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CD14~+ Monocytes Promote the Immunosuppressive Effect of Human Umbilical Cord Matrix Stem Cells

Author: WangDing
Tutor: HanZhongChao
School: Peking Union Medical College , China
Course: Internal Medicine
Keywords: Human umbilical cord mesenchymal stem cells T lymphocytes Monocytes Immunosuppressive PGE2 IL-β MCF-7 cells IL-6 IL-1β Luteolin
CLC: R329
Type: PhD thesis
Year: 2010
Downloads: 422
Quote: 1
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Abstract

Background: Mesenchymal stem cells (mesenchymal stem cells, MSC) is present in a variety of organizations with self-renewal and differentiation potential of pluripotent stem cells. Because of their easy separation amplification multilineage differentiation of hematopoietic support, low immunogenicity and immunomodulatory properties, and become the ideal seed cells for tissue engineering and regenerative medicine. Currently, pilot studies and therapeutic clinical applications are adult bone marrow-derived mesenchymal stem cells (bone marrow mesenchymal stem cell, BM-MSC) as an object of study. Adult bone marrow-derived mesenchymal stem cells (hBM-MSC) there are limited sources of sampling invasive operation, and mesenchymal stem cells, the absolute number and the proliferation and differentiation capacity will decrease with increasing donor age less than urgent researchers advantages of looking for a new replacement cells. A large number of experimental studies have shown that human umbilical cord mesenchymal stem cells (human umbilical cord matrix stem cell, hUC-MSC) has the following unparalleled advantages: First, the use of the waste of the fetus after delivery, organization wide variety of sources, no ethics restrictions ; second, stronger proliferative capacity, easy-to long-term culture, rich cells can be obtained by in vitro amplification; Third, with a double surface markers of adult stem cells and embryonic stem cells (embryonic stem cell, ESC); fourth consecutive multiple passages still maintain the characteristics of stem cells. Therefore, human umbilical cord mesenchymal stem cells as cell therapy is a very promising stem cell alternative sources, has unlimited potential and broad prospects for clinical application, and its immunological characteristics is the cell therapy success decisive factor. Objective: lymphocytes (lymphocyte) and monocytes (monocyte) is an important component of peripheral blood mononuclear cells (Peripheral blood mononuclear cells, PBMC). The purpose of this study is to reveal the interaction between human umbilical cord mesenchymal stem cells, T lymphocytes, and monocytes among its mechanism and clinical application of human umbilical cord mesenchymal stem cells to provide the necessary theoretical basis. Methods: (1) the application of flow cytometry in adult bone marrow mesenchymal stem cells and human umbilical cord mesenchymal stem cells of the immune phenotype, and embryonic stem cell markers Almighty. (2) in the induction system, directional induction of human umbilical cord mesenchymal stem cells into adipocytes, osteoblasts and cartilage cell differentiation, oil red O staining, Von Kossa staining, Alizarin red staining and type II collagen staining observations lipid droplet formation, calcium deposition and type II collagen synthesis in the identification of human umbilical cord mesenchymal stem cell differentiation potential. (3) in vitro establishment of human umbilical cord mesenchymal stem cells and CD4 / CD8 T lymphocytes co-culture system as well as human umbilical cord mesenchymal stem cells, CD4 / CD8 T-lymphocytes and CD14 monocytes of the three co-culture system. Using flow cytometry (Brdu incorporation test) to detect the proliferation of T lymphocytes, and enzyme-linked immunosorbent assay kit to detect the secretion of IFN-γ (interfern-γ), which reflect the human umbilical cord mesenchymal stem cells immunosuppressive effects of activated T lymphocytes and CD14 monocytes for this role. (4) using transwell culture plate cultured human umbilical cord mesenchymal stem cells and cells in direct contact with CD4 / CD8 T lymphocytes play to the study of human umbilical cord mesenchymal stem cells immunosuppressive effects. (5) add exogenous TGF-β (transforming growth factor-β) monoclonal antibodies indoleamine 2,3 - dioxygenase (indoleamine 2,3-dioxygenase, IDO), a specific inhibitor of 1-M -Trp, nitric oxide (N0) synthase competitive inhibitor of L-NMA and prostaglandin E2 (prostaglandin E2, PGE2) synthesis inhibitor NS-398 (cyclooxygenase-2 selective inhibitor) and indomethacin Xin (non-selective cyclooxygenase inhibitor), as the evaluation index, the level of IFN-γ secretion identification mediated human umbilical cord mesenchymal stem cells immunosuppressive effects of soluble factors. (6) by the addition of exogenous PGE2 and using ELISA kit for detection of human umbilical cord mesenchymal stem cells and CD4 / CD8 T lymphocytes co-culture system of PGE2 level evidence the PGE2 whether intermediaries umbilical cord mesenchymal stem cells immunosuppressive effects. (7) Preparation of CD3/CD28 antibody cross-linked beads (anti-CD3/CD28 Dynabeads) activation of CD4 T lymphocytes, CD8 T lymphocytes and peripheral blood mononuclear cells of three conditions medium conditions cultured using ELISA kits group in the IFN-γ and TNF-α (Tumor necrosis factor-a), IL-1β (interleukin-1β) and IL-6 (inter-leukin-6) of the level. Factor ELISA method recombinant protein before and after stimulation of the medium as well as the four factors of the above three conditions, human umbilical cord mesenchymal stem cells secrete PGE2 levels for quantitative detection of clear human umbilical cord mesenchymal stem cells to produce PGE2 drive. (8) exogenous recombinant IL-1β stimulation of human umbilical cord mesenchymal stem cells, the application of the Real-time PCR was used to detect cyclooxygenase -2 (the spectrum of effects of [cyclooxygenase, COX-2) expression and PGE2 secretion using ELISA kits. (9) the use of several specific signaling pathway inhibitors: PD98059 extracellular signal-regulated kinase (extra-cellular signal-regulated kinase, ERK) specific inhibitor, SB203580 for p38 mitogen-activated protein kinase (p38mitogen- activated protein kinase, p38 MAPK)-specific inhibitor, SP600125 for c-Jun N-terminal kinase (c-jun-N-terminal kinase, JNK) specific inhibitor, H89 protein kinase A protein kinase A (PKA) specific inhibitor, Wortmannin for phosphatidylinositol 3 - kinase (phosphatidylnsitol 3-kinase, the specific inhibitors of PI3K), IKK-NBD peptide is a specific inhibitor of NF-κB, Go6983 and Go6976 for protein kinase C (protein kinase C , PKC) specific inhibitor of IL-1β stimulate human umbilical cord mesenchymal stem cells to produce PGE2 signaling pathways associated protein initially identified. (10) using ELISA kit detects The CD14 monocyte join human umbilical cord mesenchymal stem cells with CD4 / CD8 T lymphocytes before and after co-culture system, supernatant IL-1β and PGE2 content. (11) The use of flow cytometry (Brdu incorporation assay) and ELISA kits detect the IL-1 receptor antagonist (IL-1 receptor antagonist, IL-1RA) joined human umbilical cord mesenchymal stem cells, CD4 / CD8 T lymphoid The three co-culture of cells and CD14 monocytes around the system, the proliferation of T lymphocytes, the level of IFN-γ secretion, and the content of PGE2 to further study whether the mononuclear cells through the secretion of IL-1β to promote human umbilical mesenchymal stem cells to produce PGE2, thereby enhancing the immunosuppressive effects of human umbilical cord mesenchymal stem cells. Results: (1) human umbilical cord mesenchymal stem cells and adult bone marrow mesenchymal stem cell phenotype similar to human umbilical cord mesenchymal stem cells highly expressed in embryonic stem cell markers OCT4 and SSEA-4. (2) the specific inducing conditions, human umbilical cord mesenchymal stem cells have the ability to three-differentiation of fat cells, bone cells and cartilage cells. (3) human umbilical cord mesenchymal stem cells are able to suppress CD3/CD28 antibody cross-linked beads activated CD4 / CD8 T lymphocyte proliferation and secretion of IFN-γ, and CD14 monocytes can enhance the human umbilical cord mesenchymal stem cells This inhibition. (4) does not rely on direct contact between cells play an immunosuppressive effect of human umbilical cord mesenchymal stem cells. PGE2 synthesis inhibitors can significantly reverse human umbilical cord mesenchymal stem cells immunosuppressive effects; suits derived PGE2 can simulate the immunosuppressive effects of human umbilical cord mesenchymal stem cells. (5) the inflammatory cytokines IFN-γ and IL-1β can drive human umbilical cord mesenchymal stem cells of PGE2. Specific inhibitor of ERK, p38-MAPK and JNK can significantly lowered IL-1β-induced PGE2 secretion. (6) of CD14 mononuclear cells are able to significantly raised human umbilical cord mesenchymal stem cells with CD4 / CD8 T lymphocyte co-culture supernatant IL-1β and PGE2 levels, and raised the magnitude and the number of mononuclear cells was positively correlated. (7) IL-1RA able to cut human umbilical cord mesenchymal stem cells, CD4 / CD8 T-lymphocytes and CD14 single nucleated cells three co-culture supernatant PGE2 levels, while a significant reversal of the T-lymphocyte proliferation activity and IFN-γ secretion levels. Conclusion: of CD14 single nuclear cells through the secretion of inflammation factor of IL-1β via the MAPK signaling pathway significantly with raised human umbilical cord mesenchymal stem cells PGE2 of expression, and thus enhance the human umbilical cord mesenchymal stem cells on activated CD4 / CD8 T lymphocytes in the immune suppression role. Background: Mesenchymal stem cells (mesenchymal stem cells, MSC) for its superior immunomodulatory capacity, is increasingly becoming the focus of attention of the majority of researchers. Confirmed by the large number of studies in vitro and in vivo, mesenchymal stem cells are able to regulate T lymphocytes, B lymphocytes, the function of NK cells, dendritic cells, macrophages and other immune cells, for the treatment of immune-related diseases, and autoimmune diseases brought the dawn. Between the human fetal umbilical cord matrix source of mesenchymal stem cells (human umbilical cord matrix stem cell, hUC-MSC) not only has many unique advantages, like bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cell, BM-MSC), able to efficiently regulate the activity and function of T lymphocytes. Objective: human umbilical cord mesenchymal stem cells are capable of secreting a variety of growth factors and cytokines. The purpose of this study is to reveal the human umbilical cord mesenchymal stem cells to produce IL-6 mechanism, and influence in the process of tumor cell proliferation in human umbilical cord mesenchymal stem cells from IL-6. Methods: Human peripheral blood mononuclear cells (hPBMC) and human umbilical cord mesenchymal stem cell co-culture system to the interaction observed between the two cell. Preparation of a variety of conditions of the medium and the establishment of human umbilical cord mesenchymal stem cells cultured alone system, to study the impact of IL-6 secretion of soluble factors. ELISA quantitative culture supernatant IL-6, IL-1β and PGE2 levels. The Application Engineering L-1 receptor inhibitors and a variety of signal pathway inhibitor through the secretion of IL-1β to determine whether the CD14 monocyte upregulated the expression of IL-6 and the associated signaling pathway. Western blot to detect the activation of NF-KB. Using flow cytometry tumor cells Brdu incorporation, in order to confirm whether the mesenchymal stem cells of human umbilical cord can affect the proliferation of tumor cells, while assessing the role of IL-6 in this process. Results: (1) co-culture system in the concentration of IL-6 in human umbilical cord was a positive correlation between the number of mesenchymal stem cells, with a total incubation time. Showed progressive enrichment. (2) IL-6 from human umbilical cord mesenchymal stem cells secrete. (3) of CD14 mononuclear cells through the release of IL-1β-induced human umbilical cord mesenchymal stem cells to produce IL-6. (4) PGE2 is not involved in the induction of human umbilical cord mesenchymal stem cells to secrete IL-6. (5) c-Jun N-terminal kinase (JNK) signaling pathway and the transcription factor NF-κB induced expression of human umbilical cord mesenchymal stem cells with IL-1β, IL-6 closely related. (6) conditioned medium prepared from human umbilical cord mesenchymal stem cells can promote the proliferation of MCF-7 cells stimulated by IL-1β, IL-6 neutralizing antibody partially reversed the promoting effects. (7) luteolin significant inhibition of human umbilical cord mesenchymal stem cells in IL-6 secretion in a dose-dependent manner. Conclusions: (1) of CD14 monocytes release of IL-1β via c-Jun N-terminal kinase (JNK) signaling pathway activation of the transcription factor NF-kappa B, thereby inducing human umbilical cord mesenchymal stem cells to produce IL-6. (2) IL-6 intermediaries umbilical cord mesenchymal stem cells promote the proliferation of MCF-7 cells. (3) luteolin significantly inhibited in a dose-dependent manner, human umbilical cord mesenchymal stem cells in IL-6 secretion.

CD14~+ Monocytes Promote the Immunosuppressive Effect of Human Umbilical Cord Matrix Stem Cells

Abstract

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