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北京大学学报（医学版） ›› 2019, Vol. 51 ›› Issue (5): 893-899. doi: 10.19723/j.issn.1671-167X.2019.05.017

• 论著 • 上一篇    下一篇

胞内转运对根尖牙乳头干细胞表面CXC趋化因子受体4表达的影响

姚心韵,高晓敏,邹晓英,岳林()   

  1. 北京大学口腔医学院·口腔医院,牙体牙髓科 国家口腔疾病临床医学研究中心 口腔数字化医疗技术和材料国家工程实验室 口腔数字医学北京市重点实验室,北京 100081
  • 收稿日期:2018-06-03 出版日期:2019-10-18 发布日期:2019-10-24
  • 通讯作者: 岳林 E-mail:kqlinyue@bjmu.edu.cn
  • 基金资助:
    国家自然科学基金(81650005);国家自然科学基金(81200773)

Role of endocytosis in cell surface CXC chemokine receptor 4 expression of stem cells from apical papilla

Xin-yun YAO,Xiao-min GAO,Xiao-ying ZOU,Lin YUE()   

  1. Department of Cariology and Endodontology, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2018-06-03 Online:2019-10-18 Published:2019-10-24
  • Contact: Lin YUE E-mail:kqlinyue@bjmu.edu.cn
  • Supported by:
    Supported by National Natural Science Fundation of China(81650005);Supported by National Natural Science Fundation of China(81200773)

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摘要：

目的:探讨胞内转运途径受到抑制后,根尖牙乳头干细胞(stem cells from apical papilla, SCAP)表面CXC趋化因子受体4(CXC chemokine receptor 4, CXCR4)表达的变化,为了解SCAP迁移机制提供实验依据。方法:采用免疫荧光共染色方法和原位邻近连接技术(proximity ligation assay, PLA)检测SCAP胞内CXCR4与细胞胞内转运相关细胞器标记蛋白的共定位,检测指标包括早期胞内体标记蛋白Rab5、循环胞内体标记蛋白Rab11A、溶酶体标记蛋白Lamp1。分别采用80 μmol/L的内吞抑制剂Blebbistatin、80 μmol/L的内吞抑制剂Dyanasore对SCAP进行预处理1 h,流式细胞分析方法检测表面阳性表达CXCR4的SCAP所占百分比,使用单因素方差分析进行统计学分析。结果:免疫荧光共染色结果显示,CXCR4在SCAP胞内的表达位置与早期胞内体标记物Rab5、循环胞内体标记物Rab11A的表达位置重合,小部分与溶酶体标记物Lamp1的表达位置重合。PLA结果显示,CXCR4在SCAP胞内与Rab5、Rab11A、Lamp1均存在共定位。阴性对照组SCAP CXCR4阳性表达的细胞为 0.13%±0.10%, 经Blebbistatin、Dynasore抑制内吞后,表面CXCR4阳性表达的SCAP显著增多,分别为13.34%±1.31%、4.03%±0.92%,差异有统计学意义(F=161.762,P<0.001), 且Blebbistatin组多于Dynasore组,差异有统计学意义(P<0.001)。结论:采用内吞抑制剂抑制CXCR4的胞内转运途径,可使表面表达CXCR4的SCAP增多,提升了SCAP迁移的潜能。

关键词: CXC趋化因子受体4, 根尖牙乳头干细胞, 迁移, 胞内转运

Abstract:

Objective: To evaluate the change of cell surface CXC chemokine receptor 4 (CXCR4) expression of stem cells from apical papilla (SCAP) after the inhibition of endocytotic pathway, thus to provide experimental basis for the mechanism of SCAP migration. Methods: The immunofluorescence analysis was conducted to examine the co-expression of CXCR4 and endocytotic compartments, including early endosomes, recycling endosomes and lysosomes in SCAP. Several Rab proteins were applied as markers of organelles in the endocytotic pathway, including Rab5 for early endosomes, Rab11A for recycling endosomes, and Lamp1 for lysosomes. The co-localization of CXCR4 with these endodontic compartments was further observed by proximity ligation assay (PLA). SCAP was treated with two kinds of endocytotic inhibitors, Blebbistatin and Dynasore, at a concentration of 80 μmol/L, respectively. The conditioning time was 1 hour. Flow cytometry was carried out to evaluate the proportion of SCAP that expressed CXCR4 on cell surface. The data were analysed by analysis of variance (ANOVA). Results: The red staining of CXCR4 on immunofluorescence confocal microscopy predominantly overlapped with the green staining of Rab5 and Rab11A, and partly overlapped with Lamp1. It indicated that most CXCR4 molecules were located in early endosomes and recycling endosomes, and some were located in lysosomes. The PLA results revealed that the co-localizaiton of CXCR4 with endocytotic compartments could be observed in early endosomes, recycling endosomes and lysosomes. According to the results of flow cytometry, the proportion of SCAP that expressed CXCR4 on cell surface was as low as 0.13%±0.10%. After the inhibition of endocytosis by pretreating the cells with the following two inhibitors, Blebbistatin and Dynasore, the percentage of SCAP that positively expressed CXCR4 on cell surface was significantly increased to 13.34%±1.31% in Blebbistatin group and 4.03%±0.92% in Dynasore group (F=16.721, P<0.001). Moreover, the number of SCAP that expressed CXCR4 on cell surface in Blebbistatin group was significantly higher than that in Dynasore group (P<0.001). Conclusion: The inhibition of endocytotic pathway could increase the number of SCAP that expressed CXCR4 on cell surface, and provide potency for the migration of SCAP.

Key words: CXC chemokine receptor 4, Stem cells from the apical papilla, Migration, Endocytosis

中图分类号: 

  • R78

图1

人原代根尖牙乳头细胞的形态(×40)"

图2

CXCR4与胞内转运相关细胞器标记蛋白免疫荧光共染色(×180)"

图3

CXCR4与胞内转运相关细胞器标记蛋白的原位邻近连接技术(proximity ligation assay,PLA)染色(×60)"

表1

阴性对照和两种内吞抑制剂组细胞表面阳性表达CXCR4的SCAP的百分比"

Group The proportion of SCAP that expressed CXCR4 on cell surface x?±s
Sample 1 Sample 2 Sample 3
Negative control 0.05 0.24 0.11 0.13±0.10
Blebbistatin 12.97 12.26 14.80 13.34±1.31
Dynasore 3.69 3.34 5.07 4.03±0.91

图4

SCAP表面CXCR4表达变化流式散点图"

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