生长停滞特异性蛋白6在人牙周膜细胞迁移及成骨分化中的作用

doi:10.19723/j.issn.1671-167X.2021.01.003

北京大学学报（医学版） ›› 2021, Vol. 53 ›› Issue (1): 9-15. doi: 10.19723/j.issn.1671-167X.2021.01.003

生长停滞特异性蛋白6在人牙周膜细胞迁移及成骨分化中的作用

张胜男¹,安娜^2,^Δ(),欧阳翔英^1,^Δ(),刘颖君²,王雪奎¹

1.北京大学口腔医学院·口腔医院,牙周科, 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
2.北京大学口腔医学院·口腔医院,综合二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:2020-10-12 出版日期:2021-02-18 发布日期:2021-02-07
通讯作者: 安娜,欧阳翔英 E-mail:anna@pkuss.bjmu.edu.cn;kqouyangxy@bjmu.edu.cn
基金资助:
国家自然科学基金(81500859);国家自然科学基金(82071118);国家自然科学基金(81870772);国家自然科学基金(81570986)

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

ZHANG Sheng-nan¹,AN Na^2,^Δ(),OUYANG Xiang-ying^1,^Δ(),LIU Ying-jun²,WANG Xue-kui¹

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)

摘要/Abstract

摘要：

目的: 探讨AXL受体酪氨酸激酶配体——生长停滞特异性蛋白6(growth arrest-specific protein 6,Gas6)在人牙周膜细胞(human periodontal ligament cells,hPDLCs)迁移及成骨诱导液培养下成骨分化中发挥的作用。方法: 在对hPDLCs进行体外培养的培养液中加入不同浓度的外源性人重组Gas6(recombinant human Gas6,rhGas6), 通过细胞增殖实验(CCK-8)检测rhGas6对hPDLCs细胞增殖的影响,通过细胞划痕实验和细胞迁移实验(Transwell)检测rhGas6对hPDLCs迁移的影响。用小干扰RNA(siRNA)下调hPDLCs 中Gas6基因表达,然后进行成骨诱导,利用实时荧光定量聚合酶链式反应(real-time PCR)检测runt相关转录因子2(runt-related transcription factor 2,Runx2)和碱性磷酸酶(alkaline phosphatase,ALP)基因表达变化,用ALP染色检测其对矿化结节形成的影响。结果: 不同浓度rhGas6对24、48、72 h的hPDLCs增殖的影响与对照组差异均无统计学意义(P>0.05)。划痕后24 h,800 μg/L rhGas6组愈合面积百分比(31.06%±13.70%)大于对照组(21.79%±9.51%),但差异无统计学意义(P>0.05);迁移实验中,24 h后800 μg/L rhGas6组迁移细胞数显著多于对照组(P<0.01)。加入rhGas6并成骨诱导后,800 μg/L组Runx2、ALP基因表达量显著高于对照组(1.60±0.30 vs. 0.91±0.10, 2.81±0.61 vs. 0.86±0.12,P<0.01)。敲低Gas6后,ALP表达显著低于对照组(0.39±0.07 vs. 0.92±0.14,P<0.01),Runx2表达无明显变化(P>0.05)。成骨诱导7 d后Gas6敲低组矿化结节形成显著少于对照组(0.25±0.04 vs. 1.00±0.11,P<0.001), 14 d后Gas6敲低组矿化结节形成少于对照组,但两组间差异无统计学意义(0.86±0.04 vs. 1.00±0.16,P>0.05)。结论: 下调Gas6基因后成骨诱导早期的矿化结节形成减少,ALP表达减少,加入rhGas6后Runx2、ALP表达增多,细胞迁移数量增多,提示Gas6在牙周膜细胞迁移及成骨分化中可能存在促进作用。

关键词: 牙周韧带, 生长停滞特异性蛋白6, 细胞分化, 骨生成, 碱性磷酸酶

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

中图分类号:

R781.42

张胜男,安娜,欧阳翔英,刘颖君,王雪奎. 生长停滞特异性蛋白6在人牙周膜细胞迁移及成骨分化中的作用[J]. 北京大学学报（医学版）, 2021, 53(1): 9-15.

ZHANG Sheng-nan,AN Na,OUYANG Xiang-ying,LIU Ying-jun,WANG Xue-kui. Role of growth arrest-specific protein 6 in migration and osteogenic differentiation of human periodontal ligament cells[J]. Journal of Peking University (Health Sciences), 2021, 53(1): 9-15.

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参考文献 22

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

生长停滞特异性蛋白6在人牙周膜细胞迁移及成骨分化中的作用

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

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