摘要
前言
子痫前期是妊娠期特有的疾病，多发生于妊娠20周以后，以高血压、蛋白尿和水肿为特征，严重威胁孕产妇和围生儿的健康。对子痫前期发病机制的研究是目前围生医学研究领域内的热点。新近的研究相继发现，一些细胞因子表达的改变在子痫前期发生机制中发挥重要作用。这些细胞因子可分为二类。第一类是与炎症发生有关的细胞因子。许多研究表明，子痫前期是由未知原因诱发的全身性过度炎症反应引起的血管内皮细胞损伤所致。外周循环内炎症细胞因子的大量增加是全身性过度炎症反应的重要特征。在子痫前期患者外周血中，不仅有多种前炎症细胞因子的高表达，而且有一些抑炎性细胞因子的低表达。在这些细胞因子中，肿瘤坏死因子-α的作用备受关注。肿瘤坏死因子-α是一种重要的炎症介质和免疫调节因子。肿瘤坏死因子-α的高表达可能与子痫前期的发病机制有关。子痫前期患者在临床症状出现之前就已有外周血及羊水肿瘤坏死因子-α及肿瘤坏死因子-α受体含量的显著增加。肿瘤坏死因子-α在血清中的含量与病情呈正相关，病情越重，肿瘤坏死因子-α的含量越高。肿瘤坏死因子-α可以造成血管内皮损伤，抑制一氧化氮的生成与释放、促进细胞凋亡，导致子痫前期的发生。第二类是生长因子。一些生长因子可以通过调节胎盘细胞和母体免疫细胞的生长和功能参与子痫前期的发病。子痫前期胎盘和外周血中一些生长因子如血管内皮细胞生长因子、血小板衍生生长因子和白介素-2等的表达发生了明显的改变。
目前，关于细胞因子在子痫前期表达发生变化的原因和机制尚未完全明了，有待进一步研究。一些研究提示胎盘组织细胞因子表达水平的改变是导致子痫前期细胞因子表达变化的主要原因。在胎盘组织发现了前炎症细胞因子和生长因子表达的显著改变。但是，目前并不清楚是何种原因导致细胞因子在胎盘组织的表达发生改变。既往的研究表明，滋养层细胞侵入能力下降，导致胎盘浅着床和缺氧，可能是引起细胞因子释放的重要原因。然而，最新的研究表明胎盘滋养层细胞具有天然免疫细胞特性，产生类似于天然免疫细胞的模式识别作用。这可能是胎盘细胞因子表达改变的基础。Toll样受体-4是天然免疫系统中一类重要的模式识别受体。已经发现滋养层细胞能够表达Toll样受体-4。Toll样受体-4的主要配体就是脂多糖，一种来源于革兰氏阴性细菌细胞壁的毒素。脂多糖在感染发生时可以进入机体，引起单核巨噬细胞等天然免疫细胞合成释放大量炎症细胞因子。这是败血症、脓毒血症发生全身性过度炎症的主要原因。同期的研究还发现脂多糖与子痫前期的发生有密切关系。不仅多项研究表明妊娠期细菌性感染是子痫前期重要的诱发因素，而且动物实验也证实采用脂多糖可以制备子痫前期的大鼠模型。综合上述这些研究，可以发现脂多糖作用于滋养细胞，引起细胞因子表达的改变极有可能是子痫前期重要的发病机制。
在滋养细胞，已发现脂多糖能够诱导一些细胞因子，如肿瘤坏死因子-α、白介素-6、白介素-10和干扰素-γ等。但是，它们在细胞因子网络中仅仅是沧海一粟。除了对上述细胞因子，脂多糖有可能调节其它多种与子痫前期发生有密切关系的细胞因子在细胞滋养细胞的表达。由于细胞因子的种类繁多，作用机理纷繁复杂，单独从一种细胞因子或一个侧面孤立地来研究细胞因子与子痫前期的关系是不充分不全面的。所以，本研究将在两个层面上对脂多糖对细胞滋养细胞表达细胞因子的调节作用及其与子痫前期的关系进行探讨。首先，在“宏观”层面上，利用一种新的蛋白质组学研究工具—细胞因子抗体芯片来研究脂多糖对细胞滋养细胞表达细胞因子的调节作用。细胞因子抗体芯片能够在一次实验中高通量的研究多种细胞因子的表达。本文采用的Raybiotech Human Cytokine Antibody ArrayⅢ，能够同时检测42种细胞因子的表达。这些细胞因子涵盖了目前已知的与炎症、免疫调节和细胞生长有关的多数细胞因子。其次，考虑到肿瘤坏死因子-α在子痫前期发病机制中的特殊作用，在“微观”层面上，观察脂多糖对肿瘤坏死因子的诱导作用及其对细胞滋养细胞凋亡诱导作用的关系，并观察肿瘤坏死因子和凋亡蛋白半胱氨酸蛋白酶-3在子痫前期胎盘中的表达，进一步揭示肿瘤坏死因子-α的表达在子痫前期病理生理机制中的作用。
目的
本研究通过检测脂多糖对体外无血清条件下培养的细胞滋养细胞表达细胞因子的调节作用，探讨这些细胞因子与子痫前期的关系；观察脂多糖对细胞滋养细胞凋亡的诱导作用，以及肿瘤坏死因子在此过程中的作用。同时，观察肿瘤坏死因子-α和凋亡蛋白半胱氨酸蛋白酶-3在子痫前期患者胎盘的表达。在这些基础上，揭示细胞滋养细胞的天然免疫细胞特性，阐明脂多糖调节细胞因子在细胞滋养细胞的表达及其与子痫前期的关系。
方法
采集早孕绒毛、分离细胞滋养细胞，采用无血清培养基进行培养。采用人细胞因子抗体芯片观察脂多糖对细胞滋养细胞表达细胞因子的调节作用；采用酶联免疫吸附实验、免疫荧光、激光共聚焦、Hoechst DNA染色、透射电镜、流式细胞仪、Western blot等技术观察脂多糖对细胞滋养细胞凋亡的调节作用，以及肿瘤坏死因子-α在此过程中的作用。采用免疫组织化学方法观察子痫前期胎盘组织肿瘤坏死因子-α和半胱氨酸蛋白酶-3的表达。
结果
细胞因子抗体芯片结果表明，脂多糖对细胞因子在细胞滋养细胞的表达有调节作用。脂多糖促进CXC类趋化因子生长基因相关产物和生长基因相关-α的表达，抑制CC类趋化因子单核细胞趋化蛋白-2、巨噬细胞炎症蛋白-1δ和RANTES的表达；脂多糖促进前炎性细胞因子白介素-1β、白介素-6、白介素-12p40p70、干扰素-γ、肿瘤坏死-α和抑瘤素M的表达，抑制抑炎性细胞因子白介素-4和白介素-13的表达，但是促进抑炎性细胞因子白介素-10的表达；脂多糖促进生长因子白介素-2、白介素-7、血管内皮细胞生长因子和血小板衍生生长因子的表达，抑制表皮生长因子、白介素-15和血管紧张素原的表达；脂多糖对单核细胞趋化蛋-1、单核细胞趋化蛋-3、巨噬细胞衍化趋化因子、肿瘤坏死因子-β、胰岛素样生长因子—1、单核细胞集落刺激因子、白介素-5、上皮细胞—中性粒细胞活化肽-78、单核细胞因子、粒细胞集落刺激因子、干细胞因子、瘦素、基质细胞源性细胞因子-1、I-309、TRAC和TGF-β1的表达没有影响。
通过对形态学、免疫学等定性、定量分析表明，脂多糖在体外能够诱导细胞滋养细胞凋亡，并呈现剂量依赖关系；ELISA和激光共聚焦研究表明，脂多糖促进体外培养的细胞滋养细胞分泌肿瘤坏死因子，亦呈现剂量依赖关系；相关分析表明，肿瘤坏死因子的表达与细胞凋亡指数正相关。
对子痫前期患者的研究表明，胎盘组织肿瘤坏死因子和caspase-3的表达明显高于正常孕妇。
结论
因为细胞滋养细胞具有天然免疫细胞特性，所以脂多糖能够调节一些与子痫前期的发生有密切关系的细胞因子在细胞滋养细胞的表达；脂多糖促进CXC类趋化因子的表达，活化中性粒细胞；促进前炎性细胞因子，抑制抗炎性细胞因子的表达，诱发和加剧母胎界面和母体全身炎症；调节多种生长因子的表达，抑制细胞滋养细胞的生长，引起Th1／Th2比例失衡。本文首次发现脂多糖能够调节Oncostatin M、IL-7、IL-13、GRO、和GRO-α在细胞滋养细胞的表达。肿瘤坏死因子-α促进细胞滋养细胞凋亡，在子痫前期的发病机制中有重要作用。这些研究表明妊娠期细菌性感染，特别是革兰氏阴性细菌感染是子痫前期的重要诱发因素，其机制是由脂多糖引起细胞滋养细胞所表达的细胞因子发生改变，后者直接或间接参与子痫前期的发病机制。抗感染治疗对子痫前期的防治有重要意义。
Introduction
Pre-eclampsia is a pregnant-specific disease, which seriously endangers health ofpregnancy and perinatal fetus, and is characterized by hypertension, proteinuria andedema. The study of preeclampsia mechanism is the focus in the researching field ofperinatal medicine. Systemic inflammatory response syndrome (SIRS) is the pivotal inpreeclampsia. Pro-inflammatory cytokines increase in circulation in SIRS. It isindicated that these cytokines present in pre-eclampsia as well. In further study, it isconfirmed that placenta is the resource of those cytokines. But the cause that results incytokine releasing in placenta remains unclear. It is suggested that decreased invasionof cytotrophoblast lead to shallow placentation and hypoxia, which may be the reasonfor cytokine releasing in placenta. However, recent studies indicate thatcytotrophoblast has some properties of innate immune cells. Molecular patternrecognition in cytotrophoblast may be the cause of cytokine releasing in placenta. Tolllike receptor-4 is expressed in cytotrophoblast. Toll like receptor -4 is a type of patternrecognition receptor in innate immune system. Its ligand is lipopolysachharide, a kindof toxin from gram-negative bacterial cell wall. Lipopolysachharide enter body wheninfections occur. Innate immune cell such as mononuclear cell receiveslipopolysachharide stimulation and product pro-inflammation cytokines. It is themechanism by which system inflammatory response syndrome occurs in septicaemiaand pyamia. Lipopolysachharide is found to be associated with pre-eclampsia. It is speculated that LPS can stimulate cytotrophoblast to release cytokines, which play animportant role in pre-eclampsia.
Cytokines such as tumor necrosis factor-α、interleukin-6、interleukin-10 andinterferon-γwere found to be produced in cytotrophoblast. However, cytokine has agreat variety and complicated mechanism. It is insufficient to put up conclusionsaccording to investigating the role of one or a few cytokines in pre-eclampsia.Therefore, we will preliminarily explore the regulation effects of lipopolysachhadde oncytokine releasing in cytotrophoblast and its relationship with pre-eclampsia. Weconduct this study in two aspects. Firstly, in the macro-aspect, we use a new proteomeinvestigation tool, cytokine antibody arrays, to study the regulation effect of LPS oncytokine releasing in cytotrophoblast. Cytokine antibody arrays can simultaneouslydetermine a variety of cytokines. In this study, we will use Raybiotech HumanCytokine Antibody ArrayⅢ, which can determine 42 kinds of cytokine at the sametime. Secondly, in the micro-aspect, we will explore the effects of lipopolysachharideon tumor necrosis factor-a releasing and apoptosis in cytotrophoblast and observe theexpression of tumor necrosis factor-αin placenta.
Tumor necrosis factor-α, which mostly derived from mononuclear phagocyte, hasgeneral biologic activity. Tumor necrosis factor-a is an important pro-inflammatoryand immune-regulation media. It plays roles in pathophysiological mechanism indiseases. A low level of tumor necrosis factor-αis found in epithelium and basemembrane in endometrium and ovarian stroma in normal conditions. In normalpregnancy, tumor necrosis factor-αis expressed in fetus and deciduas, and has immuneregulation effects between mother and fetus. However, tumor necrosis factor-αismarkly elevated in pre-eclampsia. The levels of tumor necrosis factor-αand tumornecrosis factor-αreceptor have increase before clinical symptom and sign occur inpre-eclampsia. It is indicated that the level of tumor necrosis factor-αis positivecorrelation with the condition of pre-eclampsia. The mechanism of tumor necrosis factor-αresults in pre-eclampsia is that it injuries vascular endothelial cells, inhibits theproduction and releasing of nitrogen monoxidum and induced apoptosis.
Objective
Cytokine antibody arrays were employed to determine the levels of cytokinesreleased by cytotrophoblast with LPS treatment. Further, the effect of LPS on apoptosisof cytotrophoblast and the role of tumor necrosis factor-αin the procession wereinvestigated. At last, the tumor necrosis factor-at expression in placenta inpre-eclampsia was observed. On the base of the above studies, we explore therelationship between cytokine releasing induced by LPS in cytotrophoblast andpre-eclampsia.
Methods
Cytotrophoblast was isolated from early pregnant villous and cultured inserum-free media. It was treated with LPS at variety concentrations. RaybiotechHuman Cytokine Antibody ArrayⅢwas used to determine expression of cytokinesproduced in cytotrophoblast with treatment of lipopolysachharide. Hoechst DNAstaining, flow cytometry, and electron transmission microscope were used to determinethe apoptotic-inducing effect of lipopolysachharide on cytotrophoblast. To explore thetumor necrosis factor-αexpression in cytotrophoblast, confocal microscope andenzyme-linked immunosorbent assay were used. At the end, immunochemistry wasemployed to determine the expression of tumor necrosis factor-αin placenta frompre-eclampsia patients.
Results
Results of Raybiotech Human Cytokine Antibody ArrayⅢindicated thatlipopolysachharide could regulate cytokine expression in cytotrophoblast. It increasedthe expression of CXC chemokines such as growth related gene production and growthrelated gene production-α, and inhibited the expressions of CC chemokines expressionsuch as monocyte chemoattractant protein-2, macrophage inflammatory protein 1 δ,and reduced upon activation, normal T cell expressed and secreted. It promoted theexpression of interleukin-1β, interleukin-6, interleukin-12, interferon-γand of tumornecrosis factor-αand oncostatin-M, which were pro-inflammatory cytokines, andinhibited the expression of interleukin-4 and interleukin-13, which wereanti-inflammatory cytokines. But, it increased the expression of anti-inflammatorycytokine, interleukin-10. About growth factor, lipopolysachharide had a promotingeffect on the expression of interleukin-2, interleukin-7, vascular endothelial growthfactor, and platelet derived growth factor. However, it down-regulated the expressionof epidermal growth factor, interleukin-15, and angiogenin, lipopolysachhafidecould not alter the expressions of monocyte chemoattractant protein-2, monocytechemoattractant protein-3, macrophage derived chemokines, epthelial-neutrophilactivating peptide, tumor necrosis factor-β, insulin-like growth factor-1、macrophagecolony stimulating factor, interleukin-5, monokine induced by interferon gamma,granulocyte colony stimulating factor, stem cell factor, Leptin,stromal-dereivedfactor-1, I-309, TRAC and transforming growth factor-β1 incytotrophoblast.
The results of Hoechst DNA staining, electron transmission microscope and flowcytometry showed that lipopolysachharide induced apoptosis of cytotrophoblast in adose-dependent manner. The results of enzyme-linked immunosorbent assay andconfocal microscope showed that lipopolysachharide induced tumor necrosis factor-αexpression in cytotrophoblast in a dose-dependent manner, too. A positivecorrelationship was found between apoptosis and expression of tumor necrosisfactor-α.
In immunochemistry study, it was revealed that tumor necrosis factor-αexpression in pre-eclampsia was increased, as contrast to normal pregnancy.
Conclusions
Lipopolysachharide regulated cytokine expression in cytotrophoblast. Thesecytokines played important role in inflammatory reaction, vascular endothelial injuryand immune imbalance, which were important mechanism in pre-eclampsia. Tumornecrosis factor-a induced apoptosis in cytotrophoblast and was tightly connected withpre-eclampsia. All these revealed that infection in pregnancy was predisposing factorof pre-eclampsia and antibiotic treatment had great value in prevention and treatmentfor pre-eclampsia.
引文
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