高效耳蜗血管纹周细胞分选方法的建立

doi:10.3969/j.issn.1674-8115.2025.11.011

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (11): 1515-1526.doi: 10.3969/j.issn.1674-8115.2025.11.011

• 论著 · 技术与方法 • 上一篇

高效耳蜗血管纹周细胞分选方法的建立

朱玥¹^,²^,³, 李晓琴¹^,²^,³, 王文枭¹^,²^,³, 周凌云¹^,²^,³, 吴皓¹^,²^,³, 陶永¹^,²^,³(), 杜婷婷¹^,²^,³()

^1.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011
^2.上海交通大学医学院耳科学研究所，上海 200125
^3.上海市耳鼻疾病转化医学重点实验室，上海 200125

收稿日期:2025-02-13 接受日期:2025-04-25 出版日期:2025-11-28 发布日期:2025-12-03
通讯作者: 陶　永，副研究员，博士；电子信箱：taoyent@sjtu.edu.cn
杜婷婷，助理研究员，博士；电子信箱：tingtingdu@shsmu.edu.cn。
基金资助:
国家自然科学基金青年科学基金(82000975);上海市耳鼻疾病转化医学重点实验室(14DZ2260300)

An efficient isolation method for pericytes of the cochlear stria vascularis

ZHU Yue¹^,²^,³, LI Xiaoqin¹^,²^,³, WANG Wenxiao¹^,²^,³, ZHOU Lingyun¹^,²^,³, WU Hao¹^,²^,³, TAO Yong¹^,²^,³(), DU Tingting¹^,²^,³()

^1.Department of Otolaryngology, Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
^3.Shanghai Key Laboratory of Translation Medicine on Ear and Nose Disease, Shanghai 200125, China

Received:2025-02-13 Accepted:2025-04-25 Online:2025-11-28 Published:2025-12-03
Contact: TAO Yong, E-mail: taoyent@sjtu.edu.cn
DU Tingting, E-mail: tingtingdu@shsmu.edu.cn. ^#Co-correspongding authors.
Supported by:
Young Scientists Fund of National Natural Science Foundation of China(82000975);Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases(14DZ2260300)

摘要/Abstract

摘要：

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。

关键词: 耳蜗, 血管纹, 周细胞, 细胞分离, 流式细胞术

Abstract:

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.

Key words: cochlea, stria vascularis, pericyte, cell isolation, flow cytometry

中图分类号:

R764.3

朱玥, 李晓琴, 王文枭, 周凌云, 吴皓, 陶永, 杜婷婷. 高效耳蜗血管纹周细胞分选方法的建立[J]. 上海交通大学学报（医学版）, 2025, 45(11): 1515-1526.

ZHU Yue, LI Xiaoqin, WANG Wenxiao, ZHOU Lingyun, WU Hao, TAO Yong, DU Tingting. An efficient isolation method for pericytes of the cochlear stria vascularis[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(11): 1515-1526.

图/表 12

表1 RT-qPCR引物序列

Tab 1 Primer sequences used for RT-qPCR

Gene	Forward (5′→3′)	Reverse (5′→3′)
Gapdh	AGCTTCGGCACATATTTCATCTG	CGTTCACTCCCATGACAAACA
Pdgfrb	ACCTGCAGAGACCTCAAAAGTAGGT	ACCACGGTGACCTCCTGCGA
Desm	AGCCAGCGCGTGTCCTCCTA	AGCGTCGGCCAGGGAGAAGT
Vwf	TGTTCATCAAATGGTGGGCAGC	ACAGACGCCATCTCCAGATTCA
Glut1	GCTGTGCTTATGGGCTTCTC	AGAGGCCACAAGTCTGCATT
Gsta4	GCTGCGGCTGGAGTGGAGTTTG	TGCCCAACTGAGCTGGTTGCC
Adgre1	TGCATCTAGCAATGGACAGC	GCCTTCTGGATCCATTTGAA

图1 木瓜蛋白酶酶解血管纹的流式细胞分析Note： The diagrams demonstrate the proportion of living single cells in the papain-dissociated suspension. The living and dead cell populations were distinctly separated. Samples were obtained from four mice (P6), with an enzyme concentration of 10 U/mL, and a mechanical dissociation time of 1 min. A. The total number of granules, including cells and fragments. B. The proportion of cells. C. The proportion of single cells. D. The proportion of living cells.

Fig 1 Flow cytometry analysis of stria vascularis digested by papain

表2 不同条件下木瓜蛋白酶酶解效果

Tab 2 Digestion efficiency of papain under different conditions

Enzyme type	Total number of granules/n	Proportion of cells/%	Proportion of single cells/%	Proportion of living cells/%	Digestion condition
Enzyme type	Total number of granules/n	Proportion of cells/%	Proportion of single cells/%	Proportion of living cells/%	Enzyme concentration/(U·mL^－1)	Mechanical dissociation time/min
Papain	21 701	50.5	99.0	73.0	20	2
	25 809	43.1	98.5	71.3	20	1
	37 429	53.5	94.5	78.6	10	1

图2 CAP酶解血管纹的流式细胞分析Note： The diagrams demonstrate the proportion of living single cells in the CAP-dissociated suspension. The living and dead cell populations were distinctly separated. Samples were obtained from four mice (P6), with an enzyme concentration of 5 mg/mL, and a mechanical dissociation time of 1 min. A. The total number of granules, including cells and fragments. B. The proportion of cells. C. The proportion of single cells. D. The proportion of living cells.

Fig 2 Flow cytometry analysis of stria vascularis digested by CAP

表3 不同条件下CAP酶解效果

Tab 3 Digestion efficiency of CAP under different conditions

Enzyme type	Total number of granules/n	Proportion of cells/%	Proportion of single cells/%	Proportion of living cells/%	Digestion condition
Enzyme type	Total number of granules/n	Proportion of cells/%	Proportion of single cells/%	Proportion of living cells/%	Enzyme concentration/(mg·mL^－1)	Mechanical dissociation time/min
CAP	26 696	31.6	94.5	58.3	10	2
	22 715	42.8	96.5	69.1	5	2
	32 321	57.5	95.4	76.0	5	1

图3 嗜热菌蛋白酶和Accutase联合酶解血管纹的流式细胞分析Note： The diagrams demonstrate the proportion of living single cells in the thermolysin-and Accutase-dissociated suspension. The living and dead cell populations were distinctly separated. Samples were obtained from four mice (P6), under the conditions described in “1.3.4”. A. The total number of granules, including cells and fragments. B. The proportion of cells. C. The proportion of single cells. D. The proportion of living cells.

Fig 3 Flow cytometry analysis of stria vascularis digested by thermolysin and Accutase

表4 相同条件下3次嗜热菌蛋白酶和Accutase联合酶解效果

Tab 4 Digestion efficiency of thermolysin and Accutase in three replicates under the same condition

Enzyme type	Total number of granules/n	Proportion of cells/%	Proportion of single cells/%	Proportion of living cells/%
Thermolysin and Accutase	131 421	58.4	87.7	81.0
	194 163	60.8	79.9	79.2
	105 934	64.4	98.5	25.4

图4 Accutase酶解血管纹的流式细胞分析Note： The diagrams demonstrate the proportion of living single cells in the Accutase-dissociated suspension. The living and dead cell populations were distinctly separated. Samples were obtained from four mice (P6), with a digestion time of 3 min, and a mechanical dissociation time of 1 min. A. The total number of granules, including cells and fragments. B. The proportion of cells. C. The proportion of single cells. D. The proportion of living cells.

Fig 4 Flow cytometry analysis of stria vascularis digested by Accutase

表5 不同条件下Accutase酶解效果

Tab 5 Digestion efficiency of Accutase under different conditions

Enzyme type	Total number of granules/n	Proportion of cells/%	Proportion of single cells/%	Proportion of living cells/%	Digestion condition
Enzyme type	Total number of granules/n	Proportion of cells/%	Proportion of single cells/%	Proportion of living cells/%	Digestion time/min	Mechanical dissociation time/min	Repeated time/n
Accutase	34 791	48.1	93.9	53.0	3	1	3
	48 120	67.9	94.0	73.9	5	1	4
	65 398	60.5	94.9	76.0	5	2	3

图5 他莫昔芬诱导Pdgfr-β-creER+;tdTomato+ 小鼠特异性标记周细胞Note： A. The timeline of tamoxifen injection and sample collection. B. Vessels were stained with lectin (green); after tamoxifen injection, pericytes in Pdgfr-β-creER+;tdTomato+ mice expressed tdTomato (red) (×400). This was validated in both P69 and P6 mice. Scale bar=50 μm.

Fig 5 Tamoxifen-induced specific labeling of pericytes in PDGFR-β-creER+; tdTomato+ mice

图6 CD140b标记周细胞流式分选及验证Note： A. The blank group was not stained with the CD140b antibody (left). After staining with the CD140b antibody (right), both positive and negative cells were collected. B. RNA was extracted from the collected cells of eight mice (P6). mRNA levels were detected by RT-qPCR. ①P<0.001, ②P=0.002, ③P=0.005, ④P=0.003.

Fig 6 Flow cytometric sorting based on the CD140b marker and verification of pericytes

图7 CD140b/CD31双选得到周细胞及其纯度验证Note： A. The blank group was stained with neither antibodies (left). After staining with the CD140b antibody and CD31 antibody (right), both the CD140b+/CD31- cells and CD140b- cells were collected. The data indicated that the dissociation effect was relatively stable, with a subset of CD140b+ cells exhibiting CD31+. B. RNA was extracted from the collected cells of eight mice (P6). mRNA levels were detected by RT-qPCR. Due to the very low expression levels of Vwf and Adgre1 in the CD140b+/CD31- cells, these markers were undetectable in some replicates. For quantification, undetectable transcripts were assigned a CT value of 38. ①P<0.001, ②P=0.001, ③P=0.006, ④P=0.015, ⑤P=0.002.

Fig 7 Flow cytometric sorting based on CD140b and CD31 markers and verification of pericytes

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高效耳蜗血管纹周细胞分选方法的建立

An efficient isolation method for pericytes of the cochlear stria vascularis

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