牙周炎患者自身组织核酸刺激小鼠巨噬细胞后对破骨相关因子mRNA表达的影响

Effects of periodontitis patient's own tissue nucleic acid on the mRNA expression of osteoclast-related factors in murine macrophages

目的检测牙周炎患者自身组织核酸刺激巨噬细胞后破骨相关因子白细胞介素-6(IL-6)、白细胞介素-12(IL-12)p35、IL-12p40、基质金属蛋白酶-9(MMP-9)、活化T细胞核因子1(NFATc1)、核激活因子κB受体(RANK)、肿瘤坏死因子-α(TNF-α)mRNA的表达,观察牙周炎患者自身组织核酸对巨噬细胞向破骨细胞分化的影响作用。方法采集翻瓣术中慢性牙周炎患者炎症牙周组织及正畸患者健康牙拔除术获取的健康牙周组织,提取组织总RNA逆转录cDNA。培养小鼠巨噬细胞系RAW264.7,加入质量浓度1μg·mL-1的特定序列寡核苷酸MT01共孵育3 h后(以1μg·mL-1的PBS作为对照),加入已提取的炎症牙周组织及健康牙周组织cDNA(质量浓度为1μg·mL-1)。实验分4组:健康组织cDNA,炎症组织cDNA,MT01+健康组织cDNA,MT01+炎症组织cDNA。4组细胞分别孵育3、6、12、24 h,采用实时定量聚合酶链反应法检测破骨相关因子IL-6、IL-12p35、IL-12p40、MMP-9、NFATc1、RANK及TNF-αmRNA的表达,进行两两组间比较。结果牙周炎患者自身组织核酸可上调RAW264.7破骨相关因子IL-6、IL-12p35、IL-12p40、MMP-9、NFATc1、RANK及TNF-αmRNA的表达;在免疫抑制剂MT01的作用下,牙周炎患者自身组织核酸上调RAW264.7内破骨相关因子mRNA的表达状况受到抑制。结论牙周炎患者自身组织核酸可以影响小鼠巨噬细胞向破骨细胞的分化。

Objective This paper aimed to determine the mRNA expression of osteoclast-related factors interleukin-6 （IL-6）, interleukin-12 （IL-12） p35, IL-12p40, matrix metalloproteinase-9 （MMP-9）, nuclear factor of activated T-cells cyto- plasmic 1 （NFATcl）, receptor activator of nuclear factor-nB （RANK）, and tumor necrosis factor-a （TNF-α） mRNA in murine macrophages infected by a periodontitis patient＇s own tissue nucleic acid. Another aim was to investigate the effects of a perio- dontitis patient＇s own tissue nucleic acid on the differentiation of macrophages into osteoclasts. Methods Inflammatory periodontal tissue samples of chronic periodontitis patients were taken during periodontal flap surgery, and healthy gingival tissue samples were taken from orthodontic patients during tooth extractions. Total RNA from periodontal tissue was extracted and reversely transcribed into cDNA and then cryo-preserved until further use. First, specific sequence oligodeoxynucleotide MT01 at a concentration of 1 μg·mL-1 was added in murine macrophage RAW264.7, and the cells were incubated for 3 hours. Cells with PBS （1 μg·mL-1） were used as negative controls. The inflammatory periodontal tissue cDNA and healthy periodontal tissue cDNA （1 μg·mL-1） was added subsequently. There were four experimental groups： healthy periodontal tissue cDNA＋ RAW264.7, inflammatory periodontal tissue cDNA＋RAW264.7, MT01＋healthy periodontal tissue cDNA＋RAW264.7, and MT01＋inflammatory periodontal tissue cDNA＋RAW264.7. Real-time quantitative polymerase chain reaction was used to detect the mRNA expression of osteoclast-related factors IL-6, IL-12p35, IL-12p40, MMP-9, NFATcl, RANK, and TNF-α mRNA after 3, 6, 12, and 24 hours. Results The mRNA levels of osteoclast-related factors NFATc 1, MMP-9,TNF-α, IL-6, IL-12p40, IL-12p35, and RANK in RAW264.7 were markedly upregulated with the treatment of periodontitis patient＇s own tissue nucleic acid. However, the mRNA expression of osteoclast-related factors was inhibited by use of an im- mtmosuppressant MT01. Conclusion The periodontitis patient＇s own tissue nucleic acid could promote the differentiation of murine macrophage into osteoclasts.