【作者】 王舰；

【导师】 罗恩杰；

【作者基本信息】 中国医科大学 ， 病原生物学， 2009， 博士

【摘要】 目的牙周炎是人类口腔的重要疾病,在我国有着较高的患病率,它是一种以牙周微生物为始动因子、宿主免疫防御反应参与的复杂的多因素疾病。牙龈卟啉单胞菌（Porphyromonas gingivalis,P.g）是一种革兰氏阴性的厌氧菌,是目前公认的牙周炎可疑致病菌,但其致病机制尚未十分明确。牙龈上皮细胞（gingival epithelialcell,GEC）位于粘膜表面,是各种致病菌首先接触的细胞类型。牙龈卟啉单胞菌与牙龈上皮细胞之间的相互作用一直是牙周炎病因学研究的热点。基质金属蛋白酶（matrix metallo proteinases,MMPs）能直接参与牙周组织的破坏降解,在牙周病发生发展的病理生理过程中发挥作用,牙周炎的严重程度和某些MMPs正相关。此外,牙周炎发生时,在可能参与牙槽骨破坏吸收的多种细胞因子中,肿瘤坏死因子α（FNF-α）、白介素-6（IL-6）、白介素-8（IL-8）、白介素-1β（IL-1β）等起了关键作用。它们不仅直接导致牙周组织破坏,还进一步影响宿主的炎症和免疫反应进程。因此,本实验通过研究牙龈卟啉单胞菌感染牙龈上皮细胞后细胞因子表达水平的变化及基质金属蛋白酶的表达变化,同时研究TNF-α对基质金属蛋白酶表达的调节,用以探讨牙龈卟啉单胞菌在牙周炎发生发展中的作用机制,并为阐述牙龈上皮细胞在局部免疫反应中的作用提供证据,同时为牙周炎的诊断、预防和治疗提供新的思路。方法1、细菌培养:将牙龈卟啉单胞菌ATCC33277株接种于含有酵母浸出物的培养基中,置于厌氧培养箱中37℃培养,48h后达到对数期备用。2、细胞培养:取实验室冻存的第3代牙龈上皮细胞进行复苏后加入含10%胎牛血清的DMEM培养液中,置37℃CCO₂孵箱中培养。取7-10代牙龈上皮细胞,分别接种于含10%胎牛血清的DMEM培养液中,将牙龈卟啉单胞菌与牙龈上皮细胞按100:1的比例接种于上述培养液中。3、细胞因子检测:应用ELISA方法检测牙龈卟啉单胞菌接种前、接种后1h、3h、6h、12h、24h培养液上清中TNF-α、IL-6、IL-8、IL-1β的表达水平,同时提取培养液中牙龈上皮细胞总RNA,应用RT-PCR方法检测细菌接种前、接种后1h、3h、6h、12h、24h上述细胞因子表达水平。4、基质金属蛋白酶检测:采用Western blotting检测牙龈卟啉单胞菌感染前后牙龈上皮细胞表达MMP-2、MMP-3、MMP-7、MMP-9的水平变化。进一步采用实时定量PCR方法检测MMP-2、MMP-3、MMP-7、MMP-9的mRNA水平变化情况。5、TNF-α上调MMP-2和MMP-9表达检测:取三种不同浓度的TNF-α作用于牙龈上皮细胞,用实时定量PCR检测TNF-α加入前、加入后牙龈上皮细胞MMP-2和MMP-9的表达。使用抗TNF-α中和性抗体处理经牙龈卟啉单胞菌感染过的牙龈上皮细胞后,检测MMP-2和MMP-9的表达。结果1、牙龈卟啉单胞菌对牙龈上皮细胞表达TNF-α、IL-6、IL-8、IL-1β的影响ELISA结果:未经感染的牙龈上皮细胞上清液中未检测出TNF-α、IL-6、IL-8、IL-1β;经牙龈卟啉单胞菌感染后1、3、6、12及24小时,细胞上清液中检测出TNF-α,分别为11.2±1.7pg/ml、26.7±6.4pg/ml、45.2±11.3pg/ml、82.6±16.4pg/ml和128.3±21.3pg/ml;IL-1β分别为14.6±2.3pg/ml、31.4±7.9pg/ml、50.5±9.8pg/ml、72.4±14.7pg/ml和92.6±16.8pg/ml;IL-6分别为3.4±0.4pg/ml、8.3±1.1pg/ml、12.6±1.3pg/ml、23.2±3.3pg/ml和35.3±5.7pg/ml;IL-8分别为5.7±0.6pg/ml、9.4±1.3pg/ml、22.4±2.8pg/ml、34.7±4.2pg/ml和52.3±5.3pg/ml。检测以上细胞因子浓度均以时间依赖的方式增加（P＜0.01）。RT-PCR结果:未经感染的牙龈上皮细胞中未检测出TNF-αmRNA、IL-6mRNA、IL-8mRNA、IL-1βmRNA表达;经牙龈卟啉单胞菌感染后的牙龈上皮细胞均检测到上述细胞因子mRNA表达,其中TNF-αmRNA、IL-1βmRNA于感染后6小时表达最为显著,IL-6mRNA、IL-8mRNA于感染后12小时表达最为显著。2、牙龈卟啉单胞菌对牙龈上皮细胞表达MMP-2、MMP-3、MMP-7、MMP-9的影响Western blotting检测结果:未被感染的牙龈上皮细胞存在MMP-2、MMP-9较弱表达;经牙龈卟啉单胞菌感染后的牙龈上皮细胞内MMP-2、MMP-9蛋白表达显著增加,其中MMP-2于感染6小时后增加最为显著,MMP-9于感染12小时后增加最为显著。未被感染的牙龈上皮细胞及经牙龈卟啉单胞菌感染后的牙龈上皮细胞内均未检测出MMP-3和MMP-7蛋白表达。Real-timePCR结果:未被感染的牙龈上皮细胞内存在较低含量的MMP-2mRNA和MMP-9mRNA;经牙龈卟啉单胞菌感染后的牙龈上皮细胞内MMP-2mRNA含量显著增加（P＜0.01）,分别为未感染细胞的1.4、4.5、8.7、8.4和4.9倍,其中感染6小时后增加最为显著。经牙龈卟啉单胞菌感染后的牙龈上皮细胞内MMP-9 mRNA含量也显著增加（P＜0.01）,分别为未感染细胞的2.3、6.6、10.8、12.5和7.2倍,其中感染12小时后增加最为显著;未被感染的牙龈上皮细胞及经牙龈卟啉单胞菌感染后的牙龈上皮细胞内均未检测出MMP-3和MMP-7mRNA表达。3、TNF-α对牙龈上皮细胞表达MMP-2、MMP-9的调节作用Real TimePCR结果:牙龈上皮细胞MMP-2 mRNA和MMP-9 mRNA表达均出现TNF-α剂量依赖性上调（P＜0.01）,TNF-α浓度为10ng/ml时表达最多;同时结果显示,MMP-2 mRNA和MMP-9 mRNA表达在TNF-α作用3小时后增加最为显著;在10ng/ml TNF-α作用3小时后,MMP-2 mRNA和MMP-9 mRNA表达分别增加了20.8倍和36.7倍。经抗TNF-α抗体处理后,各时间段MMP-2 mRNA和MMP-9 mRNA表达水平均明显降低（P＜0.01）。结论1、牙龈卟啉单胞菌作用于牙龈上皮细胞后能显著增强TNF-α、IL-1β、IL-6、IL-8的表达。2、牙龈卟啉单胞菌作用于牙龈上皮细胞后能显著增强MMP-2、MMP-9的表达,但对MMP-3、MMP-7的表达影响不大。3、TNF-α可显著提高牙龈上皮细胞表达MMP-2、MMP-9。更多还原

【Abstract】 ObjectiveAs an important oral disease,periodotitis has a hign morbidity in China.It is a complex and multi-factors involved disease caused by periodonal microbian. Porphyromonas gingivalis（P.g） is a Gram-negative bacterium and a putative pathogen of periodotitis,while the pathogenesis is not clear yet.Gingival epithelial cell（GEC） is located in outer surface of mucosa and contacted by pathogens firstly.The interaction between P.g and GEC in periodotitis pathogenesis has been focused recently.Matrix metalloproteinases（MMPs） are involved in the degradation of periodonal tissues and play a role in the pathogenesis of periodotitis.The severity of tissue damage in periodotitis is related to some MMPs.In addition,some cytokines are found to play key roles in the tissue destruction,such as TNF-α,IL-6,IL-8 and IL-1β.And these cytokines also have an effect on host inflammation and immunal response.In this test, we measured levels of some cytokines and MMPs in GEC after they were infected with P.g and studied the regulation of TNF-αon expression of some MMPs,then explored the roles of P.g in the pathogenesis of periodotitis.This study presented evidence for GEC involved in the local immunal response and new strategies for diagnostic, prevention and therapy of periodotitis.Methods1、acterial culture:P.g strain ATCC33277 were inoculated cultrues containing yeast extract in 37℃,anaerobic incubator.When the proliferation index reached log stage,they would be used for study.2、ell culturs:Cells were cultured in DMEM with 10%fetal bovine serum in 37℃, CO₂ incubator. 3、ytokin measure:Levels of TNF-α,IL-6,IL-8 and IL-1βin cell supemant were measured by ELISA before P.g inoculation and 1h,3h,6h,12h and 24h after inoculation.And mRNA of these cytokines was extracted and measured by RT-PCR before P.g inoculation and 1h,3h,6h,12h and 24h after inoculation.4、MP measure:Protein levels of MMP-2,MMP-3,MMP-7 and MMP-9 were measured by Western blotting before and after P.g infection.And mRNA levels of these MMPs were measured by real-time PCR.5、easure of upregulation of MMP2 and MMP-9 by TNF-α.GEC was affected by exogenous TNF-αin three different concentrations.Expression of MMP-2 and MMP-9 was measured by real-time PCR.And GEC was also affected by anti-TNF-aneutralizing antibody,expression of MMP-2 and MMP-9 was also determined by real-time PCR.ResultsEffect of p.g on expression of TNF-α,IL-6,IL-8 and IL-1βin GEC.ELISA results:No cytokines was found in GEC cells without p.g infection including TNF-α,IL-6,IL-8 and IL-1β.In 1h,3h,6h,12h and 24h after infection with p.g,all these cytokines were found in cell supernant.Leves of TNF-αwere 11.2±1.7pg/ml,26.7±6.4pg/ml,45.2±11.3pg/ml,82.6±16.4pg/ml and 128.3±21.3pg/ml respectiv.Levels of IL-1βwere 14.6±2.3pg/ml,31.4±7.9pg/ml,50.5±9.8pg/ml, 72.4±14.7pg/ml and 2.6±16.8pg/ml respectively.Levels of IL-6 were 3.4±0.4pg/ml, 8.3±1.1pg/ml,12.6±1.3pg/ml,23.2±3.3pg/ml and 35.3±5.7pg/ml respectively.Levels of IL-8 were 5.7±0.6pg/ml,9.4±1.3pg/ml,22.4±2.8pg/ml,34.7±4.2pg/ml and 52.3±5.3pg/ml.concentration of these cytokines are all time-dependant（P＜0.01）.RT-PCR results:TNF-αmRNA,IL-6mRNA,IL-8mRNA and IL-1βmRNAwere not expressed in GEC without p.g infection.And all these cytokine mRNA was expressed in GEC after p.g infection.Peak values of TNF-αmRNA and IL-1βmRNA occur in 6h after infection.And the peak values of IL-6mRNA and IL-8mRNA occur in 12h after infection. Effect of p.g on expression of MMP-2,MMP-3,MMP-7 and MMP-9 in GEC.Western blotting results:GEC has weak expression of MMP-2 and MMP-9 without p.g infection.And expression of MMP-2 and MMP-9 was increased after p.g infection.Peak value of MMP-2 occurs in 6h and that of MMP-9 occurs in 12h after infection.Neigher MMP-3 nor MMP-7 was found in GEC before and after infection.Real-timePCR results:GEC has low levels of MMP-2 and MMP-9 without p.g infection.And levels of MMP-2 and MMP-9 were increased after p.g infection （P＜0.01,respectively）.Levels of MMP-2 increased 1.4,4.5,8.7,8.4 and 4.9 times. Peak value of MMP-2 occurs in 6h after infection.Levels of MMP-9 increased 2.3,6.6, 10.8,12.5 and 7.2 times.And peak value of MMP-9 occurs in 12h after infection. Neigher MMP-3 mRNA nor MMP-7 mRNA was found in GEC before and after infection.Regulation of TNF-αon expresion of MMP-2 and MMP-9 in GEC.Real TimePCR results:Expression of MMP-2 mRNA and MMP-9 mRNA was upregulated in TNF-αdose-dependant way（P＜0.01）.10ng/ml of TNF-αstimulated expression of MMP-2 and MMP-9 most significantly.And expression of MMP-2 mRNA and MMP-9 mRNA was most obvious in 3h after treatment with exogenous TNF-α.In 3h after treatment with 10ng/ml TNF-α,expression of MMP-2 mRNA and MMP mRNA increased 20.8 times and 36.7 times.After treatment with anti-TNF-αantibody,expresssion of MMP-2 mRNA and MMP-9 mRNA reduced significantly in various time（P＜0.01）.Conclusion1、Expression of TNF-α,IL-1β,IL-6 and IL-8 can be increased significantly after p.g infection in GEC.2、Expression of MMP-2 and MMP-9 can be increased significantly after p.g infection in GEC.3、TNF-αcan upregulated expression of MMP-2 and MMP-9 significantly.更多还原