3D成团培养通过诱导内质网应激介导的钙信号调控髓核干细胞进入可逆性静息状态OA

Objective Intervertebral disc degeneration is closely associated with the functional changes in nucleus pulposus stem cells(NPSCs),yet traditional planar culture fails to simulate their genuine 3D microenvironment.This study aims to investigate the endoplasmic reticulum stress(ERS)response of NPSCs under 3D spheroid culture conditions,as well as its effects on cell proliferation and cell cycle changes.Methods NPSCs were isolated from 36 male SD rats(aged 8 weeks,weighing 350±50 g),and then a 3D spheroid culture model was constructed.The ERS,stress signals,cell cycle,and proliferation status of NPSCs under different culture conditions(3D spheroid culture vs planar culture)were compared.The morphological changes in endoplasmic reticulum were observed by electron microscopy.Cell viability and apoptosis rates were assessed using live/dead cell staining and Annexin V-FITC/PI flow cytometry.The expression of stress markers such as GRP78,ATF6,and PERK was detected by qPCR and Western blotting.Cell proliferation was analyzed by EdU and Ki67 flow cytometry.Calcium ion fluorescent probe plasmids were transfected to observe the changes in intracellular calcium dynamics.Finally,the endoplasmic reticulum function inhibitor 4-PBA was used to verify the role of ERS in cell cycle regulation.Results ① Light microscopy showed that the cells formed compact spheroids in the 3D culture dishes.Live/dead cell staining demonstrated favorable cytocompatibility of 3D spheroid culture,with no statistical difference compared with the planar culture group(P>0.05).The apoptosis rate remained at an extremely low level of 1.6%,and no significant difference was observed between groups(P>0.05).② Compared with planar culture,NPSCs under 3D spheroid culture exhibited obvious expansion of the endoplasmic reticulum(observed by electron microscopy)and upregulation of GRP78,ATF6,and PERK(P<0.001),suggesting ERS activation.③ Cell cycle analysis revealed a significant decrease in proliferation,upregulation of P21 expression(P<0.001)and downregulation of PCNA,CDK4 and CDK5 expression(P<0.001)in the spheroid culture group.EdU and flow cytometry demonstrated that spheroid cells showed gradually declined proliferative capacity over time,indicating cell cycle arrest.④ Confocal microscopy showed that the intracellular calcium concentration was increased under 3D spheroid culture(P<0.01),suggesting that ERS may regulate the cell cycle through calcium signaling.⑤ 4-PBA treatment partially restored cell proliferation and decreased the expression of P21 and P27(P<0.001),further demonstrating the role of ERS in cell cycle inhibition.Conclusion 3D structural microenvironment can induce the imbalance of endoplasmic reticulum homeostasis in NPSCs.ERS induced by 3D spheroid culture drives NPSCs into a reversible low-proliferation,adaptive quiescent state.