| Microglia plays a crucial role in central nervous system immune defense during pathogen infection as resident immune cell in brain. The microglia can be quickly activated from the resting state and play its role in tissue repair, such as neurotrophic support, inflammatory or lymphocytes activation when exposed to endogenous signals, such as neuronal dysfunction or death, abnormal protein aggregation, and the interaction of immune cells, or exogenous stimuli, such as infection. Activated microglia releases anti-inflammatory factors (IL-10, TGF-β) and neurotrophic factors, such as neurotrophic factor and NGF, to protect and maintain the damaged neurons. Furthermore, it also plays important roles in the releasing of the pro-inflammatory factors (prostaglandins, TNF-α, IL-1βand other cytokines) the secretion of protease and the generation of reactive oxygen species (ROS) Inflammatory response is conductive to clear the invading pathogens, removing ecrotic cell damage and abnomal accumulation of protein deposits, and promoting the repairment of damaged tissue. But excessive activation of microglia may cause serious neurotoxic effects. Excessive inflammatory response will further aggravate tissue injury. Long-term inflammatory can develop into chronic inflammatory diseases. Research indicates that the activation of microglia is the key to the process of inflammation of many neurofegenerative diseases. Therefore, the neuronal damage caused excessive activation of microglia can be avoided through treatment and relieve the disease. All in all, microglia in central nerous system plays key roles in inflammation. It is also the important target for anti-inflammatory drugs.Microglia or macrophage recognition, phagocytosis of apoptotic cells are mainly through receptor-mediated protein complex; the molecular mechanism of which is currently a hot topic in the area of Neuroimmunology. Results show that, MFG-E8 is the key factor mediated by macrophages withered dead cells. Macrophage secrets MFG-E8 which connects to the surface of apoptotic cells in the eversion of phosphatidylserine, then anchores to the surface of macrophage integrin avP5, activates macrophages CrkⅡ-DOCK180-Racl signaling pathway, leads to cell cytoskeleton rearrangement, and thus play a role in phagocytosis. Meanwhile, it adds exogenous MFG-E8 of the isomers-D89E protein can effectively close the channel, inhibit the phagocytic activity and increase inflammation. In the year of 2004, Hanayama et al found that although macrophages of apoptotic cells has many other channels, but not phagocytosis of apoptotic cells caused by the deposition of a large number of apoptotic cells, and ultimately lead to splenomegaly and severe autoimmune disease in the MFG-E8 knockout model mouse. The study confirmed the importance and uniqueness of MFG-E8-αvβ5-Rac1 signaling pathway. Therefore, we proposed that MFG-E8 regulates the phagocytic behavior of microglial cells and plays an important role in the central nervous system, but whether the regulation mechanism of microglial phagocytosis is through the PS-MFG-E8-αvβ5-Rac1 signaling pathway is unknown, and also the activate mechanism of microglia and the inflammatory response after phagocytosis.In this study, the endogenous MFG-E8 in microglia cells was overexpressed and blocked seperately of in the phagocytosis of microglial cells and red blood apoptosis model cell of PS vagus to observe the phagocytosis of microglia. Analyzing the effect of endogenous MFG-E8 changes on the phagocytic activity of microglia. Then analyzing the relationship between microglia phagocytosis and the inflammatory responses. The study is divided into three parts:First, to build the model of red blood cells with PS valgus, phosphatidylserine was integrated into the surface of normal cells in the form of liposome. Detecting the microglia phagocytosis of apoptosis of red blood cells though co-culture technique. Results show that phagocytic microglial cells play a role in the inflammatory changes.Integrated by liposome, apoptosis rate of red blood cells was 7.4% to 47.5%. First treated by NEM (20μM, 10min), and then integrated with the PSox liposomes, the apoptosis rate of red blood cells was 35.7%, which was more stablein comparison with the apoptosis rate of 47.5% that was treated by NEM (30μM,30min) treatment. Therefore the PS valgus of 35.7% in the red blood cell phagocytosis experiment was used in the following steps. In the phagocytosis test, compared with normal controls group, the apoptosis of microglia phagocytosis of red blood cells was statistically significant (27.5%±5.4% vs 3.8±1.0%, p <0.01). Compared with normal group, Real-time PCR detection of red blood cells after phagocytosis in the interstitial cells of the inflammatory cytokines IL-1βand TNF-αfound that expression of inflammatory factors increased with the increasing of the phagocytosis.Second constructing the over-expression MFG-E8 vector and RNA interference lentiviral expression vector. Detecting its efficiency in expressing and blocking over MFG-E8. Expresson of GFP preliminary fusion protein in microglial cells has increased, which means that MFG-E8 expression vector has transfected into microglial cells. Meanwhile, after detection of MFG-E8 expression with the RNA interference vector and stable transfection by means of Real-time PCR and Western Blotting detection of microglial cells in, results showed an effective resistance against the expression of MFG-E8.Third, RNA interference expression vector was transfected into microglial cells. The expression of MFG-E8 was detected. We detected the expression of endogenous regulator of microglial cells in MFG-E8 expression, the phagocytic rate of apoptosis, and inflammatory cytokine expression of microglia after phagocytosis.Expression of MFG-E8 in microglial cells increased trasfected with overexpression and decreased after RNA interference lentiviral expression vector. Compared to the control, MFG-E8 overexpression significantly increased microglia phagocytosis of apoptotic rate of red blood cells (32.9%±9.6% vs 21.5%±6.28%, p<0.01); and comparing with the control, blocking the expression of MFG-E8 can significantly inhibited microglia phagocytosis of red blood cells (17.5%±4.1% vs 22.2%±7.8%,p<0.01). Meanwhile, the increasing expression of MFG-E8 can induce the increasing of phagocytosis in microglial cells, and the expression of microglia inflammatory factors also increasing significantly; on the contrary, blocking the MFG-E8 in microglial cells can reduce the expression of phagocytosis, microglial cells also can reduce the expression of inflammatory cytokines.Endogenous changes in expression of MFG-E8 can affect their phagocytosis. Enhanced expression of MFG-E8 could boost phagocytosis of apoptotic red blood cells of PS vaglus by microglial cell. On the contrary, blocking the expression of MFG-E8 can inhibit microglia phagocytosis, and decrease the expression of inflammatory cytokines. Results show that MFG-E8 plays an important regulatory role in microglia phagocytosis, and possiblely serve as an important signal molecule in microglial phagocytosis.
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