【作者】 张瑞丽；

【导师】 王千秋；

【作者基本信息】 北京协和医学院 ， 皮肤病与性病学（专业学位）， 2014， 博士

【摘要】 背景梅毒是由梅毒螺旋体(Treponema pallidum subsp.pallidum, Tp)感染所致慢性系统性炎症性疾病,血管内皮细胞的活化和损伤在梅毒发病中起重要作用。既往研究发现Tp可以直接活化血管内皮细胞,亦可通过其膜蛋白作用于血管内皮细胞,导致血管内皮细胞的活化和损伤。由于Tp在体外培养基上无法培养,对Tp膜蛋白的研究成为研究梅毒发病机制及免疫反应机制的重点之一。基因工程技术的发展为Tp膜蛋白的研究提供技术保障。Tp0965是一种膜融合蛋白,位于Tp的细胞周质,我们前期研究结果显示,Tp0965可与临床各病期的梅毒患者阳性血清发生抗原抗体反应。本研究利用基因重组技术获得高纯度的Tp0965,随后将重组蛋白Tp0965(recombinant Tp0965, rTp0965)与人脐静脉内皮细胞(HUVEC)混合培养,观察rTp0965对HUVEC活化及HUVEC单层通透性的影响,探讨Tp0965在Tp所致血管内皮细胞的活化和损伤中的作用,为梅毒发病机制研究提供实验基础。目的研究梅毒螺旋体膜蛋白Tp0965体外对血管内皮细胞活化及血管内皮屏障通透性的影响,探讨膜蛋白Tp0965在血管内皮细胞活化及损伤中的作用,为梅毒发病机制研究提供依据。方法1.rTp0965的表达和纯化将已构建成功的重组质粒pET-28a(+)/Tp0965转化表达宿主菌株E.coli Rosetta(DE3),超声震碎细菌,取上清液进行SDS-PAGE分析,蛋白印迹法鉴定。将鉴定成功重组质粒pET-28a(+)/Tp0965表达于DE3,大规模诱导表达,利用蛋白纯化试剂盒对获得蛋白进行纯化,BCA法检测重组蛋白的浓度,然后采用多粘菌素B柱去除大肠杆菌内毒素,获得具有生物学活性的rTp0965o2.HUVEC体外活化将HUVEC接种于细胞培养板,加入rTp0965,使终浓度为5Ong/mL.100ng/mL、200ng/mL、400ng/mL、800ng/mL,分别培养3h、6h、12h、24h、48h。取培养上清液,ELISA检测细胞因子MCP-1的分泌水平。Cell ELISA法检测粘附分子ICAM-1及E-selectin在HUVEC膜上的表达水平；荧光定量Real-time PCR检测细胞中MCP-1、ICAM-1及E-selectin的mRNA转录水平。3.THP-1细胞向HUVEC的迁移和粘附将HUVEC接种于细胞培养板上,加入rTp0965混合培养24h,加入calcein-AM (5μmol/L)标记的THP-1细胞,继续培养6h,PBS洗去未粘附的THP-1细胞,荧光显微镜下计数未粘附的THP-1细胞,按公式THP-1细胞的黏附率=(加入总THP-1细胞数-未粘附THP-1细胞数/加入总THP-1细胞数×100%),计算THP-1细胞的黏附率。将HUVEC接种于Transwell小室的下室,加入rTp0965,混合培养24h,在Transwell小室的上室加入calcein AM (5μm)标记的THP-1细胞,继续培养1h,取下室培养基20μL,荧光倒置显微镜下计数THP-1细胞,按公式THP-1细胞的迁移率=穿过的THP-1细胞数/加入总THP-1细胞数×100%,计算THP-1细胞的迁移率。4.构建HUVEC单层模型HUVEC接种于将已包被基质胶的Transwell小室,置于37℃、5%CO2孵育箱培养,观察HUVEC铺满整个小室底部,然后进行4h液面渗漏试验检测,选择4h液面渗漏试验阳性的模型,作为实验模型。5.HUVEC单层通透性改变将rTp0965加入HUVEC单层模型的Transwell小室内,混合培养后,加入HRP溶液,继续培养,于培养1h和4h时取下室培养基50gL于96孔板中置于4℃避光保存,取样全部完毕,向96孔板样本中加入四甲基联苯胺,显色后加入硫酸终止显色反应,酶标仪测量450nm波长处样本A值。6.HUVEC紧密连接蛋白表达及细胞骨架系统重排将HUVEC接种于96孔细胞培养板上,加入rTp0965,继续培养24h,胰蛋白酶消化细胞,裂解细胞,提取总蛋白,蛋白印迹法检测紧密连接蛋白Claudin-1蛋白表达水平。同时将HUVEC接种于激光共聚焦专用培养皿上,加入rTp0965,继续培养,多聚甲醛固定,加入FITC-Phalloidin工作液染色细胞,激光共聚焦显微镜下观察细胞骨架蛋白F-actin的变化。7.RhoA/ROCK信号通路的调控作用将HUVEC接种于细胞培养板上,加入RhoA/ROCK信号通路抑制剂Y-27632,培养30min,加入rTp0965,继续培养24h,提取细胞总蛋白,蛋白印迹法检测紧密连接蛋白Claudin-1蛋白表达水平。同时将HUVEC接种于激光共聚焦专用培养皿上,加入Y-27632,培养30min,加入rTp0965,继续培养,多聚甲醛固定,加入FITC-Phalloidin工作液染色细胞,激光共聚焦显微镜下观察细胞骨架蛋白F-actin的变化。将Y-27632加入HUVEC单层模型的Transwell、室内,培养3Omin,加入rTp0965,继续培养24h,向Transwell小室内加入calcein AM标记的THP-1细胞,下室内加入MCP-1,继续培养,分别于培养1h及4h时取下室培养基20μL,荧光倒置显微镜下计数THP-1细胞,按公式THP-1细胞的迁移率=穿过的THP-1细胞数/加入总THP-1细胞数×100%,计算THP-1细胞的迁移率。结果1.重组蛋白Tp0965的表达和纯化SDS-PAGE分析显示清晰的相对分子量约40kDA的条带,蛋白印迹法结果显示可与二期梅毒阳性血清发生抗原抗体反应。BCA法检测重组蛋白的浓度为1.1mg/mL,多粘菌素B柱去除大肠杆菌内毒素鲎试剂检测显示内毒素含量为2.0EU/mL。2.rTp0965体外对HUVEC活化的影响rTp0965刺激IUVEC后,培养上清中MCP-1分泌水平显著高于阴性对照组,差异有统计学意义(t=14.5P<0.05)；rTp0965刺激组的ICAM-1及E-selectin的HUVEC膜表达水平亦显著高于阴性对照组,差异有统计学意义(t1=6.6,t2=7.0,均P<0.05); Real-time PCR检测结果显示,rTp0965刺激组HUVEC中MCP-1、ICAM-1及E-selectin的mRNA转录水平均显著高于阴性对照组(t1=14.0,t2=5.6,t3=8.7,均P<0.05),差异有统计学意义。3.rTp0965对HUVEC与THP-1细胞趋化和粘附的影响实验结果显示,rTp0965刺激HUVEC后,HUVEC与THP-1细胞的粘附率为48.5±7.5%,显著高于阴性对照组(233±7.9%),经χ2检验,差异有统计学意义(χ2=13.8,P<0.05)。rTp0965刺激后,THP-1细胞的迁移率显著增加,为56.9±3.1%,显著高于阴性对照组(13.8±1.8%),经χ2检验,差异有统计学意义(χ2=40.6,P<0.05)。4.rTp0965对HUVEC单层通透性的影响ELISA检测结果显示rTp0965刺激组HUVEC单层HRP流量在1h时即显著高于阴性对照组(0.42±0.08与0.15±0.07,t=4.40P<0.05)；4h时HRP流量差距明显加大(1.2±0.11与0.52±0.06,t=9.40,P<0.05),差异有统计学意义。5.rTp0965对HUVEC紧密连接蛋白表达及细胞骨架系统的影响蛋白印迹法结果显示,rTp0965刺激组HUVEC内的紧密连接蛋白Claudin-1的表达水平显著低于阴性对照组。激光共聚焦显微镜下显示,阴性对照组HUVEC内的F-actin主要富集于细胞膜的周边,分布均匀,rTp0965组细胞周边的F-actin明显减少,胞浆中出现密集的应力纤维。6.rTp0965对THP-1细胞跨HUVEC迁移的影响实验结果显示,rTp0965刺激组THP-1细胞穿过HUVEC细胞单层的迁移率显著高于阴性对照组(34.8±1.2%与12.7±0.9%,χ2=13.5,P<0.05),差异有统计学意义。7.RhoA/ROCK信号通路的调控作用用RhoA/ROCK信号通路特异性抑制剂Y27632预处理30min后,再加入rTp0965刺激HUVEC,HUVEC的紧密连接蛋白Claudin-1的表达水平较单用rTp0965刺激组高,THP-1细胞穿过HUVEC细胞单层的迁移率较单用rTp0965刺激组低(19.0±1.0%与34.8±1.2%,χ2=6.3P<0.05),细胞骨架蛋白F-actin的重排程度较单用rTp0965刺激组低。说明RhoA/ROCK信号通路参与调节rTp0965介导的HUVEC单层通透性的增加。结论1.利用原核生物表达外源基因tp0965可获得高纯度rTp0965,此蛋白可适用于梅毒发病机制的相关研究。2.rTp0965可促进HUVEC分泌MCP-1,上调ICAM-1及E-selectin的膜表达水平及mRNA转录水平,促进THP-1细胞向HUVEC的趋化运动,提高HUVEC对THP-1细胞的粘附能力,rTp0965可体外活化血管内皮细胞。3.rTp0965可能通过改变细胞骨架蛋白重排及紧密连接蛋白表达提高血管内皮细胞的通透性,hoA/ROCK信号转导通路参与调节rTp0965介导的血管内皮通透性改变。4.梅毒螺旋体膜蛋白Tp0965在血管内皮细胞的活化及损伤中起一定作用,为梅毒发病机制研究提供依据。更多还原

【Abstract】 BackgroudSyphilis is a chronic systemic, sexually transmitted disease caused by the bacterial spirochete Treponema pallidum subsp. pallidum (T. pallidum). Many evidences demonstrated that activation and damage of endothelial cells may play an important role in pathogenic mechanism of T. pallidum infections. Previous investigations reported that T. pallidum was capable of activating directly host vascular endothelium, up-regulate the expression of adhesion molecules, and promote the adherence of T-lymphocytes to human dermal microvascular endothelial cells. Meanwhile several T. pallidum outer member proteins have been shown to regulate the expression of cell adhesion molecules and binding of T-lymphocytes to human dermal microvascular endothelial cells. T. pallidum is an obligate human pathogen and cannot be cultivated in vitro. This has severely impeded progress in understanding precise pathogenesis of this microbe. The availability of the T. pallidum genome sequence made it possible to examine predicted T. pallidum open reading frames (ORFs) for potential application as diagnostic or immunization tools. This approach permits identification of low-abundant T. pallidum antigens, since they may be expressed as recombinant proteins in much larger quantities. The Protein BLAST data revealed that Tp0965is a membrane fusion protein and is located on periplasm of T. pallidum. Previous studies suggest that Tp0965is reactive with sera from syphilitic individuals at all stages and shows strong immunoreactivity. However, reports concerning the role of Tp0965in the pathogenesis of syphilis are lacking. In the present study, we examined the effects of rTp0965on expression and mRNA levels of adhesion molecules in HUVECs. In addition, we examined the changes in permeability of HUVEC monolayers and transendothelial migration of monocytes. We also investigated effects of rTp0965on the reorganization of F-actin and expression of Claudin-1. The results indicated that rTp0965has the capability of triggering endothelial cell activation and regulates the function of the endothelial barrier. Objective To study the effects of recombinant T. pallidum membrane protein Tp0965on activating of endothelial cells and increasing the permeability of endothelial cell monolayer in vitro, in order to understand its roles in the immunopathogenesis of syphilis.MethodsThe gene of Tp0965was amplified by polymerase chain reaction (PCR) from T. pallidum genomic DNA and the nucleotide sequence was cloned into the expression plasmid pET28a. The new constructs were transformed into E. coli Rosetta (DE3) and the recombinant fusion proteins were purified on Ni-NTA chromatographic column. SDS-PAGE and immunoblot analysis using the anti-polyhistidine tag antibody were employed to identify the protein and assess its purity. Protein concentrations were determined using a BCA Protein Assay Kit. To remove LPS contamination, the recombinant protein was subsequently treated by polymyxin B-agarose and the LPS level was detected by the Limulus amebocyte lysate test kit.After co-culture of the rTp0965with HUVECs, the expression and mRNA transcription levels of ICAM-1and E-selectin were detected on HUVECs’ membrane and in HUVECs by cell enzyme-linked immunosobent assay (Cell ELISA) and fluorescent real-time quantitative PCR, respectively; meanwhile, the level of MCP-1in supernatants was determined by ELISA. To test the effects of rTp0965on monocyte adhesion to HUVECs, we pretreated confluent monolayers of HUVECs and then stimulated them with rTp0965, followed by incubation with THP-1cells and then the adhesion of monocytic THP-1cells was also observed by fluorescence microscopy. To examine the effects of rTp0965on HUVECs chemoattraction of monocytes, monocyte THP-1cells were added to the inserts of transwell systems that contained HUVECs (that had been pretreated with rTp0965) in the wells. Monocyte THP-1cells migration was monitored for2h by fluorescence microscopy.For endothelial permeability and transendothelial migration measurements, HUVECs were treated with rTp0965and then HRP was added on top of the HUVEC monolayers and50μl of samples were taken from the wells. The collected samples were analyzed for the flux of HRP with a TMB kit. For the transmigration assay, CCL-2was added into the wells as a chemoattractant. Monocyte THP-1cells stained with calcein AM were added on top of HUVEC monolayers. Next, the numbers of monocyte THP-1cells in the wells and beneath the HUVEC monolayers were counted using a fluorescence microscope. In some experiments, HUVEC monolayers were pre-incubated with the ROCK inhibitor Y-27632for30mins before the endothelial permeability and transendothelial migration assays were performed as described above.To assess the effects of rTp0965on expression of tight junction proteins of HUVECs, HUVECs were treated with rTp0965and then harvested for detection of Claudin-1expression by Western blot, In some experiments, HUVECs were pre-incubated with Y-27632for30mins before HUVECs were harvested and Western blot was performed. To analyze F-actin distribution, HUVECs were treated with rTp0965then were fixed in4%buffered paraformaldehyde and stained with rhodamine-phalloidin. Finally, the coverslips were examined using a confocal laser scanning microscope system. In some experiments, HUVECs monolayers were preincubated with Y-27632for30mins before F-actin distribution was tested as described above.ResultsAfter expression and purification, the concentrations of rTp0965was210μg/mL and the final LPS level was lower than2.0EU/mL, which is an amount that did not stimulate proinflammatory cytokine production by itself.Real-time RT-PCR study showed that the mRNA transcription levels of ICAM-1and E-selectin were increased significantly after incubation with rTp0965(800ng/ml)(P<0.05) compared to controls. Cell ELISA analysis showed similar results. The rTp0965induced a remarkable increase of ICAM-1and E-selectin, compared to the controls. Adherence assay results showed that rTp0965stimulated an increase in adherence of THP-1cells to HUVECs (48.5±7.5%versus23.3±7.9%, P<0.05).ELISA data showed that the amount of soluble MCP-1was increased significantly after incubation with rTp0965(800ng/ml) compared with the control (P<0.05). Real-time RT-PCR analysis showed similar results. A remarkable increase of MCP-1mRNA, compared to the control, was induced by rTp0965. The chemotaxis assay showed that some monocyte THP-1cells migration was evoked by HUVECs in control group, but significantly more monocyte THP-1cells migrated towards the HUVECs pretreated with rTp0965.To analyze the effects of rTp0965on the permeability of HUVECs monolayers the flux of HRP was calculated. After HRP was added to the transwell inserts for4h, the permeability in rTp0965group was1.2±0.11, and that in control was0.52±0.06(P<0.05). In an in vitro static assay of transendothelial migration, addition of rTp0965(800ng/ml) to confluent monolayers of HUVECs cultured in transwell inserts resulted in an approximately two-fold increase in the migration of monocyte THP-1cells at the4h time point (35.3%versus12.7%, P<0.001). The ROCK inhibitor Y-27632partially blocked the increase in migration of monocyte THP-1cells after24h of rTp0965exposure compared with the group treated with rTp0965but without Y-27632(19.0%versus35.3%, P<0.05).To evaluate the effect of rTp0965on F-actin reorganization in HUVECs, we preincubated HUVECs with rTp0965and then stained the HUVECs with rhodamine-phalloidin. The results showed that in the absence of rTp0965, a rim of F-actin staining was present at the margins of the treated cells, with a few randomly disoriented stress fibers within the cytoplasm. However, in the presence of rTp0965, F-actin rapidly formed organized filamentous networks. Y-27632partially prevented the F-actin reorganization and redistribution after treatment with rTp0965. Western blot proved that after treatment with rTp0965for24h, the level of Claudin-1was markedly decreased in HUVECs and the effect was significantly prevented by the ROCK inhibitor Y-27632.Conclusion1. T. pallidum membrane protein Tp0965could be successfully expressed through E. coli expression system; the protein could be used for the study of the pathogenesis of syphilis.2. The protein Tp0965could increase the levels of ICAM-1, E-selectin, and MCP-1mRNA and protein expression. These increases contributed to the adhesion and chemataxis of monocytes (THP-1cells) to HUVECs preincubated with rTp0965. The protein Tp0965may activate endothelial cells.3. The protein Tp0965induced reorganization of F-actin and decreased expression of Claudin-1in HUVECs and inhanced higher endothelial permeability. Interestingly, inhibition of the RhoA/ROCK signal pathway protected against rTp0965-induced reorganization of F-actin and expression of Claudin-1as well as transendothelial migration of monocytes, indicating that the Rho signaling was involved in the dysfunction of endothelial barrier induced by rTp0965.4. The Tp0965protein may play a certain role in the immunopathogenesis of syphilis via activation as well as demage of vasicular endothelial cells.更多还原