【作者】 杨梅；

【导师】 谢秀梅； 胡锦跃；

【作者基本信息】 中南大学 ， 内科学， 2012， 博士

【摘要】 第一章聚肌胞(PolyⅠ:C)对人脐血内皮祖细胞增殖的影响及其机制研究第一节人脐血内皮祖细胞表达Toll样受体的研究目的：分离纯化人脐静脉血来源的单个核细胞并诱导培养为内皮祖细胞,观察Toll样受体在人脐血内皮祖细胞中的表达情况。方法：采用密度梯度离心法分离人脐静脉血中的单个核细胞,用含多种生长因子的内皮祖细胞专用EBM-2培养基诱导培养,诱导单个核细胞向内皮祖细胞分化,通过形态学、免疫荧光及逆转录多聚酶链反应(RT-PCR)检测并鉴定细胞的生物学特性；采用RT-PCR法检测静息状态下人内皮祖细胞(EPCs)表达的Toll样受体亚型；检测不同浓度的PolyⅠ:C对内皮祖细胞表达TLR3受体、炎性细胞因子的影响。结果：1.分离培养的内皮祖细胞早期以集落为中心呈放射状生长,晚期形成典型的“铺路石”样改变。荧光染色示DiL-acLDL和FITC-UEA-I双染色阳性；RT-PCR法检测示细胞表达干细胞表面标志物CD34、CD133、KDR,证明为内皮祖细胞。2.静息状态下,内皮祖细胞表达较高水平的TLR1、TLR3、TLR4、TLR6,表达较低水平的TLR2、TLR5、TLR7、TLR8、TLR10,不表达TLR9。而PolyⅠ:C能上调TLR3的mRNA及蛋白表达,并呈量效关系。3. PolyⅠ:C通过活化TLR3剂量依赖性上调炎性细胞因子IL-1p、IL-6、IL-8、TNF-α、IFN-p的基因表达。结论：EPCs表达功能性TLR3；PolyⅠ:C能呈剂量依赖性上调TLR3在EPCs的mRNA及蛋白表达,并通过活化TLR3剂量依赖性上调炎性细胞因子的mRNA表达。第二节PolyⅠ:C对人脐血内皮祖细胞增殖、凋亡的影响目的：观察TLR3的配体PolyⅠ:C对人脐血内皮祖细胞增殖、凋亡的影响。方法：采用不同浓度的PolyⅠ:C持续干预人脐血内皮祖细胞(EPCs),通过CCK-8细胞增殖试验检测其对EPCs增殖的影响及时间效应；通过流式细胞术检测不同浓度的PolyⅠ:C对EPCs细胞周期时相分布及细胞凋亡的影响。结果：1.与对照组相比,较高浓度PolyⅠ:C(1,10μg/m1)持续作用于脐血内皮祖细胞3天后显著抑制内皮祖细胞增殖；终浓度10μg/ml的PolyⅠ:C呈时间依赖性抑制内皮祖细胞增殖。2.较高浓度的PolyⅠ:C(1,10μg/m1)显著减少S期和G2/M期细胞比例,并通过下调细胞周期素A,B1,D1,E的表达阻滞细胞周期于G0/G1期而改变细胞周期的时相分布,从而抑制EPCs的增殖。3. PolyⅠ:C呈剂量依赖性诱导内皮祖细胞凋亡。结论：高浓度的PolyⅠ:C通过将细胞周期阻滞于G0/G1期并诱导细胞凋亡从而抑制EPCs增殖。第三节PolyⅠ:C诱导人脐血内皮祖细胞凋亡的机制研究目的：阐明PolyⅠ:C诱导人脐血内皮祖细胞凋亡的具体机制。方法：利用Caspase8的抑制剂和Caspase9的抑制剂抑制相应的信号分子,分析其对PolyⅠ:C诱导细胞凋亡的作用。以不同浓度的TNF-α及IL-1β干预EPCs24h后,CCK-8细胞增殖试验分别检测TNF-α和IL-1β对细胞凋亡的影响。利用IL-1β受体的中和抗体Anti-IL-1R1预先封闭IL-1β的结合位点,分析其对PolyⅠ:C诱导细胞凋亡的作用。结果：1. Caspase8和9的抑制剂并不能抑制PolyⅠ:C诱导的细胞凋亡。2.随着TNF-α作用浓度的增高,EPCs的细胞凋亡率并无明显增加。3.随着IL-1p作用浓度的增高,EPCs的细胞凋亡率逐步增加,并呈剂量依赖性。使用IL-1p受体的中和抗体Anti-IL-1R1预先封闭IL-1p的结合位点后,再加入PolyⅠ:C干预24h,结果显示高浓度的Anti-IL-1R1可以部分抑制PolyⅠ:C诱导的细胞凋亡。结论：PolyⅠ:C活化TLR3后通过上调IL-1p的表达诱导EPCs凋亡,而内、外源性细胞凋亡途径及TNF-a不参与PolyⅠ:C诱导的细胞凋亡第二章Pam3CSK4在人脐静脉内皮细胞中诱导非耐受性的细胞因子表达第一节Pam3CSK4对人脐静脉内皮细胞表达炎症因子的影响及其可能机制目的：阐明TLR1/2激动剂Pam3CSK4对人脐静脉内皮细胞表达炎症因子的影响及其可能机制。方法：以不同浓度的Pam3CSK4干预人脐静脉内皮细胞(HUVECs), RT-PCR法检测Pam3CSK4对TLR1、TLR2mRNA表达的影响。用RT-PCR法、Real-time RT-PCR法、ELISA法检测Pam3CSK4对HUVECs分泌TNF-α、IL-6的时间效应及剂量效应。分别用TLR1、TLR2及TLR1阻断剂阻断相应受体的表达,观察其对Pam3CSK4诱导HUVECs分泌TNF-α、IL-6的影响。用Western-blot法检测Pam3CSK4对MAPK家族及NF-κB信号转导通路的活化情况。结果：Pam3CSK4呈剂量依赖性上调TLR2的mRNA表达,而对TLR1mRNA表达的影响不明显。Pam3CSK4呈剂量依赖性上调TNF-α、IL-6的基因及蛋白表达量。加入TLR1、TLR2、TLR1/2受体阻断剂后均能明显抑制Pam3CSK4诱导的TNF-a、IL-6的基因表达上调,其中以TLR1/2受体阻断剂的阻断作用最明显。Pam3CSK4与TLR2结合后可以不同程度活化MAPK家族和NF-κB信号传导通路。结论：HUVECs表达功能性TLR2；Pam3CSK4与TLR2结合后活化MAPK家族和NF-κB信号传导通路,从而诱导炎症因子TNF-α、IL-6的分泌,且TNF-α、IL-6的分泌依赖于TLR1及TLR2的表达。第二节Pam3CSK4在人脐静脉内皮细胞中诱导非耐受性的IL-6表达目的：阐明预处理的TLR1/2激动剂Pam3CSK4重复刺激对人脐静脉内皮细胞表达炎症因子的影响及其可能机制。方法：以不同浓度的Pam3CSK4预处理人脐静脉内皮细胞(HUVECs)后,再用Pam3CSK4重复刺激HUVECs,用RT-PCR法、Real-time RT-PCR法、ELISA法检测Pam3CSK4对HUVECs分泌TNF-α、IL-6的影响。用不同TLR受体的配体预处理HUVECs后,再用Pam3CSK4重复刺激HUVECs,以观察不同TLR受体之间的耐受情况。Western-blot法检测Pam3CSK4重复刺激对MAPK家族及NF-κB信号传导通路的活化情况。RT-PCR法检测Pam3CSK4对负性调节蛋白锌指蛋白A20基因表达的影响,然后用质粒转染试验检测A20的过度表达对Pam3CSK4及LPS诱导的IL-6分泌的影响。用Notch信号通路抑制剂抑制相关分子,观察其对Pam3CSK4诱导HUVECs分泌IL-6的影响。结果：给予HUVECsPam3CSK4预处理后,再给予Pam3CSK4重复刺激可以下调TNF-a的基因及蛋白表达,而对IL-6表达无影响。LPS预处理可以使Pam3CSK4诱导的TNF-α表达下降,而对IL-6表达无影响。Pam3CSK4重复刺激HUVECs能抑制JNK通路的活化,而对p38、ERK、NF-κB信号通路的活化无影响。Pam3CSK4呈剂量依赖性上调负性调节蛋白锌指蛋白A20的基因表达,A20的过度表达对Pam3CSK4及LPS诱导的IL-6分泌无明显影。Notch信号通路抑制剂能部分抑制Pam3CSK4诱导的IL-6分泌。结论：1.Pam3CSK4重复刺激可诱导耐受性的TNF-α表达,而诱导非耐受性的IL-6表达。2.TLR2与TLR4之间存在交叉耐受性。3.Pam3CSK4可能通过Notch信号通路诱导非耐受性的IL-6表达。更多还原

【Abstract】 Charpter I The Possible Mechanisms Responsible for the Effects of PolyⅠ:C on the Proliferation of Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsPart Ⅰ The Expression of Toll-like receptors on the Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsObjective:To observe the expression of Toll-like receptors on the human umbilical cord blood-derived endothelial progenitor cells (EPCs).Methods:Mononuclear cells were isolated from fresh cord blood by density gradient centrifugation and cultured in EBM-2medium. The biological features of the isolated cells were observed at different time point and identified by morphology, immunofluorescence staining and RT-PCR. Furthermore, RT-PCR was performed firstly to detect the mRNA expression of Toll-like receptor subtypes in EPCs at rest, secondly the expression of Toll-like receptor3(TLR3)and inflammatory cytokines induced by PolyⅠ:C at different concentrations.Results:1. Early EPCs changed from small size round cells to spindle shape cells,Late EPCs formed a typical cobblestone-like cells.Adherent EPCs were characterized by DiLDL uptake and concomitant lectin binding, and by expressing of CD34、CD133and KDR, which are markers of stem cells.2. RT-PCR results shown that Under basal condition, except TLR9, TLR1～TLR10were expressed in EPCs with high expression levels of TLR1、TLR3、TLR4. TLR6and low expression levels of TLR2、TLR5、 TLR7、TLR8、TLR10. Expression of TLR3was significantly up-regulated by treatment with Poly I:C at both the gene and protein levels and there was dose-effect relationship in experiment range.3. PolyI:C upregulated the mRNA expression of inflammatory cytokines such as IL-1β、IL-6、IL-8、TNF-α、IFN-β by activating TLR3.Conclusions:EPCs expressed functional TLR3; PolyI:C can up-regulate the expression of TLR3at both the gene and protein levels and up-regulate the gene expression of inflammatory cytokines in a dose-dependent manner by activating TLR3. Part Ⅱ The Effect of PolyⅠ:C on the Proliferation and Apoptosis of Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsObjective:To analyze the effect of TLR3agonist PolyⅠ:C on the proliferation and apoptosis of human umbilical cord blood-derived endothelial progenitor cells.Methods:EPCs were treated with different concentrations of PolyⅠ: C sequentially for3days, and then cell numbers were measured by CCK-8assay.EPCs were treated with Polyl:C at different concentrations, the phase of cell cycle was tested by PI staining and cell apoptosis was detected by the PI/Annexin V staining.Results:1.Compared with the control group, PolyⅠ:C at high concentrations of1and10μg/ml significantly inhibited the proliferation of EPCs after3days treatment, and the inhibition of cell proliferation by Poly I:C was time-dependent.2. Poly Ⅰ:C at high concentrations of1and10μg/ml significantly decreased the proportion of cells in S phase and G2/M phase. Moreover, Poly Ⅰ:C down-regulated the gene expression of cyclins A, B1, D1, and E, inducing cell cycle arrest in G0/G1phase, thus inhibiting the proliferation of EPCs.3. PolyI:C induced the apoptosis of EPCs in a dose-dependent manner.Conclusions:PolyⅠ:C at high concentrations inhibited EPCs proliferation by inducing cell cycle arrest in G0/G1phase and inducing cell anoptosis of EPCs. Part Ⅲ The Possible Mechanisms Responsible for the Effects of PolyⅠ:C on the Apoptosis of Human Umbilical Cord Blood-Derived Endothelial Progenitor CellsObjective:To elucidate the mechanism of PolyⅠ:C induced apoptosis in human umbilical cord blood-derived endothelial progenitor cells.Methods:We used specific inhibitors of Caspase8and Caspase9to inhibit the corresponding signal molecules, then tested the effect of caspase inhibition on Poly Ⅰ:C-induced apoptosis. EPCs were treated with different concentrations of TNF-α and IL-1β for24h respectively,then CCK-8assay was used to detect the apoptosis of EPCs. EPCs were pre-treated with IL-1receptor1neutralizing antibody(Anti-IL-1R1),then re-stimulated with PolyⅠ:C, cell apoptosis was measured by CCK-8assay.Results:1.Caspase8and Caspase9inhibitors did not reduce PolyⅠ:C-induced apoptosis of EPCs.2. When EPCs were treated with increasing concentration of TNF-α, cell apoptosis was not increase in TNF-α-treated groups.3. IL-1β induced cell apoptosis in a dose-dependent manner. Moreover, when EPCs pre-treated with Anti-IL-1R1, were re-stimulated with PolyⅠ:C, the cell apoptosis induced by Poly Ⅰ:C was decreased.Conclusions:PolyⅠ:C induced the apoptosis of EPCs through up-regulating the expression of IL-1β via activating TLR3. Endogenous and exogenous apoptosis pathway and TNF-α did not contribute to PolyⅠ:C-induced cell apoptosis. Charpter II Pam3CSK4Induces a Non-tolerant Up-regulation of Cytokine in Human Umbilical Vein Endothelial CellsPart I The Possible Mechanism Responsible for the Effect of Pam3CSK4on the Expression of Cytokines in Human Umbilical Vein Endothelial CellsObjective:To observe the effects of TLR1/TRL2agonist Pam3CSK4on the expression of cytokines in HUVECs and to elucidate the possible mechanism.Methods:HUVECs were treated with different concentrations of Pam3CSK4, RT-PCR was used to detect the mRNA expression of TLR1and TLR2. Then, RT-PCR、Real-time RT-PCR and ELISA were performed to detect the effect of Pam3CSK4on the expression of TNF-a and IL-6at both mRNA and protein level. We used specific inhibitors of TLR1、TLR2and TLR1/TLR2to inhibit the corresponding receptors, then tested the effect of TLR1、TLR2inhibition on the expression of TNF-a and IL-6induced by Pam3CSK4. In order to elucidate the signaling pathways involving in this process, Western-blot was performed to detect the phosphorylation of MAPK and NF-κB.Results:After treated with different concentrations of Pam3CSK4for24h, the RT-PCR results show that Pam3CSK4could significantly up-regulate the mRNA expression of TLR2,the effect of Pam3CSK4on TLR1mRNA expression was not obvious. Pam3CSK4up-regulated the expression of TNF-α and IL-6at both mRNA and protein level in a dose-dependent manner.TLR1、TLR2and TLR1/TLR2inhibitors could significantly inhibit the up-regulation of TNF-α and IL-6induced by Pam3CSK4,and the inhibition induced by TLR1/TLR2inhibitor was most obvious. The Western-blot results showed that Pam3CSK4could induce the phosphorylation of MAPK and NF-κB signal transduction pathways via TLR2activation.Conclusions:HUVECs expressed functional TLR2.Pam3CSK4induced the phosphorylation of MAPK and NF-κB signal transduction pathways via TLR2activation,thus significantly up-regulate the expression of TNF-a and IL-6at both mRNA and protein level which was TLR1and TLR2dependent. Part II Pam3csk4Induces a Non-tolerant Up-regulation of Cytokine IL-6in Human Umbilical Vein Endothelial CellsObjective:To observe the effect of Pam3CSK4pre-treatment on cytokine expression in HUVECs induced by Pam3CSK4re-treatment and to elucidate the possible mechanism.Methods:HUVECs, pre-treated with1μg/ml Pam3CSK4for indicated hours, were re-treated with1μg/ml Pam3CSK4for1hour. The gene and protein expression of cytokine IL-6and TNF-a were detected by Real-time RT-PCR and ELISA respectively.Then Real-time RT-PCR and ELISA were performed to detect the effect of various PAMP pre-treatment on cytokine gene and protein expression induced by Pam3CSK4re-treatment,observing the cross-tolerance between different TLRs. Then we used western-blot to detect the phosphorylation of MAPK and NF-κB signal transduction pathways after Pam3CSK4repeating stimulation. The effect of Pam3CSK4on the gene expression of ubiquitin-editing enzyme A20was detected by RT-PCR, and the effect of A20over-expression on cytokine IL-6expression induced by Pam3CSK4and LPS was detected by plasmid transfection. We used inhibitors of Notch signal transduction pathway to inhibit the corresponding signal molecules, then tested the effect of Notch signal transduction pathway inhibition on the expression of IL-6induced by Pam3CSK4. Results:Pam3CSK4pre-treatment could down-regulate the expression of TNF-a induced by Pam3CSK4at both gene and protein level,but had no effect on the expression of IL-6. LPS pre-treatment could down-regulate the expression of TNF-a induced by Pam3CSK4,but had no effect on the expression of IL-6.The Western-blot results showed that Pam3CSK4repeating stimulation inhibited activation of JNK, without influencing the phospholyration of p38、ERK、NF-κB. Pam3CSK4down-regulated the mRNA expression of ubiquitin-editing enzyme A20in a dose-dependent manner,but A20over-expression had no effect on the the expression of IL-6induced by Pam3CSK4and LPS. Inhibitors of Notch signal transduction pathway could partly inhibit the expression of IL-6induced by Pam3CSK4.Conclusions:Pam3CSK4induced a tolerant up-regulation of cytokine TNF-a, but induced a non-tolerant up-regulation of cytokine IL-6.There was cross-tolerance between TLR2and TLR4. Pam3CSK4probably induced the non-tolerant up-regulation of cytokine IL-6via Notch signal transduction pathway.更多还原