【作者】 赵华强；

【导师】 魏奉才；

【作者基本信息】 山东大学 ， 外科学， 2011， 博士

【摘要】 慢性牙周炎(chronic periodontitis)是危害人类健康的口腔疾病,是目前成年人牙齿缺失的主要原因。口腔内龈下区以格兰氏阴性菌占主导地位的病原菌及其分泌的内毒素脂粘多糖(lipopolysaccharide, LPS)不仅能对牙周组织产生直接的破坏,还能够激发宿主的免疫反应。其中,T细胞、B细胞以及单核细胞／巨噬细胞等免疫活性细胞大量浸润至牙周组织内并分泌产生炎性因子,如肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α),白细胞介素-1β(interleukin-1β, IL-1β),IL-6,以及前列腺素E2 (prostaglandin E2, PGE2)等,造成进一步的牙周组织损害。其中,TNFα和IL-1β被认为是与牙周破坏密切相关的两种最重要的炎性因子[1]。免疫活性细胞向局部炎性牙周组织内的迁移(migration)、浸润(infiltration)和驻留(residence)是慢性牙周炎最重要的病理变化之一[2,3]。这一病理过程依赖于血管内皮细胞(vascular endothelial cell)和牙龈成纤维细胞(gingival fibroblast)各种粘附分子(adhesion molecules)的表达[4,5]。其中,血管细胞粘附分子(vascular cell adhesion molecule-1, VCAM-1),细胞间粘附分子(intercellular adhesion molecule-1, ICAM-1),内皮细胞白细胞粘附分子(endothelial leukocyte adhesion molecule 1, E-selectin)已被研究证实为与炎性反应密切相关的最重要的粘附分子[4,5]。牙龈成纤维细胞是牙周组织中含量最多的细胞[6]。研究发现,牙龈成纤维细胞不仅能维持牙周组织的整体性,在局部牙周免疫炎性反应中也具有重要意义[6,7]。在炎性环境中,血管内皮细胞表面的粘附分子表达增强,粘附分子作为配体与免疫活性细胞表面的受体结合,导致免疫活性细胞粘附继而浸润至血管周围的牙龈组织中,并继续产生和分泌炎性因子,如TNF-α和IL-1β[8]。在多种炎性因子的刺激下,牙龈成纤维细胞表面的粘附分子也出现表达增强,导致浸润的免疫活性细胞更长时间的驻留于局部炎性组织中[5,9,10],浸润细胞不断产生的炎性因子进一步强化了局部的免疫应答,延长了炎性反应时间,对组织造成了更加严重的损害。因此,降低牙龈成纤维细胞粘附分子的表达,从而减少浸润细胞进一步在牙周组织中的驻留成为牙周病治疗中的一个有效措施。研究表明,粘附分子的表达依赖于核因子κB (nuclear factorκB, NF-κB)的转录调控,抑制NF-κB的转录活性可以降低粘附分子的表达[5,11]。一氧化碳(carbon monoxide, CO)是血红素(heme)代谢的产物。血红素在血红素氧化酶(heme oxygenase, HO)的催化作用下,在体内代谢产生一氧化碳,铁和胆绿素[12]。适量的一氧化碳已被证实在诸多细胞生命过程中具有重要意义,如抑制细胞增殖(proliferation)[13,14],抑制免疫炎性反应[15,16],减少肾脏、肝脏和心脏移植过程中的缺血/再灌注损伤(ischemia/reperfusion injury)[17-20],调节血管活性[21]等。近几年,一类新型的化合物一氧化碳释放分子(carbon monoxide releasing molecue, CORM)引起了广泛的关注[22]。CORM包括多种不同的能够可控制地释放CO的化合物。其中,CORM-3为水溶性化合物,一旦溶于水便可快速地释放CO[22]。研究显示, CORM作为CO的替代物质,不仅具有与CO相似的生物学行为,而且克服了应用CO剂量难以控制,应用途径单一的缺点。因此在科研和临床上都具有更加光明的前景。在对已有文献的综合分析基础上,我们首次将CORM引入牙周炎相关研究中。本实验中,我们以人牙龈成纤维细胞为体外(in vitro)实验模型,以TNFα和IL-1β的共同存在模拟体外炎性环境,研究CORM-3对炎性环境下牙龈成纤维细胞粘附分子表达的调节作用,并探讨其可能的作用机制,旨在为探索牙周病的新型治疗药物和方法提供科学的理论基础和实验依据。材料和方法第一部分：CORM-3对炎性环境下人牙龈成纤维细胞粘附分子表达的影响我们首先收集临床上因需拔除埋伏牙而获得的健康牙龈组织,用组织块法培养人牙龈成纤维细胞(human gingival fibroblasts, HGF),以此建立了细胞模型；通过细胞乳酸脱氢酶(lactate dehydrogenase, LDH)的测定来检测500μM的CORM-3对细胞生长的影响,从而确定其是否对细胞产生毒性反应；以50ng.ml-1的TNF-α和10ng.ml-1的IL-1β共同刺激细胞,以模拟体外的炎性环境。同时在部分培养瓶中加入浓度为500μM的CORM-3。在刺激的不同时间点收集细胞,用常规Trizol方法提取细胞总RNA,然后用RT-PCR测定细胞ICAM-1,VCAM-1和E—Selectin的mRNA表达,以研究两种炎性因子在mRNA水平上对粘附分子表达的影响以及CORM-3的调控作用；在不同时间点收集细胞后提取细胞总蛋白,用Western Blot方法测定ICAM-1, VCAM-1和E-Selectin的蛋白表达,以研究两种炎性因子在蛋白水平上对粘附分子表达的影响以及CORM-3的调控作用；根据CORM-3的动态释放曲线,研究完全释放掉CO的CORM-3对炎性环境下细胞粘附分子的调节作用,以说明CORM的作用是否来自于CO；为研究CORM-3对细胞粘附分子的调控作用是否真正降低了免疫活性细胞对细胞的粘附作用,我们进行了功能性粘附实验,采集健康志愿者的外周血,用梯度密度离心方法提取了外周血单核细胞(peripheral blood mononuclear cell, PBMC),然后以细胞荧光标记物CSFE-DA标记PBMC。将PBMC置于HGF上,共同孵育3小时后,以磷酸盐缓冲盐水(phosphate-buffered saline, PBS)轻轻冲洗上层细胞,荧光显微镜下观察不同条件下PBMC对HGF的粘附情况的改变。第二部分:CORM-3对炎性环境下人牙龈成纤维细胞粘附分子表达影响的作用机制为研究CORM-3的作用是否通过先前多有报道的鸟苷酸环化酶(guanylate cyclase, GC)系统,将GC抑制剂ODQ用于CORM对粘附分子的调节作用实验；为检测CORM-3对炎性因子刺激下转录因子核因子κB (nuclear factorκB, NF-κB)活性的影响,我们用DEAE-dextran为主要转染试剂将含有NF-κB反应元件的报告基因载体(6-κB.luc)瞬时转染至HGF中,同时转染的还有泛素依赖的Renilla荧光素酶报告基因,通过Dual-Glo荧光素酶检测系统检测荧光素酶的活性,以此说明NF-κB的结合活性；NF-κB家族中两个最主要的亚型为NF-κB-p50和NF-κB-p65,在TNF-α和IL-1β共同刺激细胞4小时后,对加入或不加入CORM-3的细胞提取核蛋白,通过Western Blot方法检测NF-κB-p50和NF-κB-p65的核内表达情况,以检测CORM-3是否对此具有调节作用；在炎性因子刺激的不同时间点,用Western Blot检测细胞外调节激酶(extracellular regulated protein kinases, ERK), p38和jun N末端激酶(c-Jun N-terminal kinase, JNK)的表达及磷酸化情况,以明确丝裂原激活的蛋白激酶(mitogen-activated protein kinase, MAPK)系统在CORM-3的调节作用中的地位。结果第一部分：根据以往研究及预实验结果,选定500μM为本实验中CORM-3的工作浓度。LDH的实验结果显示500μM的CORM-3刺激细胞HGF或PBMC24小时后,细胞的数量及形态与正常培养液中生长的细胞无明显差异,提示该浓度的CORM-3对HGF或PBMC未见毒性作用；以50ng.ml-1的TNF-a和10ng.ml-1的IL-1β共同刺激细胞12小时、24小时后,粘附分子ICAM-1, VCAM-1和E-Selectin的mRNA表达水平均有明显增强,条带定量分析结果显示差异具有显著性(p<0.01),而500μM的CORM-3可显著性抑制这一表达的增强(p<0.01)；Western Blot的结果显示,TNF-a和IL-1β共同刺激细胞8小时、24小时后,ICAM-1, VCAM-1和E-Selectin的蛋白表达水平均出现显著增强(p<0.01),CORM-3同样表现出对上述粘附分子蛋白表达的抑制作用(p<0.01)；根据CORM-3的动态释放曲线,将完全释放掉CO的CORM-3进行上述实验,结果显示CORM-3失去了对ICAM-1蛋白表达的调节作用,说明CORM的调节作用来源于CO; PBMC功能性粘附实验的结果表明,两种炎性因子的共同刺激可导致PBMC对HGF的粘附作用增强(p<0.01),以ICAM-1, VCAM-1的中和性抗体同时作用HGF后,该粘附增强现象消失,说明炎性因子导致的粘附增强现象主要通过ICAM-1和VCAM-1介导。CORM-3可显著抑制TNF-α和IL-1β导致的PBMC对HGF的粘附增强(p<0.01),表现为粘附于HGF的绿色荧光标记的PBMC数量的减少。第二部分：ODQ为GC的抑制剂。蛋白检测结果显示ODQ不能改变CORM-3对炎性刺激下ICAM-1的调节作用,说明CORM的调节作用不是通过GC系统来实现的；瞬时转染和报告基因的检测结果显示,自TNF-α和IL-1β共同刺激细胞4小时后,即出现转录因子NF-κB的活性显著性增强(p<0.01),而CORM-3可显著性抑制NF-κB的活性(p<0.01)；TNF-α和IL-1β共同刺激细胞4小时后核蛋白的Western Blot检测结果显示,NF-κB-p50的核内表达无明显变化,而NF-κB-p65的核内表达有显著性增强(p<0.01),CORM-3同样能够显著性抑制NF-κB-p65的核内表达(p<0.01),提示NF-κB-p65在CORM-3对HGF粘附分子的调节作用中可能具有重要作用；在TNF-a和IL-1β共同刺激HGF10分钟、20分钟后收集细胞,Western Blot检测结果表明,两种炎性因子的共同刺激可显著增强p38,ERK和JNK的磷酸化水平,而CORM-3能够显著性抑制炎性介质导致的p38磷酸化水平(p<0.01),但对ERK和JNK的磷酸化水平无显著影响。结论正常情况下,人牙龈成纤维细胞(HGF)只能表达极其微弱的ICAM-1, VCAM-1和E-Selectin。然而在炎性因子TNF-a和IL-1β的共同刺激下,这些粘附分子的表达无论在mRNA水平还是蛋白水平均出现显著增强,CORM-3通过其释放的CO可显著抑制三种粘附分子的表达,显示出一定的抗炎作用。CORM-3对粘附分子的表达抑制导致了炎性环境下由ICAM-1和VCAM-1为主要介导的人外周血单核细胞(PBMC)对HGF的粘附作用的显著降低,表明其在免疫活性细胞对HGF的粘附作用中的功能性调节作用。CORM-3对HGF粘附分子的调节作用并非通过以往多有报道的鸟苷酸环化酶(GC)通路,而是通过其对转录因子NF-κB的调节作用来实现的,表现为CORM-3可显著抑制两种炎性因子共同刺激下细胞转录因子NF-κB的活性,这一作用可能是通过其降低NF-κB家族的主要亚型NF-κB-p65的核内表达来完成的。CORM-3还能够抑制TNF-α和IL-1β导致的p38磷酸化水平的升高,提示CORM对MAPK系统的调节作用可能部分参与了其对HGF粘附分子的调节作用。HGF粘附分子的表达上调在免疫活性细胞向炎性牙周组织的浸润和驻留过程中具有重要作用,并且被认为是疾病预后的决定因素之一,因此,降低免疫活性细胞对HGF的粘附,从而控制牙周炎的病理性炎症反应,是牙周炎治疗中的重要环节。CORM-3对炎性环境下HGF粘附分子的抑制作用及其对转录因子NF-κB的调节作用使其成为极具潜力的牙周炎治疗新方法。更多还原

【Abstract】 Chronic periodontitis (CP) is a kind of destructive oral disease which is the main reason for tooth loss in adults at present. CP is usually characterized by chronic inflammation associated with pathogenic bacteria in sub-gingival plaque, resulting in soft tissue destruction, alveolar bone resorption and tooth loss eventually. Gram-negative bacteria-dominated pathogens in subgingival plaque can not only cause direct damage to periodontal tissue, but also stimulate host immune response via the lipopolysaccharide (LPS) and the subsequent inflammatory cytokines. One of the prominent features of chronic periodontitis is dense inflammatory cell accumulation, e.g. monocyte/macrophage, lymphocyte, as well as their cytokines in extravascular periodontal connective tissues. In particular, proinflammatory cytokines TNF-a and IL-1βhave been demonstrated to be the most important ones involved in periodontal tissue destruction.The migration and infiltration of immuno-active cells is a highly regulated process that is dependent on various cell surface adhesion molecules on vascular endothelial cells, inflammatory cells and fibroblasts. In particular, the critical roles of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and endothelial leukocyte adhesion molecule (E-selectin) in inflammation have been widely studied.Fibroblasts are the most abundant cells in periodontal connective tissue. In addition to its role in maintaining periodontal tissue integrity and homeostasis, its role in regulating local inflammatory response has been well investigated. Under inflammatory condition, immuno-responsive cells, e.g. leukocytes and lymphocytes, adhere to and then infiltrate through vascular wall as a consequence of up-regulation of surface adhesion molecules on endothelial cells. Similarly, ICAM-1, VCAM-1 and E-selectin are also highly regulated in gingival fibroblasts in response to inflammatory chemokines, such as TNF-α, IL-1βand bacterial endotoxin LPS. The up-regulation of these adhesion molecules on gingival fibroblasts seems to play a crucial role in further leukocyte extravasations into the inflammatory periodontal connective tissues. Gingival fibroblast therefore represents an important target for intervention in periodontical disease therapy.Carbon monoxide (CO), an endogenous by-product of heme metabolism by heme oxygenase (HO), was shown to play important roles in multi-cellular events, including inhibition of cell proliferation, cell apoptosis and suppression of inflammation. The effect of CO was mediated by induction of HO-1, regulation of p38 MAPK signalling pathway and activation of guanylate cyclase. Recently, Motterlini et al reported a class of compounds, termed CO releasing molecules (CORMs), which are able to release CO in a controllable manner under physiological conditions. In particular, CORM-3 is fully water-soluble, can rapidly liberate CO once dissolved in physiological solutions. These molecules might therefore be of therapeutic interest to exert important pharmacological activities by carrying and delivering CO in a controllable fashion.In the present study, we investigated the influence of CORM-3 on the expression of adhesion molecules on human gingival fibroblasts stimulated by TNF-αand IL-1βconcurrently, and the possible mechanism by which CORM-3 exerts this effect.Material and methodsPartⅠ: Influence of CORM-3 on the expression of adhesion molecules on human gingival fibroblasts stimulated with TNF-αand IL-1βHuman gingival fibroblasts (HGF) were established from the explants of normal gingival tissues obtained during routine clinical procedures. Explants were washed and then minced with scissors into small pieces (1-3mm2) and cultured in DMEM supplemented with 10% FBS, penicillin 50IU/ml and streptomycin 50IU/ml in humidified air with 5% CO2 at 37℃. Cellular lactate dehydrogenase (LDH) assay was used to test the cytotoxicity of CORM-3 at 500μM to HGF or PBMC. To mimic the inflammatory environment, TNF-a at 50ng.ml-1 and IL-1βat lOng.ml-1 were used to stimulate HGF concurrently in the presence or absence of CORM-3 for different time periods. Expression of ICAM-1, VCAM-1 and E-selectin at mRNA and protein level was studied by means of RT-PCR and Western blot, respectively. To demonstrate that the regulation of CORM-3on the expression of adhesion molecules on HGF is via CO, degassed CORM-3 was also included in the experiment. Functional attachment assay was designed to check whether the down-regulation of CORM-3 on the expression of adhesion molecule can lead to a decreased attachment of peripheral blood mononuclear cells (PBMC) to HGF. PBMC were isolated from freshly drawn healthy human blood by volunteers by gradient density centrifugation. CFSE-DA was applied to label PBMC and the attachment of PBMC to HGF in the presence or absence of CORM-3 was checked by fluorescence microscopy.Part II:The mechanism of the down-regulation of the adhesion molecules on HGF by CORM-3To elucidate whether guanylate cyclase (GC) pathway was involved in the regulatory effect of CORM-3, an inhibitor of GC, ODQ was included in the experiment. HGF were co-transfected with a reporter construct containing NF-κB response elements (6-κB.luc) and renilla luciferase reporter, Dual-Glo Luciferase Assay System was used to assess the NF-κB activity of HGF in the response to inflammatory cytokines in the presence or absence of CORM-3. Nuclear protein was extracted and Western blot was applied to check the nuclear expression of NF-KB-p50 and NF-KB-p65. The possible role of mitogen-activated protein kinase (MAPK) system, including extracellular regulated protein kinases (ERK), p38 and JNK, was also studied in the experiment.ResultsPart I:Based on the previous study and the preliminary results, CORM-3 at 500μM was set as the working concentration in the experiment. Results from LDH assay showed that 500μM CORM-3 was no cytotoxicity to either HGF or PBMC. Western blotting analysis and PT-PCR results showed that the protein and mRNA expression of ICAM-1, VCAM-1 or E-selectin was up-regulated dramatically by 12hr concurrent treatment of TNF-a (50ng/ml) and IL-1β(10ng/ml), respectively. While induction of the adhesion molecules by these cytokines was significantly attenuated by CORM-3 at a concentration of 500μM. The modulatory effect of CORM-3 was mediated via CO since the degassed CORM-3 lost its effect. The down-regulation of adhesion molecules by CORM-3 also led to a decreased attachment of PBMC to HGF, evidenced by the decreased number of green-fluorescence PBMC to CORM-3 pretreated HGF in the functional attachment assay. Pre-treatment of the HGF with TNF-a and IL-1βin the presence of neutralizing antibody against ICAM-1 and VCAM-1 also significantly inhibited the PBMC adhesion.Part II:Down-regulation of ICAM-1 was not mediated via cGMP since ODQ, a specific inhibitor of GC by oxidation of enzyme heme, did not abrogate the effect of CORM-3. Dual-Glo luciferase activity assay showed that within 2 hr of TNF-a and IL-1βco-stimulation, NF-KB-binding activity was not significantly influenced by CORM-3. While NF-κB binding activity was significantly reduced at later time points of TNF-a and IL-1βco-stimulation in CORM-3-treated cells. In the mean time, the nuclear expression of NF-κB p65, rather than p50, was significantly decreased by CORM-3 treatment. TNF-a and IL-1βconcurrent stimulation also induced the expression of phosphorylated p38 dramatically, which was significantly attenuated by CORM-3 treatment. Though the expression of phosphorylated ERK and JNK was also augmented by TNF-αand IL-1βconcurrent stimulation, it was not influenced by CORM-3.ConclusionsIn the present study, we investigated the anti-inflammatory effect of CORM-3 on human gingival fibroblasts. The main findings of this study were the following. First, CORM-3, via its released CO, inhibited the up-regulation of ICAM-1, VCAM-1 and E-selectin after TNF-αand IL-1βconcurrent stimulation on human gingival fibroblasts, which also resulted in a decreased mononuclear cell adhesion to these cells. Second, initial activation of the NF-κB pathway by TNF-αand IL-1β co-stimulation was not influenced by CORM-3. Instead, CORM-3 suppressed sustained activation of this pathway, which was reflected by a decreased nuclear NF-KB-p65 expression. Third, CORM-3 inhibited MAPK p38 phosphorylation in response to proinflammatory cytokine stimulation.Since up-regulation of adhesion molecules on HGF plays a key role in recruitment and retention of immuno-active cells at sites of periodontal inflammatory disease, which has been regarded as one of the most important determinants of disease progression. The results of this study bode well for the application of CORM-3 as an anti-inflammatory agent to inhibit NF-κB activity and to suppress the expression of adhesion molecules on HGF, which suggests a promising potential of CORM-3 in inflammatory periodontal disease treatment.更多还原