大鼠脑源性间充质干细胞分离培养方法的建立及其生物学特性研究OA

AIM:To establish a protocol for the in vitro isolation and culture of primary rat brain-derived mes-enchymal stem cells(Br-MSCs)and to investigate their biological characteristics.METHODS:Six 7-day-old Sprague-Dawley rats were randomly assigned to three groups(two rats per group)for three independent primary cultures of Br-MSCs.Under aseptic conditions,cerebral tissue was dissected,rinsed and minced,followed by sieving to obtain larger-di-ameter vascular segments with an adventitial coat.After digestion with type II collagenase,the segments were seeded for culture.Upon reaching confluence,primary cells underwent serial passaging,cryopreservation,and subsequent thawing for expansion.Passage-5(P5)cells were analyzed by flow cytometry for surface CD-marker expression and induced toward adipogenic,osteogenic and chondrogenic lineages to verify their trilineage differentiation potential.Cells at P5,P10 and P15 were assessed for senescence using β-galactosidase staining and for migratory capacity via scratch-wound assay.RE-SULTS:Within 48 h post-inoculation,short spindle-shaped cells migrated from the margins of adherent tissue and formed clusters that expanded and coalesced by 72 h.A confluent monolayer with a characteristic swirling,tightly packed mor-phology was achieved by 96 h.The cells maintained stable expansion in vitro up to at least P15 post-thawing.Flow cyto-metric analysis revealed that P5 cells exhibited high expression of MSC surface markers CD90,CD73,and CD44,and low expression of hematopoietic stem cell surface markers CD45,CD34,and CD11b/c.In differentiation assays,P5 cells demonstrated trilineage potential by generating lipid-laden droplets,calcified nodules,and cartilaginous micromasses un-der adipogenic,osteogenic,and chondrogenic conditions,respectively.β-Galactosidase staining and scratch wound assay revealed that P5 cells exhibited a low proportion of senescent cells and robust migratory capacity.In contrast,P10 and P15 cells showed a significantly increased senescence ratio(P<0.01)and markedly reduced migratory ability(P<0.01).CONCLUSION:This study successfully isolated and cultured rat Br-MSCs in vitro,providing a practical animal-derived stem cell model for preclinical studies in central nervous system regenerative medicine.