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

免疫球蛋白G II型和III型受体（FcgammaRII和FcgammaRII）是多形核白细胞（PMNs）吞噬所必需的。为了确定这种受体下调是否有助于PMNs对牙周宿主的防御，我们检测了FcgammaRII和FcgammaRII表达与GCF-PMNs吞噬能力的相关性。为了验证FcgammaR的丢失发生在细胞事件的哪个水平，我们量化了mRNA水平来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积液。以自体外周血（PB） pmn为对照。流式细胞术分析FcgammaRs细胞表面表达及吞噬能力。通过逆转录聚合酶链反应（RT-PCR）评估GCF-和PB-PMNs之间FcgammaR mRNA水平的差异。通过琼脂糖凝胶电泳显示扩增产物，并通过计算机图像分析定量扩增产物的产率。GCF-PMNs中FcgammaRII和FcgammaRII的表达及吞噬能力均显著低于PB-PMNs （p<0.001）。GCF-PMNs中FcgammaRIII mRNA表达水平明显低于PB-PMNs。因此，GCF-PMNs的特征是FcgammaRs的表面表达和mRNA水平降低，吞噬功能受损。ii .本研究采用逆转录聚合酶链式反应（RT-PCR）和原位杂交（ISH）方法测定GCF中补体受体1和3 （CR1, CR3）在PMNs上的mRNA水平。采用龈沟冲洗法采集11例成人牙周炎患者的GCF样本，并以静脉穿刺PB作为对照。使用CR1、CR3和β -actin的引物进行RT-PCR分析。利用RT-PCR产物合成地高辛标记RNA探针。与β -肌动蛋白相关的CR1和CR3 mRNA水平在GCF-PMNs中均显著低于PB-PMNs。在ISH中，绝大多数PB-PMNs表现出CR1和CR3 mRNA的阳性表达，而在GCF中只有少数PMNs表现出阳性信号。我们在这项研究中的数据表明，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