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Journal of Peking University (Health Sciences) ›› 2021, Vol. 53 ›› Issue (1): 9-15. doi: 10.19723/j.issn.1671-167X.2021.01.003

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Role of growth arrest-specific protein 6 in migration and osteogenic differentiation of human periodontal ligament cells

ZHANG Sheng-nan1,AN Na2,Δ(),OUYANG Xiang-ying1,Δ(),LIU Ying-jun2,WANG Xue-kui1   

  1. 1. Department of Periodontology, , 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
    2. Department of General Dentistry Ⅱ, 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:2020-10-12 Online:2021-02-18 Published:2021-02-07
  • Contact: Na AN,Xiang-ying OUYANG E-mail:anna@pkuss.bjmu.edu.cn;kqouyangxy@bjmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(81500859);National Natural Science Foundation of China(82071118);National Natural Science Foundation of China(81870772);National Natural Science Foundation of China(81570986)

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Abstract:

Objective: To investigate the role of growth arrest-specific protein 6 (Gas6) in the process of the migration and osteogenic differentiation of human periodontal ligament cells (hPDLCs).Methods: After different concentrations of recombinant human Gas6 (rhGas6) were added to hPDLCs, cell prolife-ration experiment (CCK-8) was taken to observe the effect of rhGas6 on hPDLCs cell proliferation. Scratch test and cell migration test (Transwell) were taken to analyze the migratory ability of hPDLCs in different concentrations of rhGas6 groups. After osteogenic induction, real-time quantitative polymerase chain reaction (real-time PCR) was taken to detect the expression of the Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP). ALP staining was used to detect the amount of mineralized nodules.Results: After adding different concentrations of rhGas6, there were no statistically significant differences in hPDLCs cell proliferation among the experimental groups and the control group at 24, 48 and 72 hours (P>0.05). After 24 h of scratch, the healing area in the 800 μg/L of the rhGas6 group was greater than that in the control group, but without statistically significant difference (31.06%±13.70% vs. 21.79%±9.51%, P>0.05). In the migration test, after 24 h, the number of hPDLCs cells which penetrated through the membrane in the 800 μg/L rhGas6 group was significantly higher than that in the control group (P<0.01). After rhGas6 was added and osteogenic induction, Runx2 and ALP gene expressions of hPDLCs in the 800 μg/L group were significantly higher than those in the control group (1.60±0.30 vs. 0.91±0.10, 2.81±0.61 vs. 0.86±0.12, P<0.01). After Gas6 was knocked down, the ALP expression of hPDLCs was significantly lower than that of the control group (0.39±0.07 vs. 0.92±0.14, P<0.01). There was no significant change in Runx2 expression (P>0.05). After 7 days of osteogenic induction, the mineralized nodules formed in the Gas6 knockdown group were significantly less than those in control group (0.25±0.04 vs. 1.00±0.11, P<0.001). After 14 days of induction, the staining degree of the Gas6 knockdown group was lower than that of the control group, but there was no significant difference (0.86±0.04 vs. 1.00±0.16, P>0.05).Conclusion: After downregulation of Gas6 gene, mineralized nodule formation was reduced and ALP gene expressions were decreased in the early stage of osteogenic induction (7 days). After addition of rhGas6, Runx2 and ALP gene expressions were increased and the number of cell migration was increased, suggesting that Gas6 might play a promoting role in the migration and osteogenic differentiation of human periodontal ligament cells.

Key words: Periodontal ligament, Growth arrest-specific protein 6, Cell differentiation, Osteogenesis, Alkaline phosphatase

CLC Number: 

  • R781.42

Table 1

Real-time PCR primer sequence"

Gene Forward primer (5'-3') Reverse primer (5'-3')
GAPDH TCATTTCCTGGTATGACAACGA GTCTTACTCCTTGGAGGCC
Gas6 CTGCCACAACAAGCCGGGTA TCGTCCACATCTCGGCAAGC
Runx2 CCCACGACAACCGCACCATG GCAGCACCGAGCACAGGAAG
ALP CAGAAGAAGGACAAACTGGG ATTGTATGTCTTGGACAGAGC

Figure 1

Proliferation experiment (A) and cell scratch experiment (B) of hPDLCs after rhGas6 stimulation"

Figure 2

Transwell of hPDLCs after rhGas6 stimulation A, control(0 μg/L rhGas6); B, 800 μg/L rhGas6; C, number of cells."

Figure 3

Gene expression level of Runx2 (A) and ALP (B) after rhGas6 stimulation"

Figure 4

Real-time PCR for checking efficiency of Gas6 transfection in hPDLCs si-CTR, si-RNA-control; si-Gas6, si-RNA-Gas6."

Figure 5

Gene expression level of Runx2 (A) and ALP (B) after Gas6 gene knockdown NC, normal control(osteogenic induction); si-CTR, si-RNA-control; si-Gas6, si-RNA-Gas6."

Figure 6

ALP stain and quantitative analysis NC, normal control(osteogenic induction); si-CTR, si-RNA-control; si-Gas6, si-RNA-Gas6; ALP, alkaline phosphatase. A, 7 d;B, 14 d."

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