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

I.免疫球蛋白G II型和III型受体（Fc γ RII和Fc γ RIII）对于多形核白细胞（PMN）吞噬作用是必需的。为了确定这种受体下调是否可能有助于牙周宿主防御所承担的中性粒细胞，我们研究了FcgammaRII和FcgammaRIII的表达和GCF-PMNs的吞噬能力之间的相关性。为了验证FcgammaR的损失发生在细胞事件的哪个水平，我们定量mRNA水平以评估这些受体的从头合成。采用龈沟冲洗法收集21例成人牙周炎患者的龈沟液。自体外周血（PB）PMN作为对照。通过流式细胞术分析Fc γ R的表面表达和经由Fc γ R的吞噬能力。通过逆转录-聚合酶链反应（RT-PCR）评估GCF和PB-PMN之间FcgammaR mRNA水平的差异。通过琼脂糖凝胶电泳观察扩增产物，并将末端电泳产物进行纯化。 关于我们 通过计算机图像分析定量产物产率。GCF-PMNs的Fc γ R Ⅱ和Fc γ R Ⅲ表达及吞噬功能均显著低于PB-PMNs（p<0.001）。GCF-PMNs的FcgammaRIII mRNA水平显著低于PB-PMNs。因此，GCF-PMNs的特点是FcgammaRs的表面表达和mRNA水平的下降，和受损的phagocytosis.II。在这项研究中，我们试图确定补体受体1型和3型（CR 1，CR 3）的mRNA水平在GCF中的PMNs通过使用逆转录-聚合酶链反应（RT-PCR）和原位杂交（ISH）。采用龈沟冲洗法从11例成人牙周炎患者的龈沟液中获得样本，并以静脉穿刺的牙周组织作为对照。使用CR 1、CR 3和β-肌动蛋白的引物组进行RT-PCR分析。RT-PCR产物合成地高辛标记的RNA探针，用于ISH。GCF-PMN中相对于β-actin的CR 1和CR 3 mRNA水平显著低于PB-PMN。ISH时，大多数PB-PMN显示CR 1和CR 3 mRNA阳性表达，而GCF中只有少数PMN显示阳性信号。我们的研究结果表明，PMN细胞表面CR表达的增加似乎与从头合成无关。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