p21调节牙周膜细胞影响糖尿病拔牙创中巨噬细胞的迁移和极化

doi:10.12439/kqhm.1005-4979.2025.02.003

摘要/Abstract

摘要：

目的： 探究糖尿病小鼠拔牙创愈合过程中细胞周期蛋白依赖性激酶抑制剂1A（cyclin-dependent kinase inhibitor 1A，CDKN1A/p21）的表达变化及其对巨噬细胞迁移和极化的影响。方法： 选用C57BL/6雄性小鼠构建糖尿病模型并拔除右侧上颌第一磨牙，于拔牙后第1、3、7、14天取材。通过Micro-CT观测小鼠拔牙创硬组织愈合过程，实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR）检测组织样本中p21、CXC趋化因子配体14（C-X-C motif chemokine ligand 14，CXCL14）及巨噬细胞极化标志物mRNA相对表达水平。体外实验检测高糖刺激下人牙周膜成纤维细胞（human periodontal ligament fibroblasts，hPDLFs）中p21、CXCL14 mRNA表达水平，细胞迁移实验观察巨噬细胞RAW264.7迁移能力的变化。CXCL14蛋白刺激巨噬细胞24 h后，通过RT-qPCR验证其对巨噬细胞极化的影响。结果： 与对照组相比，糖尿病组拔牙创愈合延缓，愈伤组织中p21 mRNA表达水平在术后第3天时明显升高，CXCL14表现出相同的变化趋势（P<0.05）。同时，糖尿病组小鼠拔牙创愈伤组织中M2型巨噬细胞标志物精氨酸酶1（arginase-1，ARG-1）、分化簇206（cluster of differentiation 206，CD206）的mRNA表达水平在拔牙术后第3天同步升高（P<0.01）。体外实验经高糖刺激hPDLFs后发现，p21、CXCL14 mRNA表达水平增加，且CXCL14的存在可促进巨噬细胞的迁移及其M2型极化。使用p21小分子抑制剂UC2288可降低hPDLFs中CXCL14 mRNA表达水平。结论： 糖尿病小鼠拔牙创愈合过程中，高表达p21的牙周膜成纤维细胞可通过分泌CXCL14促进巨噬细胞的迁移及M2型极化。

关键词: 拔牙窝, 糖尿病, p21, 巨噬细胞迁移, 巨噬细胞极化

Abstract:

Objective: To explore the expression of cyclin-dependent kinase inhibitor 1A (CDKN1A/p21) in the healing process of tooth extraction wounds in diabetic mice and the effect on migration and polarization of macrophages.Methods: C57BL/6 male mice were selected to construct a model of diabetes mellitus and the right maxillary first molar was removed. The changes of bone tissue at 1, 3, 7 and 14 days after tooth extraction were observed and recorded by Micro-CT. The mRNA expression changes of p21, C-X-C motif chemokine ligand 14 (CXCL14) and the macrophage polarization markers in tissue samples were detected. In addition, real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of p21 and CXCL14 in human periodontal ligament fibroblasts (hPDLFs) stimulated by high glucose in vitro, and the cell migration experiment was used to observe the changes in the migration ability of macrophages RAW264.7. CXCL14 protein was used to stimulate macrophages for 24 h, and its effect on the polarization of macrophages was verified by RT-qPCR.Results: Compared with the control group, the healing process of the tooth extraction wound was delayed in the experimental group. The mRNA expression of p21 in tissue was significantly increased on the third day after surgery, and the mRNA expression of CXCL14 showed the same trend (P<0.05). Meanwhile, the mRNA expression of M2 macrophage markers arginase-1 (ARG-1) and cluster of differentiation 206 (CD206) in tooth extraction callus tissue of diabetic mice increased synchronously on the third day after surgery (P<0.01). After high glucose stimulation of hPDLFs, the mRNA expression levels of p21 and CXCL14 increased. CXCL14 can increase the migration ability of macrophages and promote the M2 phenotype transformation. The expression of CXCL14 mRNA in hPDLFs was decreased by using the p21 small molecule inhibitor UC2288.Conclusion: During the healing process of tooth extraction wounds in diabetic mice, hPDLFs with high expression of p21 can promote macrophage migration and M2 polarization by secreting CXCL14.

Key words: tooth extraction socket, diabetes mellitus, p21, macrophage migration, macrophage polarization

中图分类号:

R782

朱佳玉, 易紫媚, 陈思艺, 丁淑雯, 严家玉, 吴珺华. p21调节牙周膜细胞影响糖尿病拔牙创中巨噬细胞的迁移和极化[J]. 《口腔颌面外科杂志》, 2025, 35(2): 98-105.

ZHU Jiayu, YI Zimei, CHEN Siyi, DING Shuwen, YAN Jiayu, WU Junhua. p21 regulates the migration and polarization of macrophages in diabetic tooth extraction wound by regulating periodontal ligament fibroblasts[J]. 《Journal of Oral and Maxillofacial Surgery》, 2025, 35(2): 98-105.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2025/V35/I2/98

图/表 5

表1 RT‐qPCR引物序列

Table 1 Primer sequences of RT‐qPCR

基因	正向序列(5'-3')	反向序列(5'-3')
p21(M)	CCTGGTGATGTCCGACCTG	CCATGAGCGCATCGCAATC
CXCL14(M)	AGTGTAAGTGTTCCCGGAAGG	GCAGTGTGGGTGTACTTTGGCTT
INOS(M)	GAGACAGGGAAGTCTGAAGCAC	CCAGCAGTAGTTGCTCCTCTTC
ARG-1(M)	CATTGGCTTGCGAGACGTAGAC	GCTGAAGGTCTCTTCCATCACC
CD206(M)	CTCTGTTCAGCTATTGGACGC	TGGCACTCCCAAACATAATTTGA
CD86(M)	ATGGACCCCAGATGCACCA	CCTTTGTAAATGGGCACGGC
GAPDH(M)	CATCACTGCCACCCAGAAGACTG	ATGCCAGTGAGCTTCCCGTTCAG
p21(H)	CGATGGAACTTCGACTTTGTCA	GCACAAGGGTACAAGACAGTG
CXCL14(H)	TCCGGTCAGCATGAGGCTCC	CACCCTATTCTTCGTAGACC
GAPDH(H)	GGAGCGAGATCCCTCCAAAAT	GGCTGTTGTCATACTTCTCATGG

图1 糖尿病小鼠拔牙创愈合延缓 A. 上颌第一磨牙拔牙创Micro-CT三维重建图；B—E. 对照组及糖尿病组拔牙创愈合过程中骨参数分析结果。*表示P<0.05，**表示P<0.01，***表示P<0.001，与对照组比较。

Figure 1 Delayed healing of tooth extraction wounds in diabetic mice

图2 糖尿病小鼠拔牙创愈合过程中衰老及巨噬细胞极化标志物表达情况 A、B. 对照组和糖尿病组小鼠拔牙创愈伤组织中p21、CXCL14基因表达水平变化；C. 拔牙术后第3、14天，对照组与糖尿病组拔牙创愈伤组织中p21免疫组织化学染色图像（×75）；D、E. 拔牙术后第3、14天，对照组与糖尿病组拔牙创愈伤组织中，p21免疫组织化学染色图像定量分析；F、G. 糖尿病组小鼠拔牙创愈伤组织中巨噬细胞极化标志物CD206、ARG-1基因表达水平变化；H. 对照组与糖尿病组小鼠拔牙创愈伤组织中巨噬细胞极化标志物ARG-1基因表达水平变化。*表示P<0.05，**表示P<0.01，***表示P<0.001，与对照组比较；##表示P<0.01，###表示P<0.001，与拔牙后第1天时比较。

Figure 2 Expression of markers of aging and macrophage polarization in the healing process of tooth extraction wounds in diabetic mice

图3 高糖环境下巨噬细胞迁移能力的变化 A、B. 高糖刺激下，hPDLFs p21、CXCL14基因表达水平变化；C. UC2288刺激下，hPDLFs CXCL14基因表达水平变化；D、E. 巨噬细胞经不同条件培养液处理后Transwell迁移实验的下室代表性图像（×20）；F、G. Transwell迁移实验的量化分析。*表示P<0.05，**表示P<0.01，与对照组比较。

Figure 3 Changes of macrophage migration ability in high glucose environment

图4 CXCL14对巨噬细胞极化标志物相关基因表达水平的影响 A—D. 依次为对照组与CXCL14组巨噬细胞极化标志物CD86、CD206、INOS、ARG-1基因表达水平变化。*表示P<0.05，***表示P<0.001，与对照组比较。

Figure 4 Effects of CXCL14 on the expression of genes related to macrophage polarization markers

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