目的·建立一种稳定、高效的技术方法，用于分离并获得耳蜗血管纹中的周细胞，同时尽可能保留周细胞的原始理化性质。方法·取6日龄野生型C57BL/6J小鼠，于显微镜下解剖获取血管纹组织，改变酶的种类、酶解时间、机械解离时间等实验条件，并使用流式细胞技术比较酶解后的单细胞占比以及活细胞占比，以求达到酶解效果最佳、对细胞损伤最小的细胞解离方案。获得的单细胞悬液使用CD140b［即血小板衍生生长因子受体β（platelet derived growth factor receptor-β，PDGFR-β）］抗体和CD31抗体孵育，经流式细胞分选得到CD140b⁺CD31^-和CD140b^-细胞，利用实时荧光定量聚合酶链反应（RT-qPCR）检测分选所得细胞的分子标志物，验证周细胞标志物［Pdgfrb、结蛋白（desmin，Desm）］的表达情况，并评估内皮细胞［标志物为血管性血友病因子（von Willebrand factor，Vwf）、葡萄糖转运蛋白1（glucose transporter 1，Glut1）］及黑色素样巨噬细胞［标志物为谷胱甘肽S-转移酶α4（glutathione S-transferase α4，Gsta4）、黏附G蛋白偶联受体E1（adhesion G protein-coupled receptor E1，Adgre1）］的污染情况，从而验证所得细胞类型和分离方法的有效性。结果·通过流式细胞分析发现：木瓜蛋白酶酶解血管纹组织对细胞损伤较大，细胞碎片率高；低温活性蛋白酶对血管纹组织分离效率不高；嗜热菌蛋白酶和细胞消化液Accutase联合使用酶解血管纹组织的酶解效果不稳定，且细胞碎片率也较高；而使用Accutase分离血管纹组织，可以较稳定地获得数量相对较多、细胞存活率相对高的单细胞悬液，故最终选用此酶解方法。通过Accutase酶解6日龄小鼠血管纹组织联合CD140b及CD31抗体分选，成功获得高纯度周细胞。RT-qPCR显示，CD140b⁺CD31^-细胞特异性高表达周细胞标志物Pdgfrb、Desm，低表达内皮细胞标志物Vwf、Glut1，以及黑色素样巨噬细胞标志物Gsta4、Adgre1。结论·成功建立一种基于流式细胞术的稳定且有效分离血管纹周细胞的方法。

Objective ·To develop a stable and efficient method for isolating and obtaining pericytes from the cochlear stria vascularis while preserving their original physicochemical properties as much as possible. Methods ·Stria vascularis tissues were dissected from 6-day-old wild-type C57BL/6J mice under a microscope. Experimental conditions such as enzyme type, digestion time, and mechanical dissociation duration were optimized by evaluating single-cell yield and viability via flow cytometry (FCM). The single-cell samples were incubated with CD140b (i.e., platelet-derived growth factor receptor-β, PDGFR-β) and CD31 antibodies, and CD140b⁺CD31^- and CD140b^- cells were obtained by fluorescence-activated cell sorting (FACS). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to assess the expression of pericyte markers [Pdgfrb and desmin (Desm)], endothelial cell markers [von Willebrand factor (Vwf)and glucose transporter 1 (Glut1)], and melanin-like macrophage markers [glutathione S-transferase α4 (Gsta4) and adhesion G protein-coupled receptor E1 (Adgre1)] to validate cell identity and the effectiveness of the isolation method. Results ·Flow cytometric analysis revealed that papain digestion of stria vascularis tissue caused significant cell damage and a high debris rate, cold-active protease showed low dissociation efficiency, and thermolysin combined with Accutase yielded unstable digestion efficiency and a relatively high debris rate. In contrast, Accutase alone consistently produced single-cell suspensions with relatively high yield and viability, and was therefore selected as the optimal enzyme. High-purity pericytes were successfully obtained by digesting stria vascularis tissue from 6-day-old mice with Accutase followed by sorting with CD140b and CD31 antibodies. RT-qPCR showed that CD140b⁺CD31^- cells specifically exhibited high expression levels of pericyte markers Pdgfrb and Desm, and low expression levels of endothelial cell markers Vwf and Glut1, as well as melanin-like macrophage markers Gsta4 and Adgre1. Conclusion ·A stable and effective flow cytometry-based method for isolating pericytes from the stria vascularis is successfully established.