【作者】 贺银燕；

【导师】 李大金； 金莉萍； 杜美蓉；

【作者基本信息】 复旦大学 ， 妇产科学， 2007， 博士

【摘要】 趋化因子是一类小分子的肝素结合蛋白，分子量通常为8-14kDa，趋化因子和其含有七次跨膜结构的G蛋白偶联受体结合，在病理生理反应中发挥重要的作用。四年前我们率先开展了母胎界面趋化因子调控机制的研究，发现趋化因子和趋化因子受体在母胎免疫调节中发挥重要的作用。我们发现，早孕滋养细胞能分泌CXCR4的配体SDF-1和CXCR6配体的CXCL16。CXCR4／SDF-1相互作用，能募集CD56^brightCD16^-NK到达蜕膜局部。CXCR6／CXCL16结合，能够趋化外周血T细胞、γδT和单核细胞到达母胎界面，并且促进这些蜕膜局部免疫活性细胞的黏附。因此，在母—胎界面，趋化因子是蜕膜免疫活性细胞和滋养细胞相互作用和对话的媒介，而且不同的趋化因子受体／配体对作用于不同的免疫活性细胞，各自发挥着不同的作用，共同参与母胎免疫调节。CCL2是CC类趋化因子中最早被鉴定的趋化因子之一，CCL2和受体CCR2结合后，在炎症、动脉粥样硬化、肿瘤等过程中发挥重要的作用。CCL2还能诱导Th2型免疫应答。本研究中筛选了人早孕蜕膜组织和蜕膜基质细胞18种趋化因子受体和相关配体的表达，发现蜕膜组织和蜕膜基质细胞高水平转录CCL2和CCR2。我们以此为切入点，展开了CCR2／CCL2在母—胎界面生物学功能的研究，以阐明母胎界面蜕膜基质细胞和蜕膜免疫活性细胞间相互对话的分子和机制，进而揭示趋化因子在母—胎界面免疫调节网络中的作用。1．人早孕蜕膜和蜕膜基质细胞趋化因子受体及配体的转录特征探讨人早孕蜕膜及蜕膜基质细胞趋化因子受体及相应配体的表达，及其在母—胎界面的调节作用。采用半定量RT-PCR分析正常早孕人蜕膜组织和原代培养的蜕膜基质细胞18种趋化因子受体及其相应配体mRNA的表达。结果显示，人早孕蜕膜组织高表达CCR2、CCR5、CCR10；中等强度表达CCR3、CCR6、CCR9、CXCR1、CXCR4，CCR1、CCR4、CCR7、CCR9、CXCR3、CXCR5-6仅在部分蜕膜组织中表达；不表达CXCR2、XCR1、CX3CR1。蜕膜基质细胞也高表达CCR2、CCR5、CCR10；中等强度表达CCR7、CCR9；低表达CCR1、CCR3、CCR4、CCR6、CXCR6，XCR1、CX3CR1；不表达CXCR1-CXCR5。同时蜕膜组织和蜕膜基质细胞均高水平转录CCR2配体CCL2（MCP-1），CCL13（MCP-4）和CCL28（CCR10配体），中等强度转录CCL7（MCP-3，CCR2配体），不转录CCL27（CCR10配体）。2．趋化因子CCL2及其受体CCR2在早—孕期母胎界面的表达分析人早孕蜕膜及蜕膜基质细胞趋化因子受体CCR2及其配体CCL2在人早孕蜕膜组织及蜕膜基质细胞的表达和分泌，以探讨CCR2／CCL2在母—胎界面的生物学作用。收集早孕期蜕膜组织，分离蜕膜基质细胞，免疫化学方法分析正常人早孕蜕膜组织和培养的人蜕膜基质细胞CCR2／CCL2表达；ELISA检测培养的蜕膜基质细胞上清中CCL2的分泌；用流式细胞术分析蜕膜基质细胞表面CCR2和细胞内CCL2的表达；流式细胞术检测蜕膜免疫活性细胞表面CCR2的表达。结果显示，原代培养DSC的纯度为98.25％±1.74％；人早孕蜕膜组织和蜕膜基质细胞均高水平转录和翻译CCR2和CCL2，10例样本中CCR2的阳性率为84.65％±4.57％，CCL2的表达率为53.11±6.03％；蜕膜基质细胞分泌高水平的CCL2，达21.72±2.34ng／ml，其分泌呈时间依赖性；约34％的蜕膜CD56⁺CD16^-CD3^-NK、50％CD4⁺T和66％左右的CD14⁺单核细胞表达CCR2。3．CCL2对人早孕蜕膜基质细胞自分泌调控作用应用MTT法分析外源CCL2对蜕膜基质细胞促增殖效应；给予ERK／MAPK抑制剂U0126和CCR2受体抑制剂RS102895不仅拮抗外源性CCL2的促增殖效应，还能抑制蜕膜基质细胞CCL2的分泌；Th2型细胞因子IL-4和Th1型细胞因子IFN-γ能诱导蜕膜基质细胞分泌CCL2，IL-4和IFN-γ对蜕膜基质细胞分泌没有协同作用，IL-4和TNF-α却显示出很强的协同作用；不同浓度的雌激素、孕激素和人绒毛膜促性腺激素能增加蜕膜基质细胞CCL2的转录，从而诱导CCL2的分泌。本研究表明，人早孕蜕膜基质细胞能通过ERK／MAPK途径自分泌CCL2，Th2和Th1型细胞因子能相互作用促进蜕膜基质细胞分泌CCL2，雌激素、孕激素和人绒毛膜促性腺激素可上调蜕膜基质细胞CCL2的分泌。4．蜕膜基质细胞通过分泌CCL2参与维持母胎界面Th2型免疫偏移经不同方法处理DIC后，用FCM法检测DIC的凋亡；FCM分析DIC细胞内Th1型和Th2型细胞因子的表达；CBA法检测DIC Th1型和Th2型细胞因子的分泌；应用Real time PCR分析DIC转录因子GATA-3和T-bet的表达。研究结果表明，rhCCL2和蜕膜基质细胞培养上清能促进DIC细胞Th2型细胞因子-IL-4和IL-10的表达和分泌，降调节Th1型细胞因子-IFN-γ和TNF-α的表达和分泌，从而诱导母胎界面向Th2型偏移。CCL2还能促进DIC细胞内GATA3转录，抑制T-bet的转录，从而介导DIC Th2细胞因子优势表达和分泌。更多还原

【Abstract】 Research Background Chemokines, chemotactic cytokines, are an array of small molecules of heparin-binding proteins that direct the trafficking of circulating leukocytes into the sites of inflammation or injury. The chemokines and their receptors are now involved in pathogenesis of a variety of diseases. To date, the materno-fetal interface has been found to produce a lot of chemokines, and express the corresponding receptors. Our previous studies have demonstrated that human first-trimester trophoblast cells can produce SDF-1 and CXCL16, and express their receptors CXCR4 and CXCR6. Moreover, decidual CD56^bright CD16^- NK cells expressed CXCR4, andγδT cells expressed CXCR6 highly. The first-trimester human trophoblast cells has been able to induce proliferation and invasion of themselves in an autocrine manner, and recruit the CD56^bright CD16^- NK cells andγδT cells homing into decidua by expressing SDF-1/CXCR4 and CXCR6/CXCL16, respectively.During pregnancy, the decidual tissue, the maternal component at the interface, is composed predominantly of decidual stromal cells （DSC）. Not only is the DSC classically considered to be nutritious to embryo because of their high glycogen content, but also involved in a series of immune regulations such as production of cytokines, and antigen phagocytosis and presentation. A large and specific population of immune cells infiltrate constitutively into the decidua. Therefore, the decidua is in closest contact with the fetal-derived trophoblasts and decidual lymphocytes, and thought to be a place where immune regulations between mother and fetus take place. So we proposed that the decidua may be involved in the materno-fetal tolerance by secreting chemokines and expressing their corresponding receptors. MCP-1, monocyte chemotaxis protein-1, CCL2, is a CC chemokine that attracts monocytes, memory T lymphocytes and natural killer cells in vitro by way of binding its receptor CCR2. Lately, CCL2 has been demonstrated to be involved in Th2 polarization, and a successful pregnancy is a Th2 phenomenon. Here we found that human first trimester decidua and decidual stromal cells expressed high levels of CCR2 and CCL2. Therefore, CCL2 may play a role in Th2 phenomenon at materno-fetal interface.Objectives Based on the research above, biological functions of human decidual stromal cells were explored in secretion of CCL2 and expression of CCR2, which might be involved in autocrine regulation on the DSC functions, and paracrine regulation on the Th2 bias at human materno-fetal interface.1. Transcriptional characteristics of chemokine receptors and the correspondent ligands in decidua and decidual stromal cell of human first trimester gestationMethods The deciduas were from women who had undergone an artificial abortion at 5-11 weeks of normal gestation for non medical cause. The total RNA was extracted by using the TRIzol reagent, from decidua or the Percoll-gradient-isolated DSC. The expression of chemokine receptors in the decidua and DSC were investigated by way of semi-quantitative RT-PCR.Results The chemokine receptors, CCR2, CCR5, CCR10, were highly expressed in human decidua and DSC, while CCR3, CCR6, CCR8-9, CXCR1, and CXCR4 were moderately expressed in human decidua. The chemokine receptors CCR1, CCR4, CCR7, CCR9, CXCR3, and CXCR5-6 were expressed only in the partly decidua, while no CXCR2, XCR1, and CX₃CR1mRNA were found in any decidua tissue. The freshly isolated DSC expressed moderately CCR7, CCR9, CCR1, CCR3, CCR4, CCR6, CXCR6, XCR, and CX3CR1. The CCL2 and CCL13, ligands of CCR2, and CCL28, that of CCR10, were expressed highly in the decidua and DSC.Conclusion The chemokine receptors CCR2 and CCR10 that were expressed by decidua and DSC may play an important role in the cell constitution at materno-fetal interface in human first trimester pregnancy.2. Expression of chemokine CCL2 and its receptor CCR2 in human decidua and decidual stromal cells in the first trimester pregnancyMethods The expressions of CCR2 and its ligand CCL2 in decidua and DSC were determined by way of semi-quantitative RT-PCR, immunostaining, flow cytometry, respectively. The secretion of CCL2 was detected by ELISA.Results The results showed that CCR2 and its ligand CCL2 were highly expressed in decidua and DSC. The DSC could secret CCL2 in a time-dependent manner, and the highest concentration was 21.72±2.34ng/ml. About 34% CD56⁺CD16^-CD3^- decidua NK, 50%CD4⁺ T cells and 66% CD14⁺ monocytes expressed CCR2.Conclusion The CCR2 and CCL2 were expressed in decidua and DSC of human first trimester gestation, which might play an important role in the decidual immune cell constitution, and the cross-talking between decidua stromal cells and decidua immunocompetent cells in human early pregnancy.3. The regulation of CCL2/MCP-1 secretion and autocrine regulation on human decidual stromal cell of the first-trimester gestationMethods The viability of DSC was analyzed with MTT. The primary DSCs were treated in vitro by the pregnancy-associated hormones, E2, progesterone and hCG, or Th1 and Th2 cytokines. The CCL2 expression and secretion was determined respectively by real time PCR and ELISA.Results All the pregnancy-associated hormones, E2, progesterone and hCG, did promote CCL2 transcription and secretion by DSC. U0126, an inhibitor of MAPK, and RS 102895, antagonist of CCR2, inhibited CCL2 secretion by DSC. Th2 cytokine, IL-4, and Th1 cytokine, IFN-γ, also increased CCL2 secretion. IL-4 and TNF-αhad synergistic effect on CCL2 production by DSC, but IL-4 and IFN-γdid not. The rhCCL2 could enhance the proliferation and viability of human DSC of the first-trimester gestation.Conclusion CCL2 may stimulate human DSC viability in autocrine manners. And Th1/Th2 cytokines, pregnancy-associated hormones could regulate CCL2 secretion of DSC4. Decidual stromal cells maintained Th2 predominance at materno-fetal interface through producing and secreting CCL2Methods DIC were treated by different stimuli, including rhCCL2, rhCCL2+CCL2 antibodies, the DSC-derived CM, CM+CCL2 antibodies, CCL2+U0126 and CpG used as positive control. Then the apoptosis and proliferation of DIC was detected respectively by Annexin V/FITC and PCNA with FCM, and the secretion and intracellular expression of Th1 cytokines, TNF-αand IFN-γ, and Th2 cytokines, IL-4 and IL-10, were determined by CBA and FCM, respectively. Transcription of GATA-3 and T-bet in DIC was evaluated by Real time PCR.Results The results showed that rhCCL2 and the DSC-derived CM could stimulate DIC proliferation and inhibit DIC apoptosis. The rhCCL2 and the DSC-derived CM promote Th2 cytokine, IL-4 and IL-10 production, and inhibit secretion of Th1 cytokine, TNF-αand IFN-γ. The CCL2 and CM could also increase GATA-3 transcription and suppress T-bet transcription in DIC.Conclusion Decidual stromal cells can secret CCL2 that in turn increases GATA-3 transcriptions of DIC, which leads to Th2 polarization at materno-fetal interface.更多还原