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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (5): 893-899. doi: 10.19723/j.issn.1671-167X.2019.05.017

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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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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

CLC Number: 

  • R78

Figure 1

Cell morphology of human SCAP (×40) A, freshly extracted tooth; B, the apical papilla; C, 7 days after culture; D, 14 days after culture."

Figure 2

Immunofluorescence staining of CXCR4 and endocytotic markers (×180) A, PBS was used as the negative control; B, coexpression of CXCR4 and early endosomal marker Rab5; C,coexpression of CXCR4 and recycling endosomal marker Rab11A; D, coexpression of CXCR4 and lysosomal marker Lamp1. Strong cytoplastic localization of CXCR4 (red) overlapped with endocytotic markers (green)."

Figure 3

Proximity ligation assay (PLA) staining of CXCR4 and endocytotic markers (×60) A, PBS was used as the negative control; B, colocalization of CXCR4 and early endosomal marker Rab5; C, colocalization of CXCR4 and recycling endosomal marker Rab11A; D, colocalization of CXCR4 and lysosomal marker Lamp1. Each red spot corresponded to a molecular interaction."

Table 1

The Proportion of SCAP that expressed CXCR4 on cell surface with or without endocytotic inhibitors %"

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

Figure 4

Change of cell surface CXCR4 expression by side scatter in flow cytometry A, negative control group; B, Blebbistatin group; C, Dynasore group."

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