LAIRs和膠原分子調(diào)節(jié)人早孕蛻膜NK細(xì)胞功能的分子機(jī)制
[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.
【學(xué)位授予單位】:復(fù)旦大學(xué)
【學(xué)位級(jí)別】:博士
【學(xué)位授予年份】:2014
【分類號(hào)】:R714
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