CN112472799A - 酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用 - Google Patents
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Abstract
本发明提供了一种酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,酰基高丝氨酸内酯酰化酶是一种链球菌可以编码的蛋白酶,它是一种水解酶,它可以抑制牙周炎中部分致病菌之间交流,并能为具有抑制牙周炎作用的药物使用。
Description
技术领域
本发明属于药物化学技术领域,具体涉及到一种酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用。
背景技术
牙周炎(periodontitis)是一种微生物相关的、宿主介导的、多因素参与并导致牙周附着丧失的炎症性疾病。药物治疗是一种牙周炎的辅助治疗方式,目前最常用于临床的药物主要是一些抗生素类药物,如:甲硝唑、阿莫西林等,它们主要通过抑制某些特定种类的细菌及细菌复合物发挥其辅助治疗的效果。但近些年来,抗生素耐药性问题已经被认为是全球健康的主要风险,而且相关研究人员已经从感染的牙髓组织和牙周组织中分离出多种耐药菌株,这表明口腔很可能作为耐药菌菌库,限制抗生素在治疗身体其他部位的感染性疾病中的有效性。
研究表明牙周炎出现的主要因素为致病菌丰度变化引起的菌群失调(参见Uriarte S等,Immunol Rev.2016,273,282-298;Chen C等,ISME J.2018,12,1210-1224.),这些致病菌属于革兰氏阴性菌(G-)(Prasanth C.S等,Photochem Photobiol 2014,90(3),628-40;Pigossi S.C等,Gene,2019,689,152-160.)。以革兰氏阴性菌中的铜绿假单胞菌为例,这种菌感染人的上皮细胞和成纤维细胞后,所产生的3OC12-HSL可以引起细胞中IL-8大量表达,以及破坏NF-κB-IκBα负反馈环,增强了宿主炎症反应。
发明内容
本发明鉴于现有国内技术的问题,提供了一种酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,该酰基高丝氨酸内酯酰化酶可以抑制牙周炎中部分致病菌之间交流,并能为具有抑制牙周炎作用的药物使用。
为了实现上述目的,本发明采用如下技术方案:
本发明提供了一种酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,所述酰基高丝氨酸内酯酰化酶可水解酰基高丝氨酸内酯。
如上述的酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,所述酰基高丝氨酸内酯酰化酶为水解酶,参与的反应式如下式:
如上述的酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,所述酰基高丝氨酸内酯酰化酶可水解酰基高丝氨酸内酯的碳氢键,将AHLs信号分子水解产生无活性的高丝氨酸内酯和脂肪酸,酰基高丝氨酸内酯酰化酶通过水解酰基高丝氨酸内酯而发挥抑制或治疗牙周炎的作用。
如上述的酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,所述酰基高丝氨酸内酯分子的典型特征是含有高丝氨酸内酯和一个酰胺链,该类信号分子对细菌的不同调控功能取决于酰胺链中的碳原子数。
如上述的酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,所述酰基高丝氨酸内酯酰化酶用于制备阻断由不同酰胺链的AHL信号分子对细菌的不同调控功能的药物。
本发明通过功能预测显示不同严重程度的牙周炎患者的唾液样本中细菌的酰基高丝氨酸内酯酰化酶相对丰度差异,结果显示,不同严重程度的牙周炎患者的唾液样本中细菌的酰基高丝氨酸内酯酰化酶差异显著;研究表明,链球菌可以编码AHL酰化酶,因此可以诱导链球菌编码酰基高丝氨酸内酯酰化酶,从而阻断其他牙周炎致病菌之间的交流,进一步抑制或改善牙周炎。
与现有技术相比,本发明具备的有益效果:
1、本发明拟采用诱导链球菌编码酰基高丝氨酸内酯酰化酶的途径,从上游端阻碍细菌间的交流,从而减少菌群产生毒素,进一步抑制或改善牙周炎的严重程度。
2、本发明避免了服用抗生素导致致病菌产生耐药性。
附图说明
图1是用于检测牙周健康人群和轻度牙周炎和重度牙周炎的口腔微生物模型的研究设计流程图;
图2是基于16S测序结果牙周健康人群和不同程度牙周炎患者的菌群OTU的alpha多样性分析;
图3是牙周健康人群和牙周健康人群和不同程度牙周炎患者的菌群OTU的beta多样性分析;
图4是牙周健康人群和不同程度牙周炎患者的菌群属水平的alpha多样性分析;
图5是牙周健康人群和不同程度牙周炎患者的菌群属水平的beta多样性分析;
图6是牙周健康人群和不同程度牙周炎患者的菌群属水平的群落结构分布图;
图7是预测牙周健康人群和不同程度牙周炎患者的菌群功能alpha、beta多样性分析;
图8是不同程度牙周炎患者的菌群中相对丰度差异最显著的功能酶。
具体实施方式
下面通过以下具体实施方式对本发明作进一步阐述,但是本发明对内容完全不局限于此。
实施例
本发明提供了一种酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,所述酰基高丝氨酸内酯酰化酶可水解酰基高丝氨酸内酯。
如上述的酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,所述酰基高丝氨酸内酯酰化酶为水解酶,参与的反应式如下式:
酰基高丝氨酸内酯酰化酶(AHL酰化酶)是一类蛋白水解酶,编码该酶的基因有pvdQ、quiP两种。
酰基高丝氨酸内酯酰化酶(AHL酰化酶)可以水解酰基高丝氨酸内酯(acylhomoserine lactone,AHL)的碳氢键,将AHLs信号分子水解产生无活性的高丝氨酸内酯和脂肪酸,且能使其彻底丧失生物活性,这个过程为不可逆反应。研究表明微生物产生的AHL含量与群体总数成正比,当微生物总数达到一定水平时,调节基因表达,释放毒力因子、而对宿主细胞的健康造成威胁。酰基高丝氨酸内酯酰化酶的出现可以改变这种环境,因此着酰基高丝氨酸内酯酰化酶可以成为抗菌治疗的靶点。
按照前瞻性研究的设计原则,收集牙周健康人群不同牙周炎严重程度患者的唾液样本
按照制造商的建议提取,使用MagPure Swab DNA试剂盒(Magen,广东,中国),处理唾液样本中的DNA。
用1.5%琼脂糖凝胶测定DNA质量,然后用安捷伦2100生物分析仪(美国加利福尼亚州圣克拉拉安捷伦)和Qubit 2.0荧光仪(美国加利福尼亚州卡尔斯巴德英杰公司)测定DNA含量。
利用从样本中分离的50-ng DNA构建16S rDNA的V3和V4结构域库序列。
正向引物Forward primers:5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGRRBGCASCAGKVRVGAAT-3'
反向引物reverse primers:5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNVGGGTWTCTAATC-3'
测序平台:Illumina MiSeq
使用QIIME工具包(data analysis package)进行16S rRNA数据分析。首先,将原始测序数据中的联合序列去除,将双端序列按重叠部分拼接成单个序列,同时控制和过滤序列质量,去除嵌合序列,得到最终可用序列。
然后根据97%的相似度阈值将序列划分为操作分类单元(operationaltaxonomic units,OTUs)。相似度小于97%,可认为属于不同种;相似度小于93%-95%,可认为属于不同属。
利用核糖体数据库程序(RDP)分类器的贝叶斯算法对OTU的代表性序列进行物种分类分析,统计不同物种分类水平下每个样本的群落组成。特别的是,为了质量控制,我们删除了少于5个重复的OTU序列。
根据秩和检验对OTUs的分析结果,采用样本序列的随机抽样。由R程序包'vegan'用于计算。
Shannon指数和Simpson指数显示细菌多样性。
基于Brary-Curtis距离矩阵和Pearson相关系数,使用R软件包(http://www.R-project.org/)进行主坐标分析(PCoA)可视化,以显示队列中微生物群落的beta多样性差异。
物种分布栈图显示了基于相对丰度的群落结构的属级差异。
通过秩和检验,箱线图显示了关键菌属水平上的差异。
使用PICRUSt(version 1.0.0)(参见Langille M.G等,Nature biotechnology2013:31,814-821)在KEGG Orthology(KO)数据库中获得微生物组和基因功能的注释结果,然后使用STAMP进行差异分析,得到不同组群中微生物组基因功能的显著差异。
试验结果,见表1和表2,其中表1是预测牙周健康人群和牙周炎患者的菌群功能差异表2是不同程度牙周炎患者的菌群功能差异。
表1是预测牙周健康人群和牙周炎患者的菌群功能差异
调整后P值 | P值 | case出现频率 | control出现频率 | case均值 | control均值 | 富集组 | 总均值 | |
EC:2.7.13.1 | 1.0000 | 0.0008 | 0.1061 | 0.0120 | 0.1667 | 0.0120 | Case | 0.0560 |
EC:4.2.1.126 | 1.0000 | 0.0029 | 1.0000 | 1.0000 | 1720.1265 | 1557.6671 | Case | 1603.8840 |
EC:5.1.99.4 | 1.0000 | 0.0029 | 0.3636 | 0.1928 | 0.9242 | 0.3413 | Case | 0.5072 |
EC:4.2.2.20 | 1.0000 | 0.0039 | 1.0000 | 1.0000 | 127.6835 | 101.1167 | Case | 108.6745 |
EC:4.2.2.21 | 1.0000 | 0.0039 | 1.0000 | 1.0000 | 127.6835 | 101.1167 | Case | 108.6745 |
EC:2.7.1.144 | 1.0000 | 0.0047 | 1.0000 | 1.0000 | 585.1468 | 677.9961 | Control | 651.5821 |
EC:6.3.5.2 | 1.0000 | 0.0051 | 1.0000 | 1.0000 | 7258.7624 | 7074.9265 | Case | 7127.2247 |
EC:2.8.4.3 | 1.0000 | 0.0057 | 1.0000 | 1.0000 | 5833.7844 | 5664.0831 | Case | 5712.3602 |
EC:2.7.7.68 | 1.0000 | 0.0061 | 0.2727 | 0.1325 | 0.4318 | 0.1506 | Case | 0.2306 |
EC:2.7.8.28 | 1.0000 | 0.0061 | 0.2727 | 0.1325 | 0.4318 | 0.1506 | Case | 0.2306 |
EC:3.1.3.7 | 1.0000 | 0.0069 | 1.0000 | 1.0000 | 6290.0762 | 6084.3039 | Case | 6142.8425 |
EC:2.1.2.2 | 1.0000 | 0.0073 | 1.0000 | 1.0000 | 6727.7841 | 6553.5102 | Case | 6603.0881 |
EC:2.4.2.7 | 1.0000 | 0.0076 | 1.0000 | 1.0000 | 5144.4235 | 4999.4293 | Case | 5040.6776 |
EC:5.1.3.2 | 1.0000 | 0.0084 | 1.0000 | 1.0000 | 10060.8189 | 9720.4733 | Case | 9817.2957 |
EC:1.1.1.22 | 1.0000 | 0.0089 | 1.0000 | 1.0000 | 4381.9429 | 4158.5283 | Case | 4222.0859 |
EC:1.2.7.8 | 1.0000 | 0.0091 | 1.0000 | 1.0000 | 1767.1902 | 1495.7962 | Case | 1573.0031 |
EC:6.3.2.31 | 1.0000 | 0.0097 | 0.4091 | 0.2470 | 0.9470 | 0.5220 | Case | 0.6429 |
EC:6.3.2.34 | 1.0000 | 0.0097 | 0.4091 | 0.2470 | 0.9470 | 0.5220 | Case | 0.6429 |
注:P<0.01.case为牙周炎组;control为牙周健康对照组。
表2是不同程度牙周炎患者的菌群功能差异
调整后P值 | P值 | case0出现频率 | case1出现频率 | case0均值 | case1均值 | 富集组 | 总均值 | |
EC:3.5.1.97 | 1.0000 | 0.0170 | 0.0244 | 0.2000 | 0.0244 | 0.2800 | Casel | 0.1212 |
EC:2.7.1.175 | 1.0000 | 0.0185 | 1.0000 | 0.9600 | 14.2805 | 8.2000 | Case0 | 11.9773 |
EC:3.6.1.25 | 1.0000 | 0.0247 | 1.0000 | 1.0000 | 99.7017 | 76.1380 | Case0 | 90.7761 |
EC:1.14.13.142 | 1.0000 | 0.0255 | 0.0000 | 0.1200 | 0.0000 | 0.2000 | Case1 | 0.0758 |
EC:3.1.3.87 | 1.0000 | 0.0255 | 0.0000 | 0.1200 | 0.0000 | 0.2000 | Case1 | 0.0758 |
EC:1.2.7.7 | 1.0000 | 0.0258 | 0.3659 | 0.1200 | 1.7561 | 0.3200 | Case0 | 1.2121 |
EC:3.5.2.5 | 1.0000 | 0.0265 | 1.0000 | 1.0000 | 179.9529 | 124.8828 | Case0 | 159.0930 |
EC:2.4.1.245 | 1.0000 | 0.0324 | 1.0000 | 0.9200 | 9.6220 | 4.6600 | Case0 | 7.7424 |
EC:5.3.1.31 | 1.0000 | 0.0399 | 1.0000 | 1.0000 | 93.9161 | 64.9052 | Case0 | 82.9271 |
EC:2.1.1.152 | 1.0000 | 0.0415 | 1.0000 | 0.9200 | 6.0671 | 4.1200 | Case0 | 5.3295 |
EC:1.1.1.333 | 1.0000 | 0.0422 | 1.0000 | 0.9200 | 6.0183 | 4.0400 | Case0 | 5.2689 |
EC:1.1.98.3 | 1.0000 | 0.0422 | 1.0000 | 0.9200 | 6.0183 | 4.0400 | Case0 | 5.2689 |
EC:2.4.2.45 | 1.0000 | 0.0422 | 1.0000 | 0.9200 | 6.0183 | 4.0400 | Case0 | 5.2689 |
EC:2.4.2.46 | 1.0000 | 0.0422 | 1.0000 | 0.9200 | 6.0183 | 4.0400 | Case0 | 5.2689 |
EC:2.4.2.47 | 1.0000 | 0.0422 | 1.0000 | 0.9200 | 6.0183 | 4.0400 | Case0 | 5.2689 |
EC:4.1.1.71 | 1.0000 | 0.0422 | 1.0000 | 0.9200 | 6.0183 | 4.0400 | Case0 | 5.2689 |
EC:1.14.13.83 | 1.0000 | 0.0429 | 1.0000 | 0.9200 | 6.0427 | 4.1200 | Case0 | 5.3144 |
EC:1.1.1.371 | 1.0000 | 0.0451 | 0.0244 | 0.1600 | 0.0244 | 0.2000 | Case1 | 0.0909 |
EC:2.4.2.4 | 1.0000 | 0.0454 | 1.0000 | 1.0000 | 138.2085 | 99.1940 | Case0 | 123.4303 |
EC:2.4.1.161 | 1.0000 | 0.0478 | 0.0488 | 0.2000 | 0.0488 | 0.4800 | Case1 | 0.2121 |
注:注:P<0.05.case0为轻度牙周炎组;case1为重度牙周炎组。
Claims (5)
1.酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,其特征在于:所述酰基高丝氨酸内酯酰化酶可水解酰基高丝氨酸内酯。
3.如权利要求1所述的酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,其特征在于:所述酰基高丝氨酸内酯酰化酶可水解酰基高丝氨酸内酯的碳氢键,将AHLs信号分子水解产生无活性的高丝氨酸内酯和脂肪酸。
4.如权利要求1所述的酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,其特征在于:所述酰基高丝氨酸内酯分子的典型特征是含有高丝氨酸内酯和一个酰胺链,该类信号分子对细菌的不同调控功能取决于酰胺链中的碳原子数。
5.如权利要求1所述的酰基高丝氨酸内酯酰化酶在治疗牙周炎上的应用,其特征在于:所述酰基高丝氨酸内酯酰化酶用于制备阻断由不同酰胺链的AHL信号分子对细菌的不同调控功能的药物。
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CN105543193A (zh) * | 2016-02-26 | 2016-05-04 | 中国科学院上海高等研究院 | 一种n-酰基高丝氨酸内酯酶及其编码基因和重组菌 |
CN110151945A (zh) * | 2019-04-26 | 2019-08-23 | 西安交通大学 | 抗牙周炎的组合物及其在制备治疗牙周炎的药物中的应用 |
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CN105543193A (zh) * | 2016-02-26 | 2016-05-04 | 中国科学院上海高等研究院 | 一种n-酰基高丝氨酸内酯酶及其编码基因和重组菌 |
CN110151945A (zh) * | 2019-04-26 | 2019-08-23 | 西安交通大学 | 抗牙周炎的组合物及其在制备治疗牙周炎的药物中的应用 |
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