Identification of specific genes related to susceptibility to periodontitis

本研究的目的是应用RNA任意引物PCR（RAP-PCR）技术筛选牙周炎易感基因。RAP-PCR能够在凝胶上显示mRNA表达差异的指纹。第一阶段，从6名健康志愿者外周血中分离中性粒细胞，并以牙龈卟啉单胞菌-LPS和大肠杆菌-LPS刺激作为对照。RAP-PCR结果表明，中性粒细胞中的牙龈卟啉单胞菌-LPS刺激引起了差异基因表达（Dentistry in Japan，1999）。在第二阶段，测序和半定量RT-PCR分析表明，与其他细菌相比，牙龈卟啉单胞菌-LPS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。（伴放线菌、中间型放线菌、大肠杆菌）LPS和合成脂质A刺激在第三阶段，我们检测了全身性早发性牙周炎患者中性粒细胞的差异基因表达以年龄和性别匹配的健康志愿者（H，n=8）和泛发性成人牙周炎患者（AP，n=6）为对照。用N-甲酰基-甲硫氨酰基-亮氨酰基-苯丙氨酸刺激从外周血分离的中性粒细胞。采用45对引物进行RAP-PCR。RT-PCR分析显示，热休克转录因子4 b的mRNA显着更大的中性粒细胞从EOP相比，从控制（H和AP）。而Kruppel样锌指转录因子9和muskelin mRNA在EOP中显著低于H对照（J Periodont Res，in press）。

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).