application of chiropractic therapy in the treatment of outlet obstructive constipation. Pathological changes of the musculotendinous meridians and abnormal spinal structures are significant pathogenic factors for outlet obstructive constipation. Therefore, in clinical practice, spinal techniques such as guided manipulation, massage, and reconstructive techniques can be used to soften muscles, correct bones, and regulate intestinal stagnation.