蛋白聚糖与小鼠牙周炎牙槽骨吸收的相关性

doi:10.12016/j.issn.2096-1456.2022.07.001

摘要/Abstract

摘要：

目的探讨小鼠牙周炎牙槽骨中蛋白聚糖含量的改变及其与牙周炎牙槽骨吸收的相关性。方法选取12只8周龄C57BL/6J雄性鼠,6-0丝线结扎右侧上颌第二磨牙建立牙周炎模型,左侧上颌未结扎处作为对照,术后14 d处死小鼠。Micro-CT扫描分析牙槽骨吸收情况;HE染色观察牙槽骨形态变化;TRAP染色观察破骨细胞阳性率的改变;RT-qPCR检测细胞外蛋白聚集蛋白聚糖（aggrecan,ACAN）、双链蛋白聚糖（biglycan,BGN）、饰胶蛋白聚糖（decorin,DCN）和多能蛋白聚糖（versican,VCAN）等蛋白聚糖相关基因表达情况,组织蛋白酶K（cathepsin K,CTSK）、基质金属蛋白酶-9（matrix metalloprotein-9,MMP-9）、核因子κB受体活化因子配体（receptor activator of nuclear factor kappa-B ligand,RANKL）等破骨细胞相关基因表达情况,白细胞介素-1β（interleukin-1β,IL-1β）、白细胞介素-6（interleukin-6,IL-6）、肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）等炎症相关基因表达情况;最后对蛋白聚糖与破骨相关基因表达量,蛋白聚糖与炎症相关基因表达量进行Pearson相关性分析。结果牙周炎侧牙槽骨吸收增多。TRAP染色结果显示牙周炎侧牙槽骨破骨细胞数量上升。RT-qPCR结果显示牙周炎侧蛋白聚糖相关基因ACAN、BGN、DCN的表达较对照侧降低,VCAN的表达较对照侧升高。牙周炎侧破骨细胞相关基因CTSK、MMP-9、RANKL及炎症相关基因IL-1β、IL-6、TNF-α的表达较对照侧升高（P<0.05）。Pearson相关性分析表明牙周炎中蛋白聚糖与破骨细胞相关基因表达量、炎症相关基因表达量均具有负相关性（P<0.05）。结论牙周炎时牙槽骨蛋白聚糖的表达与牙槽骨吸收密切相关。

关键词: 牙周炎, 牙周组织, 细胞外基质蛋白, 蛋白聚糖, 糖基化, 破骨细胞, 炎症, 牙槽骨吸收

Abstract:

Objective To analyze changes in proteoglycan and its correlation with alveolar bone resorption in periodontitis. Methods Twelve eight-week-old C57BL/6J male mice were selected, and the periodontitis model was established by ligating the right maxillary second molar with 6-0 silk thread. The nonligated part of the left maxilla was used as the control. The mice were killed 14 days after the operation. Micro-CT was used to assess alveolar bone resorption. HE staining was used to observe the alveolar bone profile, and TRAP staining was conducted to examine the positive rate of osteoclasts. The expression of proteoglycan-related genes, such as aggrecan (ACAN), biglycan (BGN), versican (VCAN), decorin (DCN), osteoclast-related genes, such as cathepsin K (CTSK), matrix metalloprotein-9 (MMP-9), and receptor activator of nuclear factor kappa-B ligand (RANKL), and inflammation-related genes, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), was detected by real-time quantitative PCR. Additionally, the correlation of the expression of proteoglycans with osteoclast-related genes and inflammation-related genes was evaluated by Pearson correlation analysis. Results The resorption of alveolar bone on the periodontitis side increased. TRAP staining showed that the number of osteoclasts was substantially increased in the maxilla with periodontitis. Real-time quantitative PCR demonstrated that compared with the control side, the expression of proteoglycan-related genes, such as ACAN, BGN, and DCN, was decreased, whereas the expression of the VCAN gene was significantly increased in the periodontitis side. Meanwhile, the expression of osteoclast-related genes, such as CTSK, MMP-9, and RANKL, and inflammation-related genes, such as IL-1β, IL-6, and TNF-α, was markedly increased in the periodontitis side (P<0.05). Pearson correlation analysis indicated a negative correlation between the expression of proteoglycans and the mRNA levels of osteoclast-related genes and inflammation-related genes (P<0.05). Conclusion The expression of proteoglycan was closely related to alveolar bone resorption in a periodontitis model.

Key words: periodontitis, periodontal tissue, extracellular matrix protein, proteoglycan, glycosylation, osteoclasts, inflammation, alveolar bone resorption

中图分类号:

王思远, 张璠, 王雪奎, 孙瑶. 蛋白聚糖与小鼠牙周炎牙槽骨吸收的相关性[J]. 口腔疾病防治, 2022, 30(7): 457-463.

WANG Siyuan, ZHANG Fan, WANG Xuekui, SUN Yao. Correlation between proteoglycan and alveolar bone resorption in a mouse periodontitis model[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2022, 30(7): 457-463.

图/表 6

表1 RT-qPCR引物序列

Table 1 Sequences of primers for RT-qPCR

Gene	5'-3'	3'-5'
GAPDH	TGTGTCCGTCGTGGATCTGA	CCTGCTTCACCACCTTCTTGA
ACAN	CGCCACTTTCATGACCGAGA	TCATTCAGACCGATCCACTGGTAG
BGN	CCTGAGACCCTGAACGAAC	GTGACCTAAGCCCAACCTGT
VCAN	CAATTACCACCTCACCTACACT	ATAGAATTGTCCTTTGCGGATGAGG
DCN	TGAGCTTCAACAGCATCACC	AAGTCATTTTGCCCAACTGC
CTSK	GAAGAAGACTCACCAGAAGCAG	TCCAGGTTATGGGCAGAGATT
MMP-9	CTGGACAGCCAGACACTAAAG	CTGGACAGCCAGACACTAAAG
RANKL	GGAAGCGTACCTACAGACTATC	AAAGTGGAATTCAGAATTGCCC
IL-1β	TGCCACCTTTTGACAGTGATG	AAGGTCCACGGGAAAGACAC
IL-6	GGCGGATCGGATGTTGTGAT	GGACCCCAGACAATCGGTTG
TNF-α	CCCTCACACTCAGATCATCTTCT	GCTACGACGTGGGCTACAG

图1 小鼠牙周炎建模

Figure 1 Construction of mouse periodontitis model a: 8-week-old mice were selected, the periodontitis model was established by silk thread ligation and 6-0 silk thread ligation at the neck of the right maxillary second molar for 14 days; b: three-dimensional reconstruction of alveolar bone by micro-CT; c: the CEJ-ABC distance of control and experimental group; d: the maxillary alveolar bone parameters of control and experimental groups; CEJ-ABC: distance between enamel-cemental junction and alveolar bone crest (t = 20.09, P<0.001); BV/TV: bone volume/total volume(t = 4.295, P = 0.013); Tb.N: trabecular number(t = 1.153, P = 0.313); Tb.Th: trabecular thickness(t = 3.839, P = 0.019); Tb.Sp: trabecular space(t = 0.8587, P = 0.439); n = 5; ns P>0.05, *P<0.05, ***P<0.001

图2 牙周炎牙槽骨组织学染色及破骨与炎症相关基因表达情况

Figure 2 Histological staining of alveolar bone and expression of genes related to osteoclast and inflammation in periodontitis a: H&E staining; b: TRAP staining; c: mRNA expressions of CTSK(t = 4.507, P = 0.011), MMP-9(t = 6.652, P<0.001) and RANKL (t = 6.730, P<0.001); d: mRNA expressions of IL-1β (t = 7.874, P<0.001), IL-6(t = 5.996, P = 0.04) and TNF-α(t = 4.642, P = 0.002); MI： first molar; M2: second molar; *P<0.05, **P<0.01, ***P<0.001; CTSK: cathepsin K; MMP-9: matrix metalloprotein-9; RANKL: receptor activator of nuclear factor-κB ligand; IL-1β: interleukin-1β; IL-6: interleukin-6; TNF-α: tumor necrosis factor-α

图3 牙周炎牙槽骨组织中的蛋白聚糖相关基因表达情况

Figure 3 Expression of proteoglycan related genes in alveolar bone of periodontitis The mRNA expressions of ACAN(t = 5.984, P<0.001), BGN(t = 5.984, P<0.001), DCN(t = 14.73, P<0.001) decreased and VCAN(t = 6.233, P<0.001) elevated. ***P<0.001; ACAN: aggrecan; BGN: biglycan; VCAN: versican; DCN: decorin

图4 牙周炎小鼠牙槽骨蛋白聚糖与破骨相关基因相关性

Figure 4 Correlation between alveolar bone proteoglycan and osteoclast related genes in periodontitis mice Pearson correlation analysis was uesd, n=3-5. MMP-9: matrix metalloprotein-9; BGN: biglycan; DCN: decorin; CTSK: cathepsin K; ACAN: aggrecan; RANKL: receptor activator of nuclear factor-κB ligand

图5 牙周炎小鼠牙槽骨蛋白聚糖与炎症相关基因相关性

Figure 5 Correlation between alveolar bone proteoglycan and inflammation related genes in periodontitis mice Pearson correlation analysis was uesd, n=3-5. DCN: decorin; IL-1β: interleukin-1β; IL-6: interleukin-6; TNF-α: tumor necrosis factor-α

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