慢性牙周炎龈下菌斑中牙龈卟啉单胞菌胶原酶水平与牙周病变程度的关系

Correlation between the level of Porphyromonas gingivalis collagenase in subgingival plaques of chronic periodontitis and the periodontal lesions

目的检测慢性牙周炎(chronicperiodontitis,CP)患者龈下菌斑中牙龈卟啉单胞菌(Porphyromonasgingivalis,Pg)胶原酶基因prtC及其产物PrtC,了解PrtC水平与牙周病变程度的关系,确定PrtC诱导细胞分泌炎性细胞因子的作用。方法采用SDSPAGE检测原核表达系统重组PrtC(rPrtC)表达和NiNTA亲和层析法提取的rPrtC纯度。rPrtC免疫家兔获得抗血清。建立多重PCR检测56例CP患者209牙位的龈下菌斑标本中Pg16SrDNA和prtC基因。建立ELISA检测上述标本中的PrtC,并分析PrtC水平与牙周病变程度的关系。采用ELISA检测rPrtC诱导人脐静脉内皮细胞EVC304分泌IL1α、IL8和TNFα的作用。结果rPrtC表达产量约占细菌总蛋白的50%。提纯的rPrtCSDSPAGE后仅见单一的蛋白条带。96.1%(201209)和92.3%(193209)的龈下菌斑标本分别Pg16SrDNA和prtC基因PCR阳性,91.4%的龈下菌斑标本(191209)PrtCELISA阳性。重度CP龈下菌斑标本中PrtC含量明显高于轻度和中度CP标本(P<0.05),但轻度和中度、中度和重度CP标本之间PrtC含量差异无统计学意义(P>0.05)。1μg的rPrtC作用EVC304细胞24h后,5和10μg的rPrtC作用EVC304细胞12h后均可使EVC304细胞分泌的IL1α、IL8和TNFα水平明显增高(P<0.05)。结论Pg有很高的prtC基因携带率和表达率。CP牙周病变程度与龈下PrtC水平密切相关。rPrtC有直接诱导细胞合成并分泌IL1α、IL8和TNFα的活性。

Objective To detect prtC gene and its expression product （PrtC） of P. gingivalis in subgingival plaques from chronic periedontitis patients and to elucidate the correlation between PrtC levels and periodontal lesions, and to identify the effect of PrtC on inducing host cell to secrete inflammatory cytokines. Methods SDS-PAGE was used to monitor the recombinant PrtC （rPrtC） expression of prokaryotic expression system and the purity of rPrtC extracted by Ni-NTA affinity chrornatography. Rabbits were imrmunized with rPrtC to obtain antiserum. A multiple PCR was established to detect 16S rDNA and prtC genes of P. gingivalis in the subgingival plaque samples from 209 teeth sites of 56 chronic periedontitis patients. ELISA method was also established to examine PrtC in the samples and the correlation between the PrtC levels and periodontal lesions. The effects of rPrtC on inducing human umbilical vein endothelial cell line EVC-304tosecreteIL-lα, IL-8and TNF-α. Results rPrtC output was approximate 50% of the total bacterial proteins. The extracted rPrtC after SDS-PAGE only showed a single fragment, 96.1% （201/209） and 92.3% （193/209） of the subgingival plaque samples were P. gingivalis 16S rDNA and prtC genes positive confirmed by PCR. 91.4% of the subgingival plaque samples （191/209） were PrtC ELISA positive. The PrtC levels in the subgingival plaque samples from advanced periedontitis were significantly higher than those in the samples from mild and moderate peridonfifis （ P 〈 0.05）. No difference of the PrtC level was found between the samples from mild/moderate periduntitis and the samples from moderate/edvanced peridontifis （ P〉0.05）. After co-incubation with 1 μg rPrtC for 24 h and with 5 or 10μg rPrtC for 12 h, the levels of IL-1α, IL-8 and TNF- α secretedby the EVC-304 cells signifieantly increased（ P 〈 0.05 ） . Conclusion P. gingivolis has high carrying and expression frequencies of prtC gene. The lesions of chronic periodontitis are clnsely relative to the subgingival PrtC levels, rPrtC has the activity to directly induce host cells to synthesize and secrete IL-1α, IL-8 and TNF-α.