Molecular and genetic analysis of polymorphonuclear luekocytes in early onset periodontitis patients

免疫球蛋白GⅡ和III型受体(FcGammaRII和FcGammaRIII)是多形核白细胞(PMN)吞噬所必需的。为了确定这种受体下调是否有助于PMN承担的牙周宿主防御，我们检查了FcGammaRII和FcGammaRIII表达与GCF-PMN吞噬能力的相关性。为了验证FcGammaR丢失发生在哪个细胞事件水平，我们量化了mRNA水平以评估这些受体的从头合成。本文收集了21例成人牙周炎患者的牙周液。以自体外周血(PB)中性粒细胞为对照。用流式细胞仪分析细胞表面FcGammaRs的表达和吞噬功能。用逆转录聚合酶链式反应(RT-PCR)检测GCF-PMN和PB-PMN间FcGammaR基因表达水平的差异。扩增产物经琼脂糖凝胶电泳法和End…分析。更多的产品产量通过计算机图像分析进行了量化。FcGammaRII和FcGammaRIII的表达和吞噬能力均显著低于PB-PMN(p&lt；0.001)。GCF-PMN的FcGammaRIII基因表达水平明显低于PB-PMN。因此，GCF-PMN的特征是表面FcGammaRs的表达和mRNA水平降低，吞噬功能受损。II.在本研究中，我们试图通过逆转录-聚合酶链式反应(RT-PCR)和原位杂交(ISH)来检测GCF中性粒细胞补体受体1和3(CR1，CR3)的mRNA水平。对11例成人牙周炎患者进行牙周冲洗，采集牙周液标本，并以静脉穿刺法采集标本作为对照。用CR1、CR3和β-肌动蛋白的引物进行RT-PCR分析。从ISH的RT-PCR产物中合成地高辛标记的RNA探针。与β-肌动蛋白相比，GCF-PMN中CR1和CR3的mRNA水平显著低于PB-PMN。在ISH中，大部分PB-PMN显示CR1和CR3mRNA阳性表达，而只有少数PMN在GCF中显示阳性信号。我们的研究数据表明，PMN细胞表面CR表达增加似乎与从头合成无关。较少

I.Immunoglobulin G type II and III receptors (FcgammaRII and FcgammaRIII) are essential for polymorphonuclear leukocytic (PMNs) phagocytosis. To determine whether this receptor downregulation may contribute to the periodontal host defense borne by PMNs, we examined the correlation between FcgammaRII and FcgammaRIII expressions and the phagocytic capacity of GCF-PMNs. In order to verify at which level of cellular events the loss of FcgammaR occurs, we quantified mRNA levels to assess a de novo synthesis of these receptors. GCF was collected from 21 patients with adult periodontitis by gingival crevicular washing. Autologous peripheral blood (PB) PMNs served as control. Surface expressions of FcgammaRs and phagocytic capacity via FcgammaRs were analyzed by flow cytometry. The difference in FcgammaR mRNA levels between GCF- and PB-PMNs was assessed by reverse transcription-polymerase chain reaction (RT-PCR). The amplified products were visualized by agarose gel electrophoresis and the end … More product yields were quantified by computerized image-analysis. Both FcgammaRII and FcgammaRIII expressions and phagocytic capacity on GCF-PMNs were significantly lower than those on PB-PMNs (p<0.001). The mRNA level of FcgammaRIII of GCF-PMNs was significantly lower than that of PB-PMNs. Thus, GCF-PMNs are characterized by the decreased surface expressions and mRNA levels of FcgammaRs, and the impaired phagocytosis.II.In this study we attempted to determine the mRNA levels of complement receptor type1 and 3 (CR1, CR3) on PMNs in GCF by using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). GCF samples were obtained from 11 adult periodontitis patients by gingival crevicular washing, and venipunctured PB was used as a control. RT-PCR analysis was performed using the primer sets for CR1, CR3 and beta-actin. Digoxigenin-labelled RNA probes were synthesized from RT-PCR products for ISH.Both CR1 and CR3 mRNA levels relative to beta-actin were significantly lower in GCF-PMNs than in PB-PMNs. In ISH,a greater majority of PB-PMNs showed positive CR1 and CR3 mRNA expressions, while only a few PMNs showed positive signals in GCF.Our data in this study suggest that increased expressions of CR on the PMN cell surface appear to be unrelated to de novo synthesis. Less