正畸过程中伴放线菌聚集杆菌的检测及其毒力因子细胞致死性膨胀毒素的研究

Detection of aggregatibacter actinomycetemcomitans and study of virulence factors cytolethal distending toxin during orthodontic treatment

目的建立PCR方法对正畸治疗患者口腔中的伴放线菌聚集杆菌(Aa)及毒力因子细胞致死性膨胀毒素进行检测,并与牙龈指数进行相关性分析。方法 选择55例戴入矫治器后产生牙龈炎性反应患者为矫治组,34例未带矫治器的牙周健康者为对照组,49例口腔内科就诊的成人牙周炎患者为成人牙周炎组。记录牙龈炎症指数,用无菌纸尖法分别采集牙周袋最深处及龈沟内液标本进行细菌DNA提取及PCR反应。结果以16S rDNA引物PCR扩增138例患者临床标本,共扩增出Aa 44株;其中正畸治疗组29株;对照组5株;成人牙周炎组10株。用Spearm an等级相关分析矫治组Aa检出率与牙龈指数G I之间存在明显的正相关关系(P<0.01)。矫治组Aa的检出率明显高于牙周炎组和对照组(P<0.01);而牙周炎组与对照组之间的Aa检出率无明显差异(P>0.05)。44例PCR扩增阳性的患者平均年龄为16.23岁,而Aa阴性的患者年龄平均为38.73岁,二者具有明显差异(t=3.598,P<0.01)。以CDT引物直接扩增44例Aa16S rDNA阳性的临床标本中的CDT基因片段,13例为阳性,其阳性率为29.54%,其中扩增出667bp的CDT1型3例,扩增出1893bp的CDT2型10例。结论 Aa在青少年患者尤其在带矫治器的青少年患者发病中起到重要的作用,但对成人牙周炎的致病不起主要作用。CDT扩增产物有两种,但检出数量较少,还有待于进一步研究。

Objective To establish a PCR（polymerase chain reaction） method for the examination of aggregatibacter actinomycetemcomitans（Aa） and lethal virulence factors of cytolethal distending toxin of orthodontic treatment of patients and conduct a correlation analysis with gingival Index.Methods Fifty-five cases of posterior fixed appliances treatment with gingivitis reaction were selected as the treatment group.34 cases with healthy periodontium without orthodontic treatment were selected as the reference group.49 cases of adult periodontitis patients of oral medicine were formed as a periodontitis group.The gingival index was recorded.Samples of deep periodontal pocket and gingival crevicular fluid（GCF） were obtained and used for bacteria DNA extraction and PCR reaction examination.Results After applying 16S rDNA miniprimer PCR to 138 clinical samples,44 amplified Aa were obtained,amongst,29 were from the treatment group,5 from the reference group and 10 from the periodontitis group.Obvious positive correlation（P0.01） was observed between GI（gingival Index） and the relevance ratio of Aa of the treatment group by using Spearman rank correlation analysis.The Aa of the treatment group showed apparent higher relevance ratio than the reference group（P0.01）,while,there was no significant variance of Aa relevance ratio between the periodontitis group and the reference group（P0.05）.44 cases with PCR positive amplification had an average age of 16.23,while,Aa negative cases had an average age of 38.73.A significant variance（t=3.598,P0.01） was observed between the two.Within the CDT gene segments of 44 cases of clinical samples applied with Aa 16S rDNA positive CDT miniprimer,13 were found with positive ratio of 29.54%,3 for 667bp amplified CDT1,10 for 1893bp amplified CDT2.Conclusion Aa shows significant effect on young patients with fixed appliance orthodontic treatment and little efficacy on adult periodontitis patients.There are two CDT amplification products discovered yet with lower relevance ratio,which will require further pertinent study.