Study On Calcium Gene-Related Peptide To Stimulate Keratinocyte Cell Secret Chemokine Ccl27

BackgroundPsoriasis is a common chronic inflammatory skin disease, its etiology and pathogenesis are still unclear, It is currently considered to be a in genetic background, influenced by a variety of internal and external environmental factors stimulation and induced chronic relapsing inflammatory skin disease.The main histopathological features were keratinocyte hyperplasia, inflammatory cell infiltration, neovascularization. In psoriatic pathological changes of dermal papillary vasculature abnormalities first occurs, then the appearance of inflammatory cell migration and epidermal hyperplasia and differentiation abnormality, thereby starting psoriasis development process. At the same time, large numbers of immunologically active cells such as:T lymphocytes, monocytes, mast cells and the secretion of cytokines Exudate into the extravascular tissue, Induce keratinocyte activation, proliferation and Secret lot of cytokines, adhesion molecules and chemokines, which in turn promotes vascular abnormalities, thereby forming a vicious spiral of cell factor regulation, promote the progress of the disease. Chemokine is a kind of specific small molecular proteints that attracting leukocyte, divided into C, CC, CXC and CX3C four subgroup. Chemokine factor by the chemokine receptors on target cell membrane. As present research thought, chemokine and its receptor binding, is the main stimulating factor of regulation in lymphocyte migration, besides, in nerve conduction, angiogenesis and inflammatory response regulator it also play an important role. A variety of chemokines and their receptors are involved in the pathogenesis of psoriasis. Research shows that, chemotactic factor in epidermal keratinocytes of psoriasis, lichen planus, atopic dermatitis and other inflammatory skin disease were in high expression, and keratinocytes are the major source of these chemokines. Recent studies have shown, in a wide range of inflammatory and autoimmune diseases, upregulation or downregulation of chemokine and chemokine receptor expression, can influence disease susceptibility, progression and severity CCL27is mainly produced by skin keratinocytes, only to cutaneous lymphocyte associated antigen (CLA) positive memory T cells have a strong chemotaxis. CCR10is the only receptor to CCL27, CCL27/CCR10interactions in T cell-mediated cutaneous inflammatory responses play a key role in. CCL27and CCR10in the lesions of patients with psoriasis was markedly increased, and the expression level and PASI score was correlated, suggesting that CCL27is involved in the pathogenesis of psoriasis, its expression levels and the severity of psoriasis have a certain correlation. The secretion and expression of CCL27play a role as a bridge in keratinocytes and between T cells, by attracting more CCR10+/CLA+memory T cell accumulation in the skin, so that psoriasis skin inflammatory reaction exacerbate and persist.Clinical observation find that severe mental stimulation, or stress can induce and aggravate psoriasis. Previous research has found that neuropeptides widely exists in the cutaneous nerve fibers and a variety of skin cells, such as keratinocytes, Lange Hans cells, melanocytes and lymphocytes, monocytes, and the cell surface distribut neuropeptides receptors. Because of the nervous system and skin immune system various target cells have close contact, sensory nerve endings extending into the skin release neuropeptides that contact with epidermal and dermal cells, can be directly modified keratinocytes, Lange Hans cells, macrophages, dermal microvascular endothelial cells and infiltration immune cells, influence the proliferation and activation and the production of cytokines and antigen presentation of these cells. The neuropeptide calcitonin gene related peptide is the most important vasodilatory peptide, containing37amino acid residues, by calcitonin gene encoding. CGRP is widely present in the central and peripheral nervous system. CGRP is a kind of important neurotransmitters in human skin tissue, strong diastolic dermal microvascular function, but also can stimulate the activation of human dermal microvascular endothelial cell and the secretion of chemokines IL-8, induces endothelial cell and monocyte and neutrophil adhesion effect. In addition, CGRP can upregulate keratinocytes express IL-1, induces monocyte synthesis IL-8and MCP-1, enhance chemotaxis of neutrophils and T lymphocyte; also can promote HaCaT cell express nNOS and NO, involving in cutaneous microvascular expansion and neurogenic inflammation. It was proved that, in certain psychic factor and related diseases such as psoriasis,skin lesions and plasma levels of neuropeptide CGRP expression significantly increased, the local skin CGRP immunoreactive nerve fibers increase, so CGRP may be involved in the pathogenesis of psoriasis and play a important role in.The effect of CGRP on CGRP receptor has a variety of signaling pathways, including cyclic adenosine monophosphate (Cyclic Adenosine monophosphatec, cAMP), intracellular free calciumion (Intracellular Ca2+) and mitogen-activated protein kinase (Mitogen-activated protein kinase MAPK) and kf-kb signal pathway. It has been demonstrated that CGRP can act as a mitogen stimulate the proliferation of KC, and promote skin lesions localized hyperplasia, and is involved in the occurrence of psoriasis process, while the research about relationship between CGRP and chemokine is still small.Objeetive1Investigate the effects of CGRP on HaCaT cell secretion of chemokines CCL-272Investigate the effect of CGRP promotes HaCaT cells on lymphocyte chemotactic.3Investigate of MAPK signaling pathway in CGRP induced HaCaT cell secretion of chemokines CCL-27 Method1.HaCaT cell cultureHaCaT cells with10%FBS DMEM37OC,5%CO2placed in an incubator for training.2. Separat peripheral blood T lymphocyte from psoriasis and healthy controls.3. Experimental design1) HaCaT cell were incubated with CGRP, or Giving CGRP receptor antagonist CGRP8-37, ERK1/2blocking agent PD98059, p38MAPK blocking agent SB203580, PDTC pretreatment, using the Transwell system for the detection of HaCaT cells on lymphocyte chemotactic function influence.2) HaCaT cells were incubated with different concentrations of CGRP, giving CGRP receptor antagonist CGRP8-37, ERK1/2blocking agent PD98059, p38MAPK blocking agent SB203580, NF-kappa B antagonist PDTC pretreatment for determination of HaCaT cell secretion of chemokines in CCL-27mRNA and protein expression;3) The HaCaT cells were incubated with CGRP, giving CGRP8-37, PD98059, SB203580or PDTC to determine phosphorylation of ERK1/2, p38and the change of I kappa B-a;3. Experiment Methods3.1Chemotaxis experimentsSeparat peripheral blood T lymphocyte,from psoriasis patients and healthy controls, join RPMI-1640culture liquid containing5%bovine serum albumin to prepare cell suspensions, then use24hole chemotaxis chamber to assay lymphocyte chemotactic activity.3.2Real-time RT-PCRTotal RNA were extracted from the collected cells, cDNA by reverse transcription reaction,get cDNA by reverse transcription reaction, beta-actin as reference, through the real-time RT-PCR technique to detect the expression of CCL-27mRNA.3.3ELISAThe cell culture supernatants were collected, using CCL27ELISA kit to measure the concentration of CCL27.3.4.Western blotThe cell total protein was extracted, after SDS polyacrylamide gel electrophoresis (SDS-PAGE), separation, membrane switch, DAB color, gel imaging system, ImageToo13for semi quantitative analysis, detect ERK1/2, p38, JNK protein expression.Results1. CGRP promotes HaCaT cell on lymphocyte chemotactic activityIn a certain extent neuropeptide CGRP can promote HaCaT cell on lymphocyte chemotactic activity (P<0.01), The CI of psoriatic group and thecontrol group of healthy were6.65±0.21and4.25±0.16; after pretreatment with CGRP3-87, PD98059and PDTC, the effect of CGRP on the chemotactic activity of lymphocytes decreased obviously.2. CGRP induced the mRNA expression and production of CCL27CCL27Mrna expression was significantly inereased byCGRP(0.1,1, and10nM). CCL27production was measured from supernatants of CGRP-treated cultured HaCaT cells collected12h after the exposure. 3. Effeetof CGRP8-37, PD98059, SB203580, or PDTC on CGRP-induced the mRNA expression and produetion of CCL27in HaCaT cellsAfter pretreatment with CGRP8-37, PD98059and PDTC, CCL27mRNA and protein expression induced by CGRP were significantly reduced; but still higher than the normal control group.4.Roles of MAPK and NF kappa B signal transduction pathway in CGRP induce HaCaT cells expressing CCL27CGRP in a certain range of time-dependent promote ERK1/2, P38phosphorylation; after pretreated by CGRP8-37p-ERK1/2and P-38expression were significantly decreased, pretreated by PD98059p-ERK1/2expression was decreased; pretreated by SB20358p-38expression was significantly decreased.IκB-α after start degrade incubated by CGRP in10min, and no express in60min, CGRP8-37and PDTC inhibited degradation of IκB-α.Conclusion1. CGRP can promote HaCaT cell on lymphocyte chemotactic activity.2. CGRP can increase the HaCaT cell CCL27mRNA and protein expression.3. CGRP can be time dependently activat EPK1/2, p38MAPK, NF kappa B signal pathway in HaCaT cells.4. ERK1/2, p38MAPK and NF kappa B signaling pathway may be involved in CGRP up-regulation CCL27expression in HaCaT cell.