| Background and objectiveAlzheimer’s disease (AD) is a progressive neurodegenerative disorder and is the leading cause of dementia in the elderly. The deposition of extracellular amyloid-beta protein (Aβ), the formation of neurofibrillary tangles within neurons and neuronal loss is the main pathogenic factor of the AD. However, there have been no effective treatment for AD.Now, accumulating evidences suggest that neuroinflammation plays a significant role in the pathogenesis of AD. Activated microglia, astrocytes, peripheral lymphocytes and inflammatory factors play an important role in their inflammatory reaction. Neuroinflammation has become an important targets for the treatment of AD. Stem cell transplantation may exert a neuroprotective effect by anti-inflammatory and immunomodulatory effects. T regulatory cells (Tregs) are a class of T cell subsets, play an immunosuppression and immune incompetence. CD4+CD25+Tregs are the main components of Tregs subsets, exert a natural immunosuppressive effect, inhibit the activation and proliferation of CD4+or CD8+T cell and play a neuroprotective effect via inhibit neuroinflammation induced by microglia. Recently, evidences indicated that abnormalities of Tregs in cell number and/or function were associated with the inflammation or pathogenesis of AD.Wharton’s Jelly-derived mesenchymal stem cells (WJ-MSCs) are pluripotent stem cells, derived from human umbilical cord Wharton’s jelly. In addition to the ability of multipotent to differentiate into a variety of cell types, WJ-MSCs exert special immunomodulatory effects. Could transplantation of the WJ-MSCs exert therapeutic effects on AD model and reduce inflammation and modulate the immunity? Could WJ-MSCs modulate the frequency and/or function of Tregs in vitro? Could systemic transplantation of CD4+CD25+Tregs after co-cultured with WJ-MSCs cure AD and Inhibit the microglia induced neuroinflammation?Based on the backgrounds mentioned above, we studied protective effect and the mechanism of WJ-MSCs tail vein injection for the APP/PS1 double transgenic mice model; The spleen lymphocytes were co-cultured with WJ-MSCs in the medium for spleen lymphocytes in vitro for 3 days, We tried to confirm whether WJ-MSCs can modulate the frequency and/or function of Tregs in vitro; The purified CD4+CD25+T regulatory cells from APP/PS1 transgenic mice spleen lymphocytes after co-cultured with WJ-MSCs were administered to APP/PS1 transgenic mice by intracardiac injection, we aimed to investigate whether systemic transplantation of Tregs after co-cultured with WJ-MSCs could improve the impaired cognition of APP/PS1 transgenic mice, an animal model of AD. In addition, we tried to study its possible mechanism.Contents1. To investigate whether WJ-MSCs exert therapeutic effects on AD and to find its mechanisms.2. To investigate whether WJ-MSCs can modulate the frequency and/or function of Tregs in vitro.3. To investigate whether systemic transplantation of Tregs after co-cultured with WJ-MSCs exert therapeutic effects on AD and to find its mechanisms.Methods1. To make clear the effect and mechanism of WJ-MSCs on AD mice model, umbilical cords were obtained under sterile conditions from full-term infants, WJ-MSCs were isolated from human umbilical cord Wharton’s jelly. We used the 6-month-old APP/PS1 double transgenic mice as AD model, and the mice was divided into two groups:the control group and WJ-MSCs group.200ul of cell suspension (approximately 2×106 cells) was injected into the tail vein of APP/PS1 transgenic mice. For the control group,200ul of PBS was injected into the tail vein. Three weeks later, we used the modified Morris water maze to test the mice behavior, the hidden platform test was used to test learning ability and the spatial probe test was used to test mice memory. After the behavior test, thioflavin S staining was used to analysis the number and area of the senile plaques in the mice brain slices. Elisa was used to quantitate soluble Aβ4O and Aβ42 levels in the brain. First days and 4 weeks after treatment, Elisa was used to quantitate the protein expression levels of IL-10, IL-1β, TNF-a in brain homogenates and IL-10 in serum-derived samples, Iba-1 Immunohistochemical staining was used to test activation of microglial cells, Quantitative real-time PCR assay was used to test the mRNA expression levels of proinflammatory cytokines IL-1β,IL-6 and TNF-α in the brain of AD mice.2. To make clear whether WJ-MSCs can modulate the frequency and/or function of Tregs in vitro, umbilical cords were obtained under sterile conditions from full-term infants, WJ-MSCs were isolated from human umbilical cord Wharton’s jelly. Spleen lymphocytes were isolated from the spleen of the 6-month-old APP/PS1 double transgenic mice. The lymphocytes were co-cultured in the 12-well plate at the density of 5×105/well/ml with WJ-MSCs at the ratio of 1:5 (WJ-MSCs:spleen lymphocytes) or without WJ-MSCs in the medium for spleen lymphocytes in vitro for 3 days. The proportion of Tregs was measured by flow cytometry. To investigate whether Tregs after co-cultured with WJ-MSCs had the immunosuppressive function, the CD4+CD25+T regulatory cells were purified after co-cultured with WJ-MSCs or no co-cultured,and then, the purified CD4+CD25+T regulatory cells were co-cultured with CFSE labeling allogenic spleen lymphocytes in the presence of PHA (10ug/ml) in vitro for 3 days. After co-culture for 3 days in vitro, we did flow cytometry and analyzed the proliferation index by the software ModFit.3. To make clear the effect and mechanism of the purified CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs on AD mice model, the purified CD4+CD25+T regulatory cells from APP/PS1 transgenic mice spleen lymphocytes after co-cultured with WJ-MSCs were administered to APP/PS1 transgenic mice by intracardiac injection at the dose of 0.5×106 cells/1 OOul PBS for the first time, followed by a second injection at the dose of 0.2×106 cells/100μl PBS one week later. Another APP/PS1 transgenic mice were injected the same volume of PBS as the control. Three weeks after the first injection, we used the modified Morris water maze to test the mice behavior, the hidden platform test was used to test learning ability and the spatial probe test was used to test mice memory. After the behavior test, Elisa was used to quantitate the levels of plasma proinflammatory cytokines IFN-y and anti-inflammatory cytokines IL-10, TGF-β1. Four weeks after the first injection, thioflavin S staining was used to analysis the number and area of the senile plaques in the mice brain slices, Elisa was used to quantitate soluble Aβ4O and Aβ42 levels in the brain, Iba-1 immunohistochemical staining was used to test activation of microglial cells.RESULTSl.The effect and mechanism of WJ-MSCs on APP/PS1 transgenic mice model1.1 WJ-MSCs rescues learning and memory deficits in APP/PS1 mice. The morris water maze test shown that WJ-MSCs significantly reduced the escape latency compared with the control group. WJ-MSCs-treated mice also had significantly higher number of platform location crosses and longer time spent in the target quadrant than the PBS-treated groups in spatial probe test. Similar swimming speeds between the two groups were observed. Thus, our data indicated that tail vein injection of WJ-MSCs could rescue learning memory impairments in APP/PS1 mice.1.2 WJ-MSCs significantly reduces Aβ deposition and decreases soluble Aβ levels. We analyzed Aβ deposition in the mice by thioflavin S staining 4 weeks after treatment. Compared with PBS control group, WJ-MSCs treatment reduced Aβ deposition in both cortex and hippocampus of the mice. We examined cerebral soluble Aβand Aβ42 levels by ELISA assay. WJ-MSCs treatment significantly decreased Aβ40 and AP42 levels in the mice. These data seemed to support that WJ-MSCs treatment decreased Aβ level in the brain of APP/PS1 double transgenic mice.1.3 Study on mechanism of WJ-MSCs protective effect on APP/PS1 transgenic mice model.1.3.1 WJ-MSCs increased the expression of IL-10 in the serum and brain of the APP/PS1 double transgenic mice and inhibited the inflammatory reaction. IL-10 levels in serum and brain were both significantly increased in WJ-MSCs group compared with PBS group at 1 day after transplantation. Four weeks after treatment, there was no difference in the expression of IL-10 in serum between the groups. However, the WJ-MSCs treatment still remarkably increased IL-10 expression of the brain at 4 weeks post-injection.1.3.2 WJ-MSCs decreased pro-inflammatory microglial activation. At 1 day after injection, the area occupied by Iba-1-positive microglia was increased in the hippocampus of the mice in the WJ-MSCs group than in the PBS group. However, the area occupied by Iba-1-positive microglia was lower in the WJ-MSCs group compared with the PBS group at 4 weeks post-injection. It is suggested that WJ-MSCs may decrease the activation of pro-inflammatory microglia in the late stage of treatment.1.3.3WJ-MSCs significantly decreased the contents of pro-inflammatory cytokines IL-1(3 and TNF-a in the brain of APP/PS1 transgenic mice, but did not change the content of IL-6.At 1 day and 4 weeks after treatment, the expression of IL-1β and TNF-α of brain at gene level as revealed by real-time RT-PCR was all significantly decreased in WJ-MSCs group as compared with the PBS group.However, there was no difference in the expression of IL-6 in brain between the groups. Our ELISA data showed that the expression of IL-1β and TNF-α of brain was also significantly decreased in WJ-MSCs group as compared with the PBS group.2. WJ-MSCs improved the frequency and function of CD4+CD25+T regulatory cells in spleen lymphocytes from APP/PS1 transgenic mice.2.1 WJ-MSCs increased the frequency of CD4+CD25+T regulatory cells in spleen lymphocytes from APP/PS1 transgenic mice. The lymphocytes were co-cultured with WJ-MSCs at the ratio of 1:5 (WJ-MSCs:spleen lymphocytes) in vitro for 3 days. Flow cytometry data revealed that the frequency of CD4+CD25+T regulatory cells in the total cell population in the presence of WJ-MSCs in vitro for 3 days was significantly increased compared to those in the absence of WJ-MSCs.2.2 WJ-MSCs improved the immunosuppressive function of CD4+CD25+T regulatory cells in spleen lymphocytes from APP/PS 1 transgenic mice. The CD4+CD25+T regulatory cells were purified after co-cultured with WJ-MSCs or no co-cultured,and then, the purified CD4+CD25+T regulatory cells were co-cultured with CFSE labeling allogenic spleen lymphocytes in the presence of PHA (10ug/ml) in vitro for 3 days. After co-culture for 3 days in vitro, we did flow cytometry and analyzed the proliferation index by the software ModFit. We found that the purified Tregs after co-cultured with WJ-MSCs significantly reduced the proliferation index of PHA stimulated spleen lymphocytes compared to those no co-cultured by statistic analysis. These data suggested that WJ-MSCs could improve not only the frequency but also suppressive function of CD4+CD25+T regulatory cells in vitro.3.The effect and mechanism of the purified CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs on AD mice model3.1 Transplantation of CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs improved the impairments of cognition in APP/PS 1 transgenic mice. The morris water maze test shown that systemic transplantation of CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs significantly reduced the escape latency in the last 3 days and the pathway to find the hidden platform. The data also showed that transplantation of CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs significantly increased the number of platform crossing as well as the time in the target section during the 60s probe trial. These data indicated that systemic transplantation of CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs could ameliorate the cognitive impairments of APP/PS 1 transgenic mice.3.2 Transplantation of CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs significantly reduces AP deposition and decreases soluble Aβ levels. At the end of the fourth week of the initial cell transplantation, we measured the total area of the cortex and hippocampus by Thioflavin-S staining. In the cortex and hippocampus, statistic analysis showed that the area and the number of Aβ plaque were significantly reduced after transplantation of CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs. The levels of the soluble Aβ4O and Aβ42 were measured by ELISA kits. The result revealed that transplantation of CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs could significantly reduce the level of the total soluble Aβ4O and Aβ42 in the brain.These data seemed to support that CD4+CD25+T regulatory cells treatment decreased Aβ level in the brain of APP/PS1 double transgenic mice.3.3 Study on mechanism of CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs protective effect on APP/PS1 transgenic mice model.3.3.1 CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs inhibited the inflammatory reaction by increased the plasma levels of the cytokine TGF-β1 and IL-10 and decreased the plasma levels of the cytokine IFN-y. We measured the levels of plasma pro-inflammatory (IFN-y) and anti-inflammatory cytokines (IL-10 and TGFβ1) by ELISA kits at the end of Morris water maze. We found that the plasma levels of the cytokine TGF-β1 and IL-10 were both significantly increased in the plasma of the APP/PS1 transgenic mice receiving CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs compared to the APP/PS1 transgenic mice receiving PBS. In contrast, we observed that CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs exerted a significant adverse tendency in the plasma level of IFN-y compared to those receiving PBS.3.3.2 CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs decreased microglial activation. Four weeks after the first injection, we used Iba-1 antibody to label the microglia by flouresecent immunohistochemistry to analyze the status of microglia cells in the brain of APP/PSl transgenic mice. We observed that most of microglia cells exerted small bodies and thin and long processes in the cortex after treatment with CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs, compared to those exerting enlarged cell bodies and short processes in the cortex after with PBS treatment. In addition, we found that transplantation of CD4+CD25+ T regulatory cells after co-cultured with WJ-MSCs significantly reduced the number of activated microglia cells.Conclusion1. Tail vein injection of WJ-MSCs alleviates memory deficits and reduces amyloid-P deposition in an APP/PS1 transgenic mouse model.2. The mechanism of WJ-MSCs-mediated beneficial effects was related to the inhibition of inflammatory response, which increased the expression of anti-inflammatory cytokines, decreased the expression of pro-inflammatory cytokines and decreased pro-inflammatory microglial activation.3. WJ-MSCs improved the frequency and function of CD4+CD25+T regulatory cells in spleen lymphocytes from APP/PS1 transgenic mice.4. Transplantation of CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs alleviates memory deficits and reduces amyloid-β1 deposition in an APP/PS1 transgenic mouse model.5. The protective effect of CD4+CD25+T regulatory cells after co-cultured with WJ-MSCs was related to the inhibition of inflammatory reaction, which increased the expression of anti-inflammatory cytokines, decreased the level of pro-inflammatory cytokines and decreased microglial activation. |
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