【作者】 张睿娟；

【导师】 杨林花；

【作者基本信息】 山西医科大学 ， 内科学， 2009， 博士

【摘要】 血管内皮细胞是构成血管壁的主要细胞成分,不仅参与创伤、休克、感染、心血管疾病、肿瘤等多种疾病的发生、发展,而且在血管通透性屏障、免疫防御及炎症反应中起着极为重要的作用。生理条件下,血管内皮细胞通过释放内皮衍生松弛因子(EDRF)、血栓调节蛋白(TM)和组织型纤溶酶原激活物(t-PA)等各种活性物质,抑制血小板聚集、血液凝固以及促进纤溶,防止血栓形成,从而维持血流通畅;在IL-1β、LPS、TNF-α等炎性因子的刺激下,血管内皮细胞则可受损,内皮细胞释放的促栓物质和抑栓物质失衡,抗血栓能力减弱,促进血栓形成。人KLF4基因定位于9q31,其蛋白质由470个氨基酸构成。KLF4在体内分布广泛,参与细胞凋亡、增殖、分化以及肿瘤的发生、发展,其调节机制可能与乙酰化修饰有关。1998年,Yet等证实KLF4在人主动脉和脐静脉的内皮细胞中表达,影响内皮细胞炎性调节因子的释放,具有抗炎作用。因此,明确KLF4在炎性因子刺激内皮细胞中的作用,探索KLF4在内皮细胞血栓调节中的作用和可能机制,对阐明炎性因子诱导血栓形成的机制提供实验依据。目的本课题拟通过体外培养人原代脐静脉内皮细胞, IL-1β和TNF-α为炎性刺激因子,观察KLF4、vWF、PAI-1在内皮细胞中的表达变化。采用同源重组方法构建携带反义KLF4基因腺病毒,观察干扰内皮细胞中KLF4表达后,对炎性因子诱导的内皮细胞中vWF、PAI-1的影响,明确KLF4对血栓形成关键物质vWF、PAI-1基因表达的调节,阐明KLF4在内皮细胞血栓调节中的作用,并初步探讨KLF4影响内皮细胞抗栓作用的具体作用机制。方法采用胰蛋白酶消化法,从新鲜脐静脉分离和培养原代人脐静脉内皮细胞(HUVECs),通过形态学和流式细胞仪方法进行鉴定。炎性刺激因子IL-1β(2.5 ng/mL )、TNF-α(10 ng/mL)分别刺激HUVECs 2h、4h后,以正常HUVECs为对照,提取细胞RNA和蛋白。采用Real-time PCR、Western blot、激光共聚焦显微镜技术观察脐静脉内皮细胞中KLF4、PAI-1和vWFmRNA、蛋白的表达。通过同源重组方法构建Ad-反义KLF4腺病毒。将KLF4基因片段反向克隆到重组腺病毒载体Ad-Easy系统中的穿梭质粒pshuttle-CMV的多克隆位点(multiple cloning site, MCS ),构建出携带反义KLF4基因片段的重组穿梭质粒pshuttle-CMV-反义KLF4;再将pshuttle-CMV-反义KLF4与重组腺病毒载体Ad-Easy系统中的骨架质粒pAdEasy-1在大肠杆菌BJ5183内进行同源重组。酶切线性化重组载体并转染293细胞,经293细胞包装生成携带反义KLF4基因片段的重组腺病毒Ad-反义KLF4。将重组腺病毒Ad-反义KLF4按照不同浓度感染人原代脐静脉内皮细胞,确定最佳感染率。采用Real-time PCR、Westernblot方法观察重组腺病毒Ad-反义KLF4对内皮细胞中KLF4表达的抑制效果。重组腺病毒Ad-反义KLF4感染内皮细胞48h后,IL-1β刺激4h,提收取细胞RNA和蛋白。同时,采用Ad-GFP病毒作为对照。Real-time PCR检测内皮细胞中KLF4、PAI-1、vWF mRNA表达;Western blot检测KLF4、PAI-1蛋白的表达;激光共聚焦显微镜观察KLF4、vWF表达和定位。免疫共沉淀技术观察KLF4乙酰化水平变化。观察抑制KLF4表达对炎性因子刺激内皮细胞中PAI-1和vWF表达的影响,以及可能的作用机制。各组实验数据以均数±标准差( x±s)表示。两组数据间的差异用方差分析。P<0.05为差异有显著性。结果1.选用新鲜脐带作为血管内皮细胞的来源,采用0.25%胰蛋白酶消化法分离原代脐静脉内皮细胞(HUVECs),通过形态学和细胞表面特异性标记的方法进行鉴定,倒置相差显微镜下观察细胞呈梭状或鹅卵石样镶嵌排列,单层生长,胞浆丰富,胞核可见,为圆形或椭圆形;流式细胞仪观察到大部分细胞均可表达vWF抗原和CD31抗原,证实所分离和培养的细胞为HUVEC。2. KLF4在正常脐静脉内皮细胞中表达,定位于细胞核中。炎性因子增强内皮细胞中KLF4的表达。随着刺激时间的延长,KLF4表达逐渐增强。明显高于正常HUVECs中KLF4表达,差异具有统计学意义(P<0.05)。3. IL-1β、TNF-α刺激内皮细胞中vWF、PAI-1表达,与正常HUVECs中vWF、PAI-1表达比较,差异具有显著性(P<0.05)。炎性因子刺激4h组中vWF、PAI-1表达高于其在2h组中表达(P<0.05)。其中以IL-1β刺激组中vWF、PAI-1表达增加明显,均高于TNF-α刺激组,但差异无统计学意义(P>0.05)。4.成功地构建出携带反义KLF4基因片段的重组腺病毒Ad-反义KLF4,经菌液PCR、酶切和DNA测序鉴定重组腺病毒完全正确,并在HEK293A细胞中包装、扩增腺病毒,获得高浓度Ad-反义KLF4腺病毒。5.荧光显微镜下观察,以MOI值200的Ad-GFP感染HUVECs 48h后,近90%内皮细胞表达绿色荧光。故选择MOI值200的Ad-反义KLF4腺病毒感染HUVECs细胞,观察KLF4对内皮细胞中vWF、PAI-1的调节。6. MOI值200的重组腺病毒Ad-反义KLF4感染HUVECs后可使KLF4表达明显下降(0.44±0.06),与Ad-GFP组(0.61±0.01)和未感染组(0.59±0.01)比较,差异具有统计学意义(P<0.05)。而Ad-GFP组与未感染组之间差异无统计学意义(P>0.05)。7. Ad-反义KLF4感染HUVECs后,vWF、PAI-1表达随之明显增加(1.17±0.05,0.73±0.01),与感染Ad-GFP组(1.04±0.03,0.67±0.04)相比较,差异具有统计学意义(P<0.05)。IL-1β刺激Ad-反义KLF4感染HUVECs组,vWF、PAI-1表达增加显著(1.90±0.11,0.97±0.02),明显高于单纯炎性刺激组(1.22±0.06,0.86±0.04)(P<0.05)。8.炎性因子IL-1β刺激内皮细胞KLF4乙酰化水平增加。结论1.炎性因子增强内皮细胞中KLF4表达。2. IL-1β、TNF-α刺激内皮细胞vWF、PAI-1表达。3.抑制KLF4表达后,炎性因子诱导vWF、PAI-1表达增强,内皮细胞的促栓作用增强。4. KLF4具有抑栓作用,KLF4乙酰化可能是其机制之一。5. KLF4可能是内皮细胞抗栓作用的转录调节因子。更多还原

【Abstract】 The vascular endothelium plays a critical role in vascular homeostasis, maintain blood fluidity through the production of factors that promote fibrinolysis, inhibit blood coagulation, and inhibit platelet activation. Both biomechanical and biochemical stimuli can affect endothelial gene expression, induce endothelial dysfunction, and confer a pro-adhesive and pro-thrombotic phenotype. The inflammatory cytokines tumor necrosis factor-α(TNF-α)、IL-1βinduce the production of procoagulant factors, antifibrinolytic substances, while inhibiting the expression of natural anticoagulants. Pro-inflammatory cytokines can render the endothelium dysfunction, the endothelial surface loses its nonthrombogenic properties.The human KLF4 (Krüppel-like factors 4)gene localized to chromosome 9q31 and encoded a protein which consists of 470 amino acids. KLF4 might participate in the control of cell proliferation, cell differentiation, cell apoptosis, development of tumor, and acetylation or deacetylation of histone has been associated with the regulation of transcription of KLF4. Recently studies indicate that KLF4 is expressed in endothelial cells, inhibited the inflammatory cytokine-mediated induction of adhesion molecule expression, and may have an anti-inflammatory effect.The precise role of KLF4 in endothelial thrombotic function has not been elucidated. Therefore, our study wants to investigate the impossible role and related mechanisms of KLF4 in endothelium stimulated by inflammatory cytokines. The study raises the experimental evidence on the possible mechanisms that mediate the effects of these stimuli on endothelial function.ObjectiveTo investigate the effect of KLF4 under basal and IL-1β, TNF-αinflammatory cytokine on endothelial thrombotic function by stably transfecting recombinant adenoviral vector carrying antisense KLF4,and obseved differential effects of KLF4 on mRNA and protein levels of vWF,PAI-1 as endothelial key coagulant factors. At the same time, we design to demonstrate the mechanisms involved in it.MethodsFirstly, human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical vein and cultured in endothelial cell medium with 5% fetal bovine serum and 1% penicillin/streptomycin. Identified by microscopy and flow cytometry, HUVECs were treated with human IL-1β, human TNF-αat final concentrations of 2.5 ng/mL and 10 ng/mL, respectively, for 2 hours and 4 hours. HUVECs were subcultured using trypsin/EDTA solution. Total RNA was prepared from HUVECs using the Trizol RNA purification system. Meanwhile, HUVECs were suspended in ice-cold lysis buffer. To detected KLF4, plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF) mRNA expressions by Real Time-PCR, analyzed the changes of KLF4, PAI-1 protein by Western blot, observed the expression and location of KLF4, vWF by confocal laser microscopy, detected the influence of PAI-1 on migration abilities of endothelial cell by a standard wounding assay of HUVECs. Statistical analyses: all data were expressed as x±s. Differences between groups were assessed with variance analysis. P values less than 0.05 were considered to indicate a statistically significant difference.Secondly, Full-length human KLF4 cDNA was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pshuttle-CMV, with the resultant plasmid and the backbone plasmid AdEasy-l, the homologous recombination took place in the E.co1i BJ5183 and the recombinant adenoviral plasmid carrying the antisense KLF4 gene was constructed was generated. The adenoviruses (Ad-KLF4AS) were packaged and amplified in the HEK 293 ce1ls. Then the viral titer was checked by GFP. Recombinant adenoviral vector carrying antisense KLF4 infected the human HUVECs. HUVECs were infected with Ad-GFP or Ad-KLF4AS at 200 MOI for 48 h, and then harvested for total RNA, protein analysis. Study on the inhibitory action of Ad-KLF4AS to KLF4 mRNA and protein expression level in HUVEC.Finally, HUVECs, which has been transfected by recombinant adenoviral vector carrying KLF4 antisence gene at 200 MOI for 48 h, were treated with human IL-1βat final concentrations of 2.5ng/mL, for an additional 4 h. The mRNA expressions of KLF4, PAI-1 and vWF were determined by Real Time-PCR. The changes of protein of KLF4, PAI-1 were analyzed by Western blot. The expression and location of KLF4 and vWF were detected by confocal laser microscopy. The acetylation of KLF4 was performed by immunoprecipitation (IP). All data were expressed as x±s. Analysis of variance (ANOVA) was used to test for significant differences among multiple test groups. In all tests a p-value of 0.05 was considered significant.Results1. Primary human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords following trypsin procedures, and cultured in endothelial cell medium with 5% fetal bovine serum and 1% penicillin/streptomycin. Identified by microscopy and flow cytometry, the common endothelial cell features such as“cobblestone”morphology, positive staining for HUVECs are characterized by flow cytometry with antibodies to factors VIII and CD31.2. Endothelial cells can express KLF4, and usually located on the cell nuclear. Endothelial KLF4 mRNA and protein levels are induced after treatment with TNF- and IL-1β. When time was added, KLF4 expression were increased gradually (P<0.05). Endothelial KLF4 is regulated by inflammatory cytokines. Pro-inflammatory treatment HUVECs led to different localization patterns of KLF4 with an increase in cell cytoplasm.3. Pro-inflammatory mediators significantly up-regulate endothelial PAI-1 and vWF expression.Compared with control group, the expression of PAI-1, vWF mRNA steadily increased since 2 hours to 4 hours after TNF- and IL-1βstimulation(p<0.05). The PAI-1 protein are induced after treatment with TNF- and IL-1βat higer level than in the absence of TNF- and IL-1β. The protein of vWF exhibited a similar result. Immunofluorescence confocal imaging suggested an increased expression of vWF at the protein level by pro-inflammatory stimuli.4. Antisense KLF4 mediated by adenovirus was constructed successfully, the sequence and correct site of adenovirus carring an antisense KLF4 were confirmed by PCR and sequencing assay. The strong green fluorescence was observed in HEK 293 cells by fluorescence microscopy.5. HUVECs were infected with Ad-GFP at a 200 MOI,more than 90% of the cells express GFP by fluorescence microscopy. This system was subsequently used to study the effect of KLF4 on endothelial coagulant function.6. Compared with Ad-GFP infection group and non-infection group, infection group with recombinant adenoviral vector carrying antisense KLF4 at a 200 MOI can cut down the expression of KLF4 gene in HUVEC(P<0.05). But, there were no significant difference in Ad-GFP infection group and non-infection group (p>0.05).7. KLF4 affects the level of endothelial cell vWF and PAI-1 expression. The vWF, PAI-1 mRNA and protein expression in endothelial cells were increased significantly after HUVECs were infected with Ad-KLF4AS as compared with HUVECs exposed to IL-1βand non-infected HUVECs(p<0.05).8. KLF4 in HUVEC is acetylated by pro-inflammatory cytokines.Conclusions1. KLF4 is expressed in vascular endothelial cells of the adult vasculature.2. Endogenous KLF4 expression is highly sensitive to pro-inflammatory stimuli such as TNF- and IL-1β. The remarkably stable transcriptional induction of KLF4 specifically by pro-inflammatory stimuli suggests a role for KLF4 as an intermediary transcriptional regulator of endothelial inflammatory specific gene expression.3. PAI-1 and vWF expression in endothelium are induced by pro-inflammatory stimuli.4. Down-regulated of Krüppel-like factor 4 leads to increase of tumor necrosis factor-αand IL-1βinduced endothelial cell vWF and PAI-1 expression. It implicate that KLF4 inhibit cytokine-mediated induction of the pro-coagulants vWF and PAI-1 expression.5. Acetylation is important for KLF4-mediated transcription in response of pro-inflammatory cytokines.6. The ability to modulate vWF and PAI-1 expression suggest that Krüppel-like factor 4 might play an important role in regulator of endothelial coagulant function in response to pro-inflammatory stimuli and may be an important target for acetylation effects.更多还原