| PerfaceSubstance P is excitatory neuropeptides synthesised in neural and glial cells of the human central and peripheral nervous syste,one of the earliest neuropeptide,and widely distributed in the fine primary afferent nerve fibers.Physical and mental stimulation to the nervous system,can affect the direct effect of the immune system function,or through the release of hormones or other cytokines to regulate immune function,and the classical neurotransmitters,neuropeptide is a direct role in immune cells involved in immune response and other important regulators of the cell.The biological responses of SP are triggered via the wellestablished tachykinin receptors NK1,NK2 and NK3. Substance P is the major endogenous ligand for the NK1 receptor type.SP is the nerve internal secretion and the immune system common chemistry language.Being related to many kinds of clinical diseases,SP the function which causes under the dissimilar condition has the complexity and multiple.Further study to the specific mechanism of SP in the neuroendocrine-immune network could update the pathophysiology of inflammatory diseases and immunologic diseases,and may help to the prevention and cure in clinic.NK cells constitute an important component of the innate immune system,played an important role in the anti-infective,anti-tumor,immunoregulation and hematopoietic system.NK cells exert cell-mediated cytotoxicity and act to regulate innate and acquired immune responses through the release of various cytokines(such as IFN,GM-CSF and TNF)and chemokines.NK cell activity is regulated by an array of membranebond inhibitory and activating receptor.NK cell lines established especially for the establishment of NK92 their adoptive immunotherapy in the treatment of tumors in the applied research,and in-depth study of NK cell characteristics,functions,and further reveal it in the immune system to the exact role in providing a more favorable Research Materials and Methods.The neuropeptide substance P(SP)can modulate a number of immunological functions in vitro and in vivo.Here,we investigated if SP boosts proliferation and cytotoxicity of natural killer cells,and observed the effect of substance P(SP)on the expression of NKG2D/NKG2A to study the effect of SP on NK function and the possible mechanism of action.Materials and Methods1.Cell culture and treatment with SP in different concentration for 24h.2.The proliferation of human NK cells(NK92-MI)treated with SP(10-6mol/L, 10-8mol/L,10-9mol/L,10-10mol/L,10-12mol/L,10-14mol/L)was detected by MTT method and FACS.3.the cytotoxicity of NK92MI was checked through MTT assay.4.FACS was applied to measure the expression of NK cell receptors(NKG2D and NKG2A).5.RT-PCR was used for the mRNA expression of NKG2A/D.Results1.SP at the 10-6M-10-10M significantly promotes the proliferation and cytotoxicity of NK92MI,compare to the control group.2.After the co-culture with SP at 10-6mol/L,10-8mol/L,10-14mol/L for 24h,the percentage of G1 decreased and the S%increased,the proliferation index obviously raised.Otherwise,the S%descend and the G%raise up at 10-4mol/L.3.SP with diferent concentrations up-regulate the activity of NK92-MI at 4:1,and the effect of SP cut down after the concentration up.4.The membrane expression of NKG2D each concentration groups all upregulated with different levels.Compare to the control,SP upregulate the expression of NKG2A at 10-8-10-12M.5.NK92MI co-cultured with SP for 24h,RT-PCR measured the mRNA level of the receptors NKG2A and NKG2D.The results suggested that the mRNA expression of NKG2A/D up-regulated with different levels.ConclusionSP in a scope of concentration could promote the proliferation of NK92MI.SP with different concentrations up-regulated the activity of NK92-MI(at 4:1);enhanced the expression of NKG2D.Substance P inhibit the expression of NKG2A at 10-14M,but enhance the expression of NKG2D,which may promote the cytotoxicity of NK92-MI.
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