【作者】 顾飞；

【导师】 葛颂；

【作者基本信息】 遵义医学院 ， 口腔临床医学， 2011， 硕士

【摘要】 目的：比较分析不同fimA型P. gingivalis菌毛蛋白诱导人脐静脉血管内皮细胞(human umbilical vein endothelial cells, HUVECs)炎症反应的相关信号转导。方法：1.厌氧培养并鉴定ⅡfimA型(WCSP 115)和IV fimA型(W83)p.gingivalis,收集菌液提取细菌DNA,克隆ⅡfimA型和ⅣfimA型P.gingivalis的fimA序列,构建fimA的原核表达系统pET-30a-FimA BL21DE3, IPTG诱导表达重组菌毛蛋白rFimA;2.体外培养HUVECs(CRL-2480),待HUVECs单层融合后,与不同终浓度的P. gingivalis-rFimA(0.5μg/ml、5μg/ml、10μg/ml)共同孵育2h、6h、24h,提取HUVECs总RNA, Real Time-PCR检测CD14、TLR2(Toll-like receptor 2)、TLR4(Toll-like receptor 4)和MyD88(myeloid differentiation factor 88)的mRNA表达水平,流式细胞术(FCM)检测HUVECs膜表面CD14、跨膜受体TLR2和TLR4蛋白表达量。结果：1.Ⅱ型或Ⅳ型rFimA蛋白刺激HUVECs, CD14 mRNA表达量增加,且在2h时,Ⅳ型rFimA蛋白诱导HUVECs表达CD 14 mRNA的水平高于Ⅱ型rFimA蛋白(P<0.05)；24h时,Ⅳ型rFimA蛋白在三种浓度下诱导的CD14蛋白水平较阴性对照组的CD14蛋白水平升高(P<0.05),但2h时,5μg/ml与10μg/ml的Ⅳ型rFimA蛋白诱导的CD14蛋白水平降低,与基因表达水平不一致,Ⅳ型rFimA在2h的0.5μg/ml浓度组与在6h时的三个浓度组、Ⅱ型rFimA各浓度组在三个时间点的蛋白水平与阴性对照组比较差异均无统计学意义(P>0.05)。2.Ⅱ型或Ⅳ型rFimA均能诱导HUVECs表达TLR2 mRNA水平升高(P<0.05),且Ⅳ型rFimA诱导HUVECs的TLR2 mRNA表达水平高于Ⅱ型rFimA; 24h时,Ⅳ型rFimA的刺激使TLR2 mRNA水平升高并具有浓度依赖性,且各浓度组诱导的TLR2蛋白水平升高(P<0.05),与TLR2基因水平相符；但2h时,Ⅳ型rFimA 5μg/ml与10μg/ml浓度组诱导的TLR2蛋白水平均降低,与其基因表达水平不一致,Ⅳ型rFimA在2h的0.5μg/ml浓度组与6h时的三个浓度组、Ⅱ型rFimA各浓度组在三个时间点的TLR2蛋白水平与阴性对照组比较差异均无统计学意义(P>0.05)。3.Ⅱ型或Ⅳ型rFimA均能诱导HUVECs表达TLR4 mRNA水平升高(P<0.05),且Ⅳ型rFimA的三个浓度组相比较,5μg/ml组的TLR4 mRNA水平在三个时间点都达到峰值,但各浓度组的TLR4蛋白水平在各时间点均未见表达(P>0.05)。4.Ⅱ型或Ⅳ型rFimA蛋白刺激HUVECs后,MyD88 mRNA表达量增加(P<0.05),且Ⅱ型P. gingivalis-rFimA的刺激存在着时间依赖效应。结论：ⅡfimA型和ⅣfimA型P. gingivalis-FimA均能刺激HUVECs表达膜分子CD14和跨膜分子TLR2,而且使胞内分子MyD88和跨膜分子TLR4的mRNA转录水平升高,提示P. gingivalis可通过其菌毛蛋白FimA诱导内皮细胞的炎症反应,P.gingivalis-FimA可能是动脉粥样硬化的危险因素,并且可能是牙周炎与As相关的生物学基础；CD14-TLR2系统及MyD88信号通路可能参与了P. gingivalis菌毛诱导的内皮细胞炎症反应和细胞因子产生；不同fimA基因型P. gingivalis-rFimA对HUVECs功能的影响存在着差异,且Ⅳ型P. gingivalis-FimA诱发HUVECs炎症反应的能力强于Ⅱ型P. gingivalis-FimA。更多还原

【Abstract】 Objective:To explore the intracellular signaling pathways involved in inflammatory response in HUVECs under the induction of fimbrillin from different strains of P. gingivalis with different fimA genotypes.Methods:1. P. gingivalis WCSP 115 (typeⅡfimA gene) and W83 (typeⅣfimA gene)were cultured anaerobically in standard condition and identified,then P. gingivalis cells were collected to isolate their DNA.After the fimA gene sequence was cloned, we established fimA prokaryotic expression system pET-30a-FimA BL21DE3 which expressing recombinant fimbrillin under the induction of IPTG..2. HUVECs were cultured in vitro and different concentrations of recombinant fimbrillin from P. gingivalis with different fimA genotypes were added to confluent HUVECs monolayers and co-cultured for 2h,6h and 24h. At different time periods, HUVECs were collected, total RNA was extracted from part of them and used for RT-PCR to investigate the levels of mRNA of cluster of differentiation 14(CD14), Toll-like receptor 2(TLR2), Toll-like receptor 4(TLR4) and myeloid differentiation factor 88(MyD88).The other cells were detected by FCM analysis for CD14,TLR2,TLR4 expression.Results:1. When HUVECs were stimulated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of CD 14 mRNA expressed by HUVECs were higher than that of the negative control group(P<0.05). At 2h,the up-regulating effects on CD 14 mRNA expression by P. gingivalis-rFimA withⅣfimA genotype were stronger than that by P. gingivalis-rFimA withⅡfimA genotype. After HUVECs were exposed to different concentrations(0.5μg/ml,5μg/ml,10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype for 24h, the production of CD 14 protein by HUVECs was higher than that of the negative control group(P<0.05). At 2h, however, when the higher dose (5μg/ml or lOμg/ml) of P. gingivalis-rFimA withⅣfimA genotype was used, the level of CD14 protein producted by HUVECs was lower, and there was a discrepancy between the level of CD14 protein and the level of its mRNA. When HUVECs were stimulated with 0.5μg/ml rFimA from P. gingivalis withⅣfimA genotype at 2h,0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅣfimA genotype at 6h, and 0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅡfimA genotype at 2h,6h and 24h, the protein expression of CD14 were similar to that of the negative control group (P>0.05).2. The expression of TLR2 mRNA in HUVECs induced by P. gingivalis-rFimA withⅡorⅣfimA genotype were higher than that of the negative control (p<0.05). The up-regulating effects on TLR2 mRNA by P. gingivalis-rFimA with IV fimA genotype were stronger than that by P. gingivalis-rFimA withⅡfimA genotype.The up-regulating effects of TLR2 mRNA induced by P. gingivalis-rFimA with IV fimA genotype were in a dose-dependent manner at 24h. In addition, the level of TLR2 protein expressed by HUVECs stimulated with rFimA was higher than that of the negative control group(P<0.05),which was correspond to the gene level; At 2h, however, when the higher dose (5μg/ml or 10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype was used,the level of TLR2 protein was lower, and there was a discrepancy between the level of TLR2 protein and the level of its mRNA. When HUVECs were stimulated with 0.5μg/ml rFimA from P. gingivalis withⅣfimA genotype at 2h,0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅣfimA genotype at 6h,and 0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅡfimA genotype at 2h,6h and 24h, the protein expression of TLR2 were similar to that of the negative control group(P>0.05).3. When HUVECs were stimulated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of TLR4 mRNA expressed by HUVECs was higher than that of the negative control(P<0.05). As compared with the stimulation of other concentrations (0.5μg/ml and 10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype, the level of TLR4 mRNA reached its maximum with the stimulation of 5μg/ml P. gingivalis-rFimA withⅣfimA genotype at each time point. The level of TLR4 protein expressed by HUVECs were analyzed at 2h,6h and 24h, we observed that P. gingivalis-rFimA failed to stimulate TLR4 expression in HUVECs in each concentration group(p>0.05).4. When HUVECs were treated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of MyD88 mRNA expressed by HUVECs was higher than that of the negative control group(P<0.05). The up-regulating effects on MyD88 mRNA by P. gingivalis withⅡfimA genotype were in a time-dependent manner.Conclusion:Based on these findings, we concluded that P. gingivalis-FimA withⅡorⅣfimA genotype could active HUVECs via expressing CD14 and TLR2 and up-regulating the level of MyD88 mRNA and TLR4 mRNA. It indicated that the inflammation response in HUVECs induced by P. gingivalis may be mediated through its FimA,A,P. gingivalis-FimA is likely a potential risk factor for atherosclerosis and the biological basis underlying the relationship between periodontitis and atherosclerosis.Our data suggest that singnaling pathways of CD14-TLR2 system and MyD88 may be involved in inflammatory response and cytokine production in HUVECs.The difference of P. gingivalis-FimA with fimA genotypes may lead different effects to HUVECs, and the stimulation of P. gingivalis-FimA withⅣfimA genotype was stronger than that of p. gingivalis-FimA withⅡfimA genotype.更多还原