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Vaccination with Evodiamine Stimulated Dendritic Cells Pulsed with Homogenate Protein of Spinal Cord Promotes Functional Recovery from Spinal Cord Injury in Mice

Author: WangKe
Tutor: ZhaoJianHua
School: Third Military Medical University
Course: Surgery
Keywords: Spinal cord injury Dendritic cells Spinal cord homogenate protein Evodiamine Autoimmune reactions Neuroprotective
CLC: R651.2
Type: Master's thesis
Year: 2010
Downloads: 35
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Abstract

Divided into primary injury and secondary injury, spinal cord injury (spinal cord injury, SCI) primary injury is caused by mechanical injury, secondary injury caused by a series of biochemical, immunological mechanisms and the spinal cord of the original a cascade of re-injury of the spinal cord after onset damage caused due to various factors, increase the primary injury, leading to a more severe clinical symptoms. The traditional theory is that inhibit the activity of immune cells to reduce secondary spinal cord injury. Therefore, methylprednisolone, dexamethasone, reducing edema, protect cell membranes, while inhibiting the activity of immune cells, reducing secondary injury in the early stages after SCI. However, a lot of experimental evidence that: central nervous system (central nervous system, CNS) autoimmune reaction of endogenous antigen neuroprotective effect. CNS antigen-specific T cells (such as myelin basic protein-specific T cells) play a key role in physiological processes in the CNS protection and repair. Based on the previous experimental studies in rodents, Schwartz neurodegenerative condition \including spinal cord injury. Dendritic cells (dendritic cell, DC) are professional antigen-presenting cells, play a key role in the stimulate and regulate the adaptive immune response. DC-based immunotherapy in recent years, has received a lot of attention. DC which has been successfully used to produce tumor antigen-specific cytotoxic T cells, to induce immune tolerance and T cell polarization. Therefore, DCs as a vaccine to stimulate protective immune response. Previously, we reported local or systemic injection load the spinal cord homogenate protein (homogenate protein of spinal cord hp) of DCs (hpDC), can significantly promote functional recovery of SCI mice. Better efficacy compared with the DC load myelin basic protein load spinal cord homogenate protein. Evodiamine (Evodiamine, EVO), is an alkaloid extracted from Evodia. It has a variety of biological effects, such as anti-tumor growth and angiogenesis, anti-inflammatory role. The findings of our group can raise the capacity of mature DC induce antigen-specific T cell responses in vitro EVO. Then, load evodiamine stimulation of spinal cord homogenate protein the DC (hpDCevo), whether than load spinal cord homogenate protein the DC (hpDC), stimulate stronger against CNS antigen-specific T lymphocytes in the immune response, and further promote SCI animal functional recovery, at home and abroad has not been reported. MATERIALS AND METHODS: Firstly, by intraperitoneal injection of PBS, DC, load spinal cord homogenate protein load evodiamine stimulation of spinal cord homogenate protein DC treatment T9 spinal cord injury in animals, the use of BMS score, histopathological examination assessment evodiamine stimulation load DC can promote spinal cord homogenate protein SCI mouse hindlimb functional recovery. After using ELISA, MTT, immunofluorescence assay mouse spleen T lymphocyte proliferative activity of Th cells secrete cytokines IFN-γ and IL-4 to explore the Th cell polarity changes, as well as the nerve trophic factor BDNF and NT-3 and neural stem cell marker Nestin, axon marker NF200 expression. Spinal cord homogenate protein load further explore evodiamine stimulation DC to promote the SCI mouse hindlimb functional recovery of possible mechanisms. Results: 1. Mice modeling showed complete paraplegia, 2w the BMS score gradually restored about. Which, load evodiamine stimulation of spinal cord homogenate protein the DC (hpDCevo), mice fastest recovery, enter 28dpi plateau the BMS score as high as 6.29 ± 0.25 and 6.92 ± 0.2 final spinal cord homogenate protein was significantly higher than the load DC group (6.00 ± 2.7) and PBS group (3.75 ± 0.27) (p lt; 0.05). Spinal cord homogenate protein load that intraperitoneal injection of evodiamine stimulation DC to promote the recovery of hindlimb motor function in spinal cord injury in mice. 2. Histopathological detection evodiamine stimulation spinal cord homogenate protein load DC (hpDCevo) group of the damage area (1.93 ± 0.16mm2) and local void range (0.22 ± 0.01mm2) and load spinal cord homogenate protein the DC (hpDC) group damage area (2.18 ± 0.12mm2) and local empty range (0.25 ± 0.02mm2) showed a statistically significant difference (p lt; 0.05). That the spinal cord homogenate protein load intraperitoneal injection of evodiamine stimulation the DC significantly reduce the area and empty area of ??spinal cord injury. 3 vitro MTT assay confirmed that, evodiamine, DC evodiamine stimulated DC same species animals were not promote T lymphocyte proliferation; However, the load of ovalbumin and evodiamine stimulation DC (ovaDC) load ovalbumin the DC (ovaDCevo) could promote the same species of animal T lymphocyte proliferation OD value of 0.62 ± 0.03,0.99 ± 0.04, respectively, evodiamine stimulation load ovalbumin DC than load ovalbumin DC stimulated more strongly T cell proliferation (p lt; 0.05). That evodiamine upregulated antigen-loaded DC ability to stimulate the proliferation of T cells. Vitro experiments confirmed intraperitoneal injection of evodiamine stimulation load spinal cord homogenate protein the DC (hpDCevo), group splenic T cells stronger proliferative capacity (OD value ratio the load a spinal cord homogenate protein the DC (hpDC), splenic T cells were 0.33 ± 0.03,0.25 ± 0.04) (p lt; 0.05). At the same time, the load evodiamine stimulation of spinal cord homogenate protein damage local DC group (673.33 ± 167.17/mm2), the number of T cells was significantly higher than the DC load spinal cord homogenate protein group (500 ± 146.42/mm2) (p lt; 0.05 ). Step intraperitoneal injection load evodiamine stimulate spinal cord homogenate protein DC excited more strongly T cell immune response. 5 In vitro experiments confirmed, intraperitoneal injection evodiamine load spinal cord homogenate protein stimulation DC (hpDCevo) group of T cells in the spleen of the secretion of IFN-γ was significantly higher than the DC load spinal cord homogenate protein (HPDC) group of, and IFN- γ concentration is gradually increased with time. Cultured in vitro for 72 hours, groups of IFN-γ concentration was 2792.54 ± 162.99 pg / ml 932.94 ± 60.24pg/ml (p LT; 0.05). The intraperitoneal injection of evodiamine stimulation load spinal cord homogenate protein the DC (hpDCevo) injury local concentration of IFN-γ (1882.85 ± 50.67ng / g) was significantly higher than the load of the DC protein of spinal cord homogenate (HPDC) group (970.56 ± 19.9 4ng / g) (p lt; 0.05), and IL-4 in vitro and in vivo experiments there was no statistical difference. Tips, load intraperitoneal injection cornel alkali stimulation of spinal cord homogenate protein the DC (hpDCevo) excitation dominated by a Th1 immune response. 6 In vitro experiments confirmed, intraperitoneal injection evodiamine stimulation load spinal cord homogenate protein the DC (hpDCevo) group splenic T cells secrete BDNF (393.80 ± 48.99 pg / ml) and NT-3 (32.17 ± 2.48 pg / ml) of concentration was significantly higher than the DC load spinal cord homogenate protein group (BDNF (HPDC): 300.10 ± 14.34 pg / ml and NT-3: 7.95 ± 4.08 pg / ml) (p LT; 0.05). Group injury local load evodiamine stimulation of spinal cord homogenate protein the DC (hpDCevo) of BDNF (27.71 ± 0.35ng / g) and the concentration of NT-3 (1.47 ± 0.11ng / g) are also significantly higher than that of the DC load spinal cord homogenate protein (HPDC) group (BDNF: 25.39 ± 0.55 ng / g, and S-3: 0.98 ± 0.16 ng / g) (p LT; 0.05) predator staining found the load evodiamine stimulation of spinal cord homogenate protein (hpDCevo) DC group, the area of ??spinal cord injury the Nestin cell (187.5 ± 18.73/mm2) and NF200 (88 ± 12.9/mm2) the number of the cells were significantly more than the load spinal cord homogenate protein the DC (hpDC), group (Nestin cells: 101.75 ± 15.44/mm2 , NF200 cells: 61.75 ± 20.12/mm2) (p lt; 0.01). Conclusion: 1. Experiment is the first to show, intraperitoneal injection of evodiamine stimulation of spinal cord homogenate protein load DC than load spinal cord homogenate protein DC significantly promote T9 SCI mice hind limb motor function recovery, reduce the damage area . Evodiamine enhanced load spinal cord homogenate protein DC neuroprotection. 2 evodiamine can upregulate antigen-loaded DC to stimulate the ability of the T cell immune response. Load spinal cord homogenate protein intraperitoneal injection of evodiamine stimulation the DC promotion of Th0 to Th1 offset. Intraperitoneal injection of evodiamine stimulation load spinal cord homogenate protein DC than the DC load spinal cord homogenate protein significantly promote local damage neurotrophic factor BDNF and NT-3 expression, and raised the number of Nestin cells and NF200 cells. This to show that hpDCevo induced neural stem cells / precursor cells differentiate into neurons, or by promoting T cells to secrete neurotrophic factors. So as to promote the recovery of hindlimb motor function in spinal cord injury in mice.

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