坏死性凋亡对牙周炎免疫微环境的影响机制初探

doi:10.12439/kqhm.1005-4979.2023.05.004

摘要/Abstract

摘要：

目的：探索坏死性凋亡对牙周炎免疫微环境的影响及机制初探。方法：筛选出牙周炎中差异表达的坏死性凋亡相关基因，通过机器学习算法计算其中的核心基因并构建诊断模型。分别分析牙周炎与健康组中的免疫细胞相对丰度，并计算其与坏死性凋亡相关基因的相关性。收集人健康牙龈与炎症牙龈组织，并提取其RNA，通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction，RT-qPCR) 验证坏死性凋亡核心基因的组间表达。结果：共有10个坏死性凋亡相关基因差异表达，其中核心基因共有8个，炎症组织相对于健康组织，浆细胞、B细胞、中性粒细胞与NK细胞含量升高，而巨噬细胞、休眠树突状细胞含量下降。其中，混合系激酶域样伪激酶（mixed lineage kinase domain like pseudokinase，MLKL)、黑色素瘤缺乏因子2 (absent in melanoma 2，AIM2)与浆细胞表达量呈显著正相关。结论：坏死性凋亡对牙周炎免疫微环境存在影响。

关键词: 坏死性凋亡, 牙周炎, 基因芯片, 免疫微环境

Abstract:

Objective: To explore the effect and mechanism of necroptosis on the immune microenvironment of periodontitis. Methods: We screened out the differentially expressed necroptosis-related genes in periodontitis, first calculated the hub genes through machine learning algorithms, and constructed a diagnostic model. Then the relative abundance of immune cells in the periodontitis and healthy groups was analyzed and its correlation with necroptosis-related genes was calculated. Collect healthy and inflammatory gingival tissues from individuals and extract RNA, then validate the inter-group expression level of necroptosis-related genes using real-time quantitative polymerase chain reaction (RT-qPCR). Results: A total of 10 necroptosis-related genes were differentially expressed, including 8 hub genes. Compared with healthy tissues, the relative infiltration of plasma cells, B cells, neutrophils and NK cells in periodontal inflammatory tissues were increased. The relative infiltration of macrophages and resting dendritic cells decreased. Among them, absent in melanoma 2 (AIM2), mixed lineage kinase domain like pseudokinase (MLKL) were significantly positively correlated with the expression of plasma cells. Conclusion: Necroptosis affects the immune microenvironment of periodontitis.

Key words: necroptosis, periodontitis, gene microarray, immune microenvironment

中图分类号:

R782

郑章龙, 李家, 姜吉蕊, 单铮男, 李生娇. 坏死性凋亡对牙周炎免疫微环境的影响机制初探[J]. 《口腔颌面外科杂志》, 2023, 33(5): 298-304.

ZHENG Zhanglong, LI Jia, JIANG Jirui, SHAN Zhengnan, LI Shengjiao. Mechanism and relevance of necroptosis to immune microenvironment of periodontitis: A pilot study[J]. 《Journal of Oral and Maxillofacial Surgery》, 2023, 33(5): 298-304.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2023/V33/I5/298

图/表 7

表1 患者临床信息

Table 1 Patients′ clinical information

临床信息	健康组(n=4)	牙周炎组(n=4)
平均年龄/岁	34.33	39.25
性别
男	1	1
女	3	3
平均牙龈指数	0	3.75
平均探诊深度/mm	2.63	5.75
系统性疾病
有	0	0
无	4	4
吸烟史
有	0	0
无	4	4
药物史
有	0	0
无	4	4
牙槽骨吸收
有	0	4
无	4	0
探诊后出血
有	0	4
无	4	0
6个月内有无接受牙周治疗
有	0	0
无	4	4

表2 引物序列

Table 2 Primer sequence

基因名称	正向引物序列(5′-3′)	反向引物序列(3′-5′)
GAPDH	GTCTCCTCTGACTTCAACAGCG	ACCACCCTGTTGCTGTAGCCAA
CASP6	GCATGGTGGATCACCAGACAGAC	GGAGCCATTCACAGTTTCTCGG
PELI1	AACCAGATCGGCTCAGCAGAGA	ATGCTTCACGGTAGGAGTGTGG
AIM2	GCTGCACCAAAAGTCTCTCCTC	CTGCTTGCCTTCTTGGGTCTCA
CFLAR	AGTGAGGCGATTTGACCTGCTC	CCTCACCAATCTCTGCCATCAG
CTSB	GCTTCGATGCACGGGAACAATG	CATTGGTGTGGATGCAGATCCG
MAPK14	CCGAACGATACCAGAACCTGTC	ACGCAACTCTCGGTAGGTCCTT
FAS	GGACCCAGAATACCAAGTGCAG	GTTGCTGGTGAGTGTGCATTCC
MLKL	AGGAGGCTAATGGGGAGATAGA	TGGCTTGCTGTTAGAAACCTG

图1 坏死性凋亡相关基因在牙周炎中的表达变化及其之间的相互作用 A. 坏死性凋亡基因在健康和牙周炎样本之间的表达变化火山图；B. 坏死性凋亡核心基因相关性热图，红色代表正相关，蓝色代表负相关，×代表相关性不显著；C. MLKL与AIM2表达量呈显著正相关；D. CTSB与PELI1表达量呈显著负相关。

Figure 1 The alterations in expression and interactions of necroptosis-associated genes in periodontitis

图2 识别坏死性凋亡核心基因并构建诊断模型 A. LASSO回归系数；B. 确定最佳参数(λ)；C. 坏死性凋亡核心基因的PCA；D. 8个核心基因的多因素逻辑回归森林图。

Figure 2 Identification of central hub genes of necroptosis and development of a diagnostic framework

图3 评估坏死性凋亡核心基因在牙周炎中的诊断价值 A、B. GSE16134 数据集（训练集）和 GSE10334 数据集（测试集）的ROC；C. GSE16134 数据集中疾病（牙周炎）和健康样本的风险评分。

Figure 3 Assessment of the diagnostic significance of necroptosis-related genes in periodontitis

图4 牙周炎免疫微环境初探 A. 免疫细胞在牙周炎组与健康组中的相对浸润程度，*表示P<0.05，**表示P<0.01，***表示P<0.001，与健康组比较；B. NRGs中的核心基因表达量与免疫细胞浸润的相关性散点图，其中AIM2与MLKL表达量与浆细胞浸润程度呈显著正相关；C、D. AIM2与MLKL在牙周炎症中表达量升高。

Figure 4 An initial investigation into the immune microenvironment in periodontitis

图5 RT-qPCR验证核心基因在牙周炎症组中的表达变化 A. FAS、MAPK14、MLKL、PELI1的相对表达水平；B. AIM2、CASP6、CFLAR、CTSB的相对表达水平。* 表示 P<0.05，与健康组比较。

Figure 5 Validation of differential expressions of hub genes in periodontitis samples by RT-qPCR

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