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Therapeutic Effect and Mechanism: Experimental Study of FTY-720in the Treatment of EAE in Mice

Author: ZhuDeSheng
Tutor: GuanYangTai
School: Second Military Medical University
Course: Neurology
Keywords: MS EAE FTY-720 effect mechanism
CLC: R744.51
Type: PhD thesis
Year: 2013
Downloads: 39
Quote: 0
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Abstract


Multiple sclerosis (MS) is a T cell-mediated autoimmune disease and is the mostcommon chronic inflammatory demyelinating disorder in the central nervoussystem(CNS), Inflammatory abnormalities always show in the brain and spinal cord.Nerve pathology mainly includes focal demyelination, oligodendrocyte death, axonaldegeneration as well as the proliferation of astrocytes, the pathogenesis of MS is not yetclear.In China, with the change of people’s habits and the ecological environment, as wellas the raising level of awareness and diagnosis of MS, It has become a common clinicaldisease of the nervous system. The treatment dependent on drug, includingGlucocorticoids, Beta interferon and Acetyl of Gray behalfketone, but these drugs areeffective only on a part of MS patients, there is still a considerable part of patientssuffering from the pain,It is urgent to solve the problem. Therefore, it is currentlypressing medical research to investigate the pathogenesis and clinical treatment of MSthrough the animal model of MS.Experimental autoimmune encephalomyelitis (EAE) is the prime animal model ofMS, the animal is susceptible to CD4+helper T-Lymphocyte. The clinical andpathological features of EAE are similar to multiple sclerosis, therefore,EAE isrecognized as the ideal model of MS.Chronic relapsing EAE (Cr-EAE) is also similar tothe "relapsing–remitting” MS. It is valuable for us to investigate the pathologicalfeatures of MS and therapeutic effect by EAE experimental.FTY-720is an immunosuppressant drug that is developed in recent years. Itelectively reduces the number of T lymphocytes in the peripheral blood circulation, Themechanism of the drug is unique, It has been proved as a powerful immunosuppressiveand little side effects drug.FTY-720showed immunosuppressive activity in organtransplantation of experimental animal by oral. There is few research on FTY-720thathow to induce the immune tolerance. There is a report in the literature on the molecularmechanisms of FTY-720in transplantation in2012. Based on this theoreticalfoundation,We explore the therapeutic effect and mechanism of FTY-720.We are intends to build the animal model of Multiple sclerosis and carry out twomajor experimental researchs including animal intervention experiments and discussion on the mechanism of action on this platform.Then, we will to respectively explore theclinical efficacy of early immunosuppressive therapy and the mechanism of FTY-720..We hope to further understanding the immune pathogenesis and pathologicalcharacteristics of demyelinating disease in central nervous system by these experimentaland wish to explore new ways to study the basic research and clinical treatment of MS.Objective:Part I:The experiment is to explore the typical clinical and pathological features of EAE bybuilding EAE mouse model. By observing the changes in the course of disease andpathologic features of EAE after early treatment with immunosuppressant--FTY-720, wehope to explore the intervention effect of immunosuppressive therapy on the incidence ofEAE.Part II:This experiment is to carry out cell culture experiments in vitro to observe theproliferation and apoptosis of CD4+T lymphocyte which is dealed with FTY-720.we alsowant to carry out intervention experiments in vivo to observe the inhibition of CD4+Tlymphocytes after treatment with FTY-720. then to detect some tissue cytokines of EAEmice in spinal cord and to detect the expression of intracellular signaling molecules. Wedetect the expression of inflammatory and screen the cytokines signaling pathway andsignaling proteins to explore the action mechanism of FTY-720.Methods:Part I: Animal intervention experiment.In this study, we design a prospective cohort study, C57BL/6mouse will be inducedto build chronic EAE model via MOG35-55initiatively, then they will be divided into thecase group and different intervention groups, the case group is treated with saline andintervention groups are treated with different dose of immunosuppressant FTY-720.Clinical indicators such as body weight, clinical score, incidence will be observed in thecourse of the disease in four groups. Spinal cord pathological features of EAE mice wereobserved at the end of the experiment time. At last,we comparatively analysiz these datesbetween these groups.Part II: Biochemistry detection experiment.We will observe the proliferation and apoptosis of CD4+T lymphocyte in vitro by “FCM” to see whether FTY-720show the immunosuppressive action on EAE or not? Weobserve the change of CD4+T lymphocyte in spleen of EAE mice by “FCM” to value theeffect of FTY-720on EAE in vivo. We detect the expression of inflammatory factor inspinal of EAE mice by “Rt-PCR” to value the effect of FTY-720on inflammatory factor.We detect the expression of decapentaplegic in CNS of EAE mice by “Western blot” toscreen the signal path and decapentaplegic that affect the expression of inflammatoryfactor in spinal.Results:Part I:1. The case group: clinical neurological score, body weight and incidence of these EAEmice showed chronic multiple performance, the phenomenon is manifest by"chronicity monophase", with typical process of MS disease; The pathologicalobservations also showed the typical pathological characteristics of demyelinationwith inflammatory cell infiltration.2. The intervention group: clinical neurological score, weight and the incidence of EAEmice were single-phase performance with "chronicity monophase", although we canobserve the pathology characteristics with inflammatory cell infiltration,demyelination, but compared with case group, their clinical symptoms andpathological features are not typical.Part II:1. Cell culture experiment in vitro: the proliferation of CD4+T lymphocyte reduced andthe apoptosis of CD4+T lymphocyte increased after intervention with FTY-720.2. Drug interference experiment in vivo: the number of CD4+T lymphocyte in spleen ofEAE mice reduced, it shows that FTY-720can inhibit the activation of CD4+Tlymphocyte.3. Cytokine detection experiment: FTY-720can restrain the CD4+T lymphocyte tosecrete specificity cytokine such as IL-10and IL-17, it also can restrain the CD4+Tlymphocyte to secrete non-specificity cytokine such as IL-2,IL-6,TNF-α,iNOS andINF-γ.4. Signaling molecules detection experiment: the expression of cPLA2in spinal cord ofEAE mice reduced after intervention with FTY-720. Conclusion:Part I:1. Model assessment: C57BL/6mice can be successfully induced “chronicitymonophase” EAE model via MOG35-55.2. Intervention assessment: The early use of immunosuppressive agents of FTY-720can reduce EAE’s clinical neurological score and the occurrence of EAE, it also canchange the course of EAE.3. Dose assessment: FTY-720(0.3mg/kg/d) can effectively reduce the incidence ofEAE.Part II:1. FTY-720can inhibit the activation of CD4+T lymphocyte and reduce the number ofCD4+T lymphocytes in peripheral blood of EAE mice.2. FTY-720can inhibit the secretion of specific and non-specific inflammatory cytokinein CNS of EAE mice and downregulate the inflammatory response;3. FTY-720may inhibit the secretion of inflammatory cytokine through downregulationthe expression of cPLA2, the path of cPLA2may participate in theimmunosuppressive action.

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CLC: > Medicine, health > Neurology and psychiatry > Neurology > Spinal cord disease > Demyelinating disease > Multiple sclerosis
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