LAIRs和胶原分子调节人早孕蜕膜NK细胞功能的分子机制
发布时间：2018-08-01 09:14
【摘要】：妊娠是一个独特的免疫学事件。妊娠成功需要通过精细的母-胎对话,在母-胎界面建立独特的免疫耐受微环境。母-胎界面多种细胞和分子之间的对话是母-胎调节的核心内容,解析其中的免疫细胞,滋养细胞和蜕膜基质细胞通过它们表面膜分子和分泌的细胞因子之间的相互作用,是阐明母-胎免疫耐受机制的重要研究内容,不仅对生殖免疫学,而且对肿瘤和移植免疫学都具有重要的理论和临床意义。白细胞相关免疫球蛋白样受体(LAIR)是新近鉴定的以胶原为配体的免疫受体。LAIR-1广泛表达在造血细胞和各类免疫细胞表面,胞质区有ITIM基序,LAIR-1对外周血免疫细胞功能的调节作用及其在肿瘤免疫和移植免疫中的意义是免疫学研究的热点。然而,在生殖免疫学中,目前还没有任何相关研究。胶原蛋白作为母-胎界面含量丰富的重要细胞外基质,在妊娠维持以及免疫调节中的作用研究也甚少。蜕膜NK细胞约占蜕膜免疫细胞总数的70%,其表型和生物学行为对母-胎免疫耐受的形成发挥着至关重要的作用。本研究在分析了LAIRs和胶原分子在母-胎界面的表达及其和自然流产关系的基础上,从LAIR-1和胶原分子的相互作用对在母-胎界面占绝对组成优势的早孕期NK细胞的调控入手,以解析滋养细胞和蜕膜基质细胞通过表达的胶原蛋白和LAIRs的相互作用对NK细胞表型和生物学行为的调节,并进一步研究JAK-STAT信号通路在此调节中的角色。研究成果可以为临床上全面理解自然流产等母-胎免疫调节紊乱性疾病提供新的视角,同时对相关疾病的诊断和防治也可提供新的靶点和思路。第一部分LAIR-1和胶原分子的表达水平与自然流产相关目的分析比较LAIR家族成员(LAIRs)和胶原分子在正常和异常妊娠组织中的表达；以及在体外培养的正常和流产的滋养细胞,蜕膜基质细胞和NK细胞中的表达。方法收集正常和异常妊娠早孕期妇女的绒毛和蜕膜组织,采用马松染色法(Masson)和免疫组化技术(IH)分析LAIR-1和胶原分子在其中的表达情况；分离各种早孕期滋养细胞,蜕膜基质细胞(DSC)和蜕膜免疫细胞(DIC),采用多聚酶链式反应(PCR),酶联免疫吸附(ELISA)等技术分析以上细胞中多种LAIRs和胶原分子表达的情况；用流式细胞术(FCM)分析正常和流产组织蜕膜NK细胞LAIR-1的表达。结果获得了较全面的LAIRs和胶原分子在正常妊娠和自然流产组织中的表达情况。对比分析结果显示：正常妊娠的绒毛和蜕膜组织相对于自然流产的绒毛和蜕膜组织,都有更高水平胶原的表达；体外培养的正常滋养细胞、蜕膜基质细胞在mRNA和蛋白水平也都比自然流产的滋养细胞、蜕膜基质细胞表达分泌更高水平的胶原蛋白；正常妊娠的蜕膜组织比自然流产的蜕膜组织表达更高的LAIR-1,并且正常妊娠的蜕膜NK也比自然流产的蜕膜NK细胞表达更高水平的LAIR-1。结论 人早孕期母-胎界面滋养细胞和蜕膜基质细胞的胶原蛋白表达下降和自然流产具有相关性；蜕膜组织和蜕膜NK细胞LAIR-1的低表达与自然流产也有相关性。第二部分LAIRs和胶原分子相互作用调节NK细胞表型目的解析LAIR-1和胶原分子相互作用的特点,及其对外周NK细胞及蜕膜NK细胞表面受体和细胞内细胞因子表达的调节作用。方法常规无菌分离外周血单个核细胞(PBMC)和蜕膜免疫细胞(DIC)后,磁珠分选其中的外周NK细胞和蜕膜NK细胞,并在其体外培养体系中加入不同浓度的胶原分子,用多聚酶链式反应(PCR)和流式细胞(FCM)技术分析胶原分子受体LAIR-1的表达情况；进一步在早孕期NK细胞培养体系加入胶原蛋白分子,并且设立LAIR-2处理组,用FCM分析NK细胞表面的激活性受体和抑制性受体以及细胞内Thl、Th2型细胞因子和杀伤活性相关分子的表达。结果 胶原蛋白分子可以剂量依赖性的方式诱导其受体LAIR-1在NK细胞mRNA和蛋白水平表达增加。胶原蛋白分子可以通过和LAIR-1的相互作用降低蜕膜NK细胞表面激活性受体NKp30的表达,上调抑制性受体KIR2DL1的表达；胶原蛋白分子和LAIR-1的相互作用也可以降低蜕膜NK细胞Thl型细胞因子IFN-γ口TNF-a的表达,而不影响蜕膜NK细胞Th2型细胞因子IL-4和IL-10的表达；高浓度胶原蛋白分子可以降低蜕膜NK细胞穿孔素的表达,且后者的表达和]LAIR-1表达的荧光强度成反比,这种作用以及胶原蛋白分子对NK细胞表型和Thl型细胞因子表达的调节都可以被LAIR-2所拮抗。结论胶原分子上调NK细胞中LAIR-1的表达,并且通过和LAIR-1的相互作用可以降低NK细胞Th1型细胞因子和穿孔素的表达,诱导蜕膜NK细胞的耐受表型。第三部分 滋养细胞和蜕膜基质细胞通过胶原分子参与调节蜕膜NK细胞的功能目的解析人早孕期母-胎界面的滋养细胞和蜕膜基质细胞通过表达的胶原蛋白分子对蜕膜NK细胞表型的调节作用。方法在体外建立人早孕期滋养细胞,蜕膜基质细胞单独培养、以及滋养细胞和蜕膜基质细胞共培养体系来模拟早孕期母-胎界面微环境,然后与纯化的蜕膜NK细胞共培养,并设立LAIR-2处理组,用流式细胞术(FCM)分析蜕膜NK细胞的表面激活性受体和抑制性受体以及细胞内细胞因子和杀伤活性相关分子的表达。进一步用P4H shRNA质粒转染滋养细胞与蜕膜基质细胞来模拟自然流产的母-胎界面胶原蛋白分子低水平表达的微环境,用多聚酶链式反应(PCR)、酶联免疫吸附(ELISA)方法和氨基酸分析仪检测转染处理后,滋养细胞、蜕膜基质细胞单独培养和共培养体系中胶原蛋白的表达水平；然后用FCM分析蜕膜NK细胞表面的激活性受体和抑制性受体以及细胞内细胞因子和杀伤活性相关分子的表达。结果滋养细胞和蜕膜基质细胞可以通过分泌胶原分子与LAIR-1相互作用降低蜕膜NK细胞表面激活性受体NKp30的表达,上调抑制性受体KIR2DL1的表达；也可以降低蜕膜NK细胞Thl型细胞因子IFN-y和]TNF-a,以及穿孔素的表达。用LAIR-2阻断和P4H shRNA质粒转染以基因沉默胶原合成,都能够抑制滋养细胞与蜕膜基质细胞对蜕膜NK细胞的表面受体、细胞内细胞因子以及杀伤活性的调节作用。值得一提的是,用P4H shRNA质粒转染滋养细胞与蜕膜基质细胞会使蜕膜NK细胞Th2型细胞因子IL-4和IL-10的表达水平下降。结论人早孕期滋养细胞和蜕膜基质细胞可以通过表达的胶原分子参与调节蜕膜NK细胞的功能,从而有利于正常妊娠的维持。第四部分JAK-STAT信号通路参与LAIR-1对NK细胞的调节作用目的解析LAIR-1调节NK细胞功能的信号通路。方法在纯化的外周NK细胞(pNK)和蜕膜NK细胞(dNK)的体外培养体系中加入胶原蛋白分子；用shRNA转染的滋养细胞和蜕膜基质细胞共培养的上清(Culture Medium, CM)培养NK细胞后,用免疫蛋白印迹实验(Western Blot)分析LAIR-1和AK-STAT信号通路中STAT1、p-STAT1、STAT4、p-STAT4的表达,进一步用Western Blot和流式细胞术(FCM)分析JAK-STAT信号通路下游相关转录因子T-bet和Helios等的表达；用免疫共沉淀技术(CoIP)分析JAK-STAT信号通路上游相关分子JAK1、JAK2和SHP-1的相互关系,以及SHP-1和LAIR-1的相互作用关系。用病毒质粒构建LAIR-1过表达的稳转细胞株NK-92-LAIR-1,并且通过QPCR、Western Blot和荧光显微镜进行鉴定,进一步对其在活化状态下STATs和LAIR-1的表达进行初步分析。结果在胶原蛋白分子的刺激下,LAIR-1可以募集SHP-1,并通过后者和JAK1、JAK2的直接作用,对JAK-STAT信号通路中STAT1、STAT4的活化发挥抑制作用,进而影响转录因子T-bet和Helios等的表达；成功地构建了LAIR-1过表达的稳转细胞株NK-92-LAIR-1.结论JAK-STAT信号通路参与LAIR-1和胶原分子相互作用对NK细胞的调节作用。LAIR-1过表达稳转细胞株的建立为我们后期进行深入系统的相关研究工作打下了坚实的基础。
[Abstract]:Pregnancy is a unique immunological event. Pregnancy success requires a delicate mother fetal dialogue to establish a unique immune tolerance microenvironment at the mother fetal interface. The dialogue between cells and molecules at the mother fetal interface is the core of the maternal fetal regulation, parsing the immune cells, trophoblastic cells and decidual stromal cells through their tables. The interaction between the mask molecules and the secreted cytokine is an important study of the mechanism of maternal fetal immune tolerance, not only for reproductive immunology, but also of important theoretical and clinical significance for both tumor and transplantation immunology. The leukocyte related immunoglobulin like receptor (LAIR) is a newly identified collagen free ligand. The pestilence receptor.LAIR-1 is widely expressed in the surface of hematopoietic cells and various immune cells, the cytoplasmic region has ITIM motif, the role of LAIR-1 in the immunological function of peripheral blood and its significance in the immunology and transplantation of the tumor are the hot spots of immunology. However, there is no related research in the reproductive immunology. The role of an important extracellular matrix, rich in maternal fetal interface, is rarely studied in pregnancy maintenance and immunoregulation. Decidua NK cells account for about 70% of the total number of decidual immune cells. Their phenotypic and biological behavior plays a vital role in the formation of maternal fetal immune tolerance. This study analyzed the LAIRs and collagen molecules in this study. On the basis of the relationship between mother fetal interface and spontaneous abortion, the interaction between LAIR-1 and collagen molecules begins with the regulation of the early pregnancy NK cells with the absolute dominance of the mother fetal interface, in order to analyze the phenotype and biology of the trophoblast and the decidual stromal cells through the interaction of the collagen and LAIRs expressed by the NK cells. Regulation of behavior and further study of the role of JAK-STAT signaling in this regulation. The results can provide a new perspective for a comprehensive understanding of maternal fetal immunomodulatory disorders, such as spontaneous abortion, and also provide new targets and ideas for the diagnosis and prevention of related diseases. Part 1, LAIR-1 and collagen molecules Expressions of LAIR family members (LAIRs) and collagen molecules in normal and abnormal pregnancy tissues, and the expression of normal and abortive trophoblastic cells, decidual stromal cells and NK cells in vitro. Methods to collect the villus and decidua of normal and abnormal pregnancy women. Tissue, the expression of LAIR-1 and collagen molecules were analyzed by Masson staining (Masson) and immunohistochemical technique (IH), and various early pregnancy trophoblastic cells, decidual stromal cells (DSC) and decidual immune cells (DIC), polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA) were used to analyze a variety of LAIRs in the above cells. The expression of collagen molecules and the expression of LAIR-1 in normal and aborted tissue decidua NK cells by flow cytometry (FCM). Results obtained more comprehensive expression of LAIRs and collagen molecules in normal pregnancy and spontaneous abortion tissues. Comparative analysis showed that the villus and decidua tissues of normal pregnancy were relative to spontaneous abortion. The villi and decidual tissues all have higher level of collagen expression; in vitro cultured normal trophoblastic cells, decidual stromal cells also express higher levels of collagen than spontaneous abortion trophoblastic cells and decidual stromal cells, and the decidual tissue of normal pregnancy is more expressive than natural abortion decidual tissue. High LAIR-1, and the decidual NK in normal pregnancy also expressed higher levels of LAIR-1. than decidual NK cells in spontaneous abortion. Conclusion the decrease of collagen expression in mother fetal interface trophoblast and decidual stromal cells in early pregnancy was associated with spontaneous abortion, and the low expression of LAIR-1 in decidual and decidual NK cells was also associated with spontaneous abortion. Correlation. Second part LAIRs and collagen molecules interact to regulate the phenotype of NK cells to analyze the interaction between LAIR-1 and collagen molecules, and the regulation of the expression of surface receptors and intracellular cytokines in peripheral NK cells and decidua NK cells. Methods routine aseptic isolation of peripheral blood mononuclear cells (PBMC) and decidua immunization After DIC, the magnetic beads were selected for the separation of peripheral NK cells and decidua NK cells, and the collagen molecules were added to the culture system in vitro. The expression of collagen molecular receptor LAIR-1 was analyzed with polymerase chain reaction (PCR) and flow cytometry (FCM), and collagen molecules were added to the early pregnancy NK cell culture system. The LAIR-2 treatment group was set up to analyze the expression of activator and inhibitory receptor on the surface of NK cells and the expression of Thl, Th2 type cytokine and cytotoxic activity related molecules on the surface of NK cells. Results collagen molecules can induce the expression of LAIR-1 in mRNA and protein levels in NK cells in a dose-dependent manner. The expression of NKp30 on the surface of the decidua NK cell can be reduced by interaction with LAIR-1, and the expression of the inhibitory receptor KIR2DL1 is up-regulated, and the interaction of collagen molecules and LAIR-1 can also reduce the expression of IFN- gamma TNF-a in the decidua NK cell Thl cell factor, without affecting Th2 cytokine IL-4 and NK cells in the decidua NK cells. The expression of IL-10, high concentration collagen molecules can reduce the expression of perforin in decidual NK cells, and the expression of the latter is inversely proportional to the fluorescence intensity of]LAIR-1 expression. This action, and the regulation of collagen molecules on the phenotype of NK cells and the expression of Thl type cytokines, can be antagonized by LAIR-2. Conclusion collagen molecules up regulation of NK cells. The expression of medium LAIR-1 and the interaction with LAIR-1 can reduce the expression of Th1 type cytokine and perforin in NK cells and induce the tolerance phenotype of decidual NK cells. Third trophoblast and decidual stromal cells are involved in regulating the function of decidua NK cells through collagen molecules to analyze the nourishment of mother fetal interface in early pregnancy. Cell and decidual matrix cells regulate the phenotype of decidual NK cells by expression of collagen molecules. Methods human early gestational trophoblastic cells were established in vitro, decidual stromal cells were cultured separately, and trophoblast and decidual stromal cells co culture system was used to simulate the microenvironment of mother fetal interface in early pregnancy, and then the purified decidua NK was fine. The LAIR-2 treatment group was established and the expression of the surface activator and inhibitory receptor and the cytokine and cytokine related molecules in the decidua NK cells were analyzed by flow cytometry (FCM). The P4H shRNA plasmid was further transfected to the trophoblast and decidua matrix cells to simulate the mother fetal interface collagen eggs of natural abortion. The low level expressed microenvironment of the white molecule was detected by polymerase chain reaction (PCR), enzyme linked immunosorbent assay (ELISA) and amino acid analyzer to detect the expression of collagen protein in the cultured and co cultured system of trophoblast and decidual stromal cells, but then FCM was used to analyze the activation receptors and inhibition of the surface of the decidua NK cells. The expression of the cytokine and cytokine and cytokine in the cell can be expressed in the trophoblast and decidual stromal cells to reduce the expression of the active receptor NKp30 of the decidual NK cell, up the expression of the inhibitory receptor KIR2DL1, and to reduce the Thl type of the decidual NK cells by the interaction of collagen molecules with LAIR-1 Cytokine IFN-y and]TNF-a, and the expression of perforin. The use of LAIR-2 blocking and P4H shRNA plasmid transfection by gene silencing collagen synthesis can inhibit the regulating effect of trophoblast and decidual matrix cells on the surface receptor, cytokine and cytotoxic activity of decidual NK cells. It is worth mentioning that the transfection of P4H shRNA plasmids is the key to the regulation of the cytokine and cytokine activity of decidual NK cells. The expression level of Th2 cytokine IL-4 and IL-10 in decidual NK cells decreased in the trophoblast and decidual matrix cells. Conclusion early pregnancy trophoblast and decidual stromal cells can regulate the function of the decidua NK cells through the expression of collagen molecules, which is beneficial to the maintenance of normal pregnancy. The fourth part of the JAK-STAT signaling pathway is useful. The regulation of LAIR-1 on NK cells aims to analyze the signaling pathway of LAIR-1 to regulate the function of NK cells. Methods the collagen molecules were added to the purified peripheral NK cells (pNK) and decidual NK cells (dNK) in vitro culture system, and the supernatant (Culture Medium, CM) cultured with shRNA transfected trophoblast and decidual matrix (Culture Medium) After the cell, the expression of STAT1, p-STAT1, STAT4, p-STAT4 in the LAIR-1 and AK-STAT signaling pathways was analyzed by immunoblotting (Western Blot), and the expression of the downstream related transcription factors and the expressions of the downstream related transcription factors were analyzed by Western Blot and flow cytometry (FCM). The relationship between JAK1, JAK2 and SHP-1, and the interaction between SHP-1 and LAIR-1, and the interaction between SHP-1 and LAIR-1. Construction of LAIR-1 over expressed stable cell line NK-92-LAIR-1 with virus plasmids and identification by QPCR, Western Blot and fluorescence microscopy to further differentiate the expression of STATs and LAIR-1 from the activation state. Under the stimulation of collagen molecules, LAIR-1 can raise SHP-1, and through the direct action of the latter and JAK1, JAK2, it inhibits the activation of STAT1 and STAT4 in the JAK-STAT signaling pathway, and then affects the expression of the transcription factor T-bet and Helios, and has successfully constructed the NK-92-LAIR-1. conclusion of the LAIR-1 overexpressed stable cell line. The JAK-STAT signaling pathway participates in the regulation of LAIR-1 and collagen molecular interaction on NK cells and the establishment of.LAIR-1 over expressed stable cell lines has laid a solid foundation for our further systematic research on the later system.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714
本文编号：2157100
[Abstract]:Pregnancy is a unique immunological event. Pregnancy success requires a delicate mother fetal dialogue to establish a unique immune tolerance microenvironment at the mother fetal interface. The dialogue between cells and molecules at the mother fetal interface is the core of the maternal fetal regulation, parsing the immune cells, trophoblastic cells and decidual stromal cells through their tables. The interaction between the mask molecules and the secreted cytokine is an important study of the mechanism of maternal fetal immune tolerance, not only for reproductive immunology, but also of important theoretical and clinical significance for both tumor and transplantation immunology. The leukocyte related immunoglobulin like receptor (LAIR) is a newly identified collagen free ligand. The pestilence receptor.LAIR-1 is widely expressed in the surface of hematopoietic cells and various immune cells, the cytoplasmic region has ITIM motif, the role of LAIR-1 in the immunological function of peripheral blood and its significance in the immunology and transplantation of the tumor are the hot spots of immunology. However, there is no related research in the reproductive immunology. The role of an important extracellular matrix, rich in maternal fetal interface, is rarely studied in pregnancy maintenance and immunoregulation. Decidua NK cells account for about 70% of the total number of decidual immune cells. Their phenotypic and biological behavior plays a vital role in the formation of maternal fetal immune tolerance. This study analyzed the LAIRs and collagen molecules in this study. On the basis of the relationship between mother fetal interface and spontaneous abortion, the interaction between LAIR-1 and collagen molecules begins with the regulation of the early pregnancy NK cells with the absolute dominance of the mother fetal interface, in order to analyze the phenotype and biology of the trophoblast and the decidual stromal cells through the interaction of the collagen and LAIRs expressed by the NK cells. Regulation of behavior and further study of the role of JAK-STAT signaling in this regulation. The results can provide a new perspective for a comprehensive understanding of maternal fetal immunomodulatory disorders, such as spontaneous abortion, and also provide new targets and ideas for the diagnosis and prevention of related diseases. Part 1, LAIR-1 and collagen molecules Expressions of LAIR family members (LAIRs) and collagen molecules in normal and abnormal pregnancy tissues, and the expression of normal and abortive trophoblastic cells, decidual stromal cells and NK cells in vitro. Methods to collect the villus and decidua of normal and abnormal pregnancy women. Tissue, the expression of LAIR-1 and collagen molecules were analyzed by Masson staining (Masson) and immunohistochemical technique (IH), and various early pregnancy trophoblastic cells, decidual stromal cells (DSC) and decidual immune cells (DIC), polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA) were used to analyze a variety of LAIRs in the above cells. The expression of collagen molecules and the expression of LAIR-1 in normal and aborted tissue decidua NK cells by flow cytometry (FCM). Results obtained more comprehensive expression of LAIRs and collagen molecules in normal pregnancy and spontaneous abortion tissues. Comparative analysis showed that the villus and decidua tissues of normal pregnancy were relative to spontaneous abortion. The villi and decidual tissues all have higher level of collagen expression; in vitro cultured normal trophoblastic cells, decidual stromal cells also express higher levels of collagen than spontaneous abortion trophoblastic cells and decidual stromal cells, and the decidual tissue of normal pregnancy is more expressive than natural abortion decidual tissue. High LAIR-1, and the decidual NK in normal pregnancy also expressed higher levels of LAIR-1. than decidual NK cells in spontaneous abortion. Conclusion the decrease of collagen expression in mother fetal interface trophoblast and decidual stromal cells in early pregnancy was associated with spontaneous abortion, and the low expression of LAIR-1 in decidual and decidual NK cells was also associated with spontaneous abortion. Correlation. Second part LAIRs and collagen molecules interact to regulate the phenotype of NK cells to analyze the interaction between LAIR-1 and collagen molecules, and the regulation of the expression of surface receptors and intracellular cytokines in peripheral NK cells and decidua NK cells. Methods routine aseptic isolation of peripheral blood mononuclear cells (PBMC) and decidua immunization After DIC, the magnetic beads were selected for the separation of peripheral NK cells and decidua NK cells, and the collagen molecules were added to the culture system in vitro. The expression of collagen molecular receptor LAIR-1 was analyzed with polymerase chain reaction (PCR) and flow cytometry (FCM), and collagen molecules were added to the early pregnancy NK cell culture system. The LAIR-2 treatment group was set up to analyze the expression of activator and inhibitory receptor on the surface of NK cells and the expression of Thl, Th2 type cytokine and cytotoxic activity related molecules on the surface of NK cells. Results collagen molecules can induce the expression of LAIR-1 in mRNA and protein levels in NK cells in a dose-dependent manner. The expression of NKp30 on the surface of the decidua NK cell can be reduced by interaction with LAIR-1, and the expression of the inhibitory receptor KIR2DL1 is up-regulated, and the interaction of collagen molecules and LAIR-1 can also reduce the expression of IFN- gamma TNF-a in the decidua NK cell Thl cell factor, without affecting Th2 cytokine IL-4 and NK cells in the decidua NK cells. The expression of IL-10, high concentration collagen molecules can reduce the expression of perforin in decidual NK cells, and the expression of the latter is inversely proportional to the fluorescence intensity of]LAIR-1 expression. This action, and the regulation of collagen molecules on the phenotype of NK cells and the expression of Thl type cytokines, can be antagonized by LAIR-2. Conclusion collagen molecules up regulation of NK cells. The expression of medium LAIR-1 and the interaction with LAIR-1 can reduce the expression of Th1 type cytokine and perforin in NK cells and induce the tolerance phenotype of decidual NK cells. Third trophoblast and decidual stromal cells are involved in regulating the function of decidua NK cells through collagen molecules to analyze the nourishment of mother fetal interface in early pregnancy. Cell and decidual matrix cells regulate the phenotype of decidual NK cells by expression of collagen molecules. Methods human early gestational trophoblastic cells were established in vitro, decidual stromal cells were cultured separately, and trophoblast and decidual stromal cells co culture system was used to simulate the microenvironment of mother fetal interface in early pregnancy, and then the purified decidua NK was fine. The LAIR-2 treatment group was established and the expression of the surface activator and inhibitory receptor and the cytokine and cytokine related molecules in the decidua NK cells were analyzed by flow cytometry (FCM). The P4H shRNA plasmid was further transfected to the trophoblast and decidua matrix cells to simulate the mother fetal interface collagen eggs of natural abortion. The low level expressed microenvironment of the white molecule was detected by polymerase chain reaction (PCR), enzyme linked immunosorbent assay (ELISA) and amino acid analyzer to detect the expression of collagen protein in the cultured and co cultured system of trophoblast and decidual stromal cells, but then FCM was used to analyze the activation receptors and inhibition of the surface of the decidua NK cells. The expression of the cytokine and cytokine and cytokine in the cell can be expressed in the trophoblast and decidual stromal cells to reduce the expression of the active receptor NKp30 of the decidual NK cell, up the expression of the inhibitory receptor KIR2DL1, and to reduce the Thl type of the decidual NK cells by the interaction of collagen molecules with LAIR-1 Cytokine IFN-y and]TNF-a, and the expression of perforin. The use of LAIR-2 blocking and P4H shRNA plasmid transfection by gene silencing collagen synthesis can inhibit the regulating effect of trophoblast and decidual matrix cells on the surface receptor, cytokine and cytotoxic activity of decidual NK cells. It is worth mentioning that the transfection of P4H shRNA plasmids is the key to the regulation of the cytokine and cytokine activity of decidual NK cells. The expression level of Th2 cytokine IL-4 and IL-10 in decidual NK cells decreased in the trophoblast and decidual matrix cells. Conclusion early pregnancy trophoblast and decidual stromal cells can regulate the function of the decidua NK cells through the expression of collagen molecules, which is beneficial to the maintenance of normal pregnancy. The fourth part of the JAK-STAT signaling pathway is useful. The regulation of LAIR-1 on NK cells aims to analyze the signaling pathway of LAIR-1 to regulate the function of NK cells. Methods the collagen molecules were added to the purified peripheral NK cells (pNK) and decidual NK cells (dNK) in vitro culture system, and the supernatant (Culture Medium, CM) cultured with shRNA transfected trophoblast and decidual matrix (Culture Medium) After the cell, the expression of STAT1, p-STAT1, STAT4, p-STAT4 in the LAIR-1 and AK-STAT signaling pathways was analyzed by immunoblotting (Western Blot), and the expression of the downstream related transcription factors and the expressions of the downstream related transcription factors were analyzed by Western Blot and flow cytometry (FCM). The relationship between JAK1, JAK2 and SHP-1, and the interaction between SHP-1 and LAIR-1, and the interaction between SHP-1 and LAIR-1. Construction of LAIR-1 over expressed stable cell line NK-92-LAIR-1 with virus plasmids and identification by QPCR, Western Blot and fluorescence microscopy to further differentiate the expression of STATs and LAIR-1 from the activation state. Under the stimulation of collagen molecules, LAIR-1 can raise SHP-1, and through the direct action of the latter and JAK1, JAK2, it inhibits the activation of STAT1 and STAT4 in the JAK-STAT signaling pathway, and then affects the expression of the transcription factor T-bet and Helios, and has successfully constructed the NK-92-LAIR-1. conclusion of the LAIR-1 overexpressed stable cell line. The JAK-STAT signaling pathway participates in the regulation of LAIR-1 and collagen molecular interaction on NK cells and the establishment of.LAIR-1 over expressed stable cell lines has laid a solid foundation for our further systematic research on the later system.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714
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