Identification of specific genes related to susceptibility to periodontitis

本研究的目的是利用RNA随机引物聚合酶链式反应(RAP-PCR)技术筛选与牙周炎易感性相关的基因。RAP-PCR技术可以在凝胶上显示不同基因表达的指纹图谱。在第一阶段，从6名健康志愿者的外周血中分离人中性粒细胞，以牙龈假单胞菌-内毒素和大肠杆菌-内毒素刺激为对照。RAP-PCR结果表明，中性粒细胞的差异基因表达是由牙龈假单胞菌-内毒素刺激引起的(日本牙科，1999)。在第二阶段，测序和半定量RT-PCR分析表明，与其他细菌(伴生放线放线杆菌、中间肺炎杆菌、大肠杆菌)和合成类脂A刺激相比，牙龈假单胞菌-脂多糖显著上调中性粒细胞中的超绒毛蛋白和血管内皮生长因子基因(J Perodont，2001)。在第三阶段，我们使用RAP-PCR检测了泛发性早发性牙周炎(EOP，n=6)患者中性粒细胞中的差异基因表达。用N-甲酰-甲硫基-亮氨酰苯丙氨酸刺激外周血分离的中性粒细胞。本研究采用45对RAP-PCR方法进行扩增。RT-PCR分析显示，EOP组中性粒细胞中热休克转录因子4b的表达水平显著高于对照组(H组和AP组)。而EOP组Kruppel样锌指转录因子9和Muskelin mRNA水平显著低于H对照组(J Perodont Res，新闻中)。

The aim of this study is to identify genes which relate to susceptibility for periodontitis by using RNA arbitrarily primed PCR(RAP-PCR). RAP-PCR enables to show finger-print of a difference of mRNA expression on a gel. In first stage, human neutrophils were separated from peripheral blood of 6 healthy volunteers, stimulated with P.gingivalis-LPS and E.coli-LPS as controls. The RAP-PCR results demonstrated that differential gene expression was caused by P.gingivalis-LPS stimulation in neutrophils(Dentistry in Japan, 1999). In the second stage, the sequencing and semiquantitative RT-PCR analyses demonstrated that supervillin and vascular endothelial growth factor genes were significantly upregulated by P.gingivalis-LPS in neutrophils compared to other bacterial(A.actinomycetemcomitans, P.intermedia, E.coli)LPS and synthetic lipid A stimulations(J Periodont Res, 2001).In the third stage, we examined differential gene expression in neutrophils from patients with generalized early-onset periodontitis patients(EOP, n=6)using RAP-PCR.Age and gender matched healthy volunteers(H, n=8)and generalized adult periodontitis patients(AP, n=6)were used as control subjects. Neutrophils separated from peripheral blood were stimulated with N-formyl-methionyl-leucyl-phenylalanine. The RAP-PCR with 45 primer pairs was performed as our previous study. The RT-PCR analyses revealed that mRNA for heat shock transcription factor 4b was significantly greater in neutrophils from EOP compared to those from the controls(H and AP). While, Kruppel-like zinc finger transcription factor 9 and muskelin mRNA were significantly lower in EOP compared to H controls(J Periodont Res, in press).