| Spinal cord injury(SCI)is a seriously disabling disease,which is one of the most serious disaster no matter for patients and families or to society,the pain and burden often accompany lifetime.The current basic.and clinical studies suggest that neuron of secondary damage after SCI is likely to be reversed,which provides a research direction for the treatment of SCI.Microglia is the resident immune cell in Central Nervous System(CNS)and it is an important part of the immunization and defense system of the CNS.Microglia can protect CNS from various pathological factors,but it sometimes leads to CNS related lesions of deteriorating even further.After the occurrence of neuropathy such as stroke,traumatic brain injuries and SCI,microglia shows the dual function of damage and repair for local neurons in the damaged region.On the one hand,microglia can make a rapid immune response,directionally migrate to lesion site and release a large amount of proinflammatory factor to start the inflammatory reaction.Excessive and repeatedly inflammation will produce toxic substances,which will not only cause severe damage to the nervous system,but also influence neuron repair and regeneration.On the other hand,after microglia cells migrate to injury,its phagocytosis can remove apoptosis neurons,reduces the release of cytokines and neurotoxin substances,thereby inhabits excessive immune inflammatory reaction,coordination and promote the reconstruction of the neural network structure and function recovery.Therefore,microglia play a very important role in the physiological and pathologic changes of CNS.Chinese herbal compound "Jisuikang?" is the empirical formula of my tutor.Our have carried out a number studies concerning "Jisuikang" promote restoration of neuron after SCI,the results show that "Jisuikang" has an important regulatory effect on the immune response after SCI,and it can effectively promote the repair and regeneration of damaged neurons.Currently,less researches of TCM were pay attention to the phagocytosis of microglia,the existing research is mainly limited to some Chinese herbs could inhibit excessive inflammatory reaction and plays a neuroprotective role in SCI.In view of those,the purpose of this study is to investigate the potential mechanism of effective intervention on SCI of "Jisuikang"and provide a scientific evidence on SCI for clinical treatment of TCM.In the first part of this research,BV2 was transfected by lentivirus to express green fluorescent protein.The neurons were labeled by fluorescent nuclear staining.A system for dynamically observing the engulfment of neuron debris by BV2 was established.We found that the "Jisuikang" can promote phagocytosis of neuron debris by BV2.We hold the opinion that,CNS is a complex system composed of a variety of glial cells and neuron,SCI is not the disease of single system or a single cell group.Therefore,on the basis of the first part study,we extract the fetal rats nerve tissue and culture it without special selection.And we preliminarily simulate a mixed-culture system with multiple factors and integrity.After using drugs to induce neurons apoptosis,we once again proved that "Jisuikang" plays an important role in promoting the phagocytosis of microglia by this mixed-culture system.It is important that this trial reflects the overall concept of TCM.Reviewing previous intervention of traditional Chinese Medicine research of microglia,we found that there are little researches about Chinese herbs regulating phagocytosis of microglia to promote neurons repair,so we think whether the protective effect of traditional Chinese medicine for damage nerve associated with regulation of phagocytosis of microglia?Both the two experimental results show that the "Jisuikang" can effectively regulate the phagocytosis of microglia,but the function whether have correlation with promote nerve repair?Based on this problem,we carried out a third part of the experiment to verify whether "Jisuikang" can promote neurons by regulating the microglia phagocytosis repair regeneration.In this experiment,we successfully separate and purify primary neurons and microglia,successfully establish microglia/damage neurons co-culture system.We found the "Jisuikang" not only can promote microglia phagocytosing apoptosis neurons,but also make survive neuron regeneration get obvious improvement.We found the "Jisuikang" not only can promote microglia phagocytosing apoptosis neurons,but also make survive neuron regeneration get obvious improvement.In conclusion,we found that TCM compound "Jisuikang" can effectively promote microglia phagocytosing neuron debris,which may improve the local microenvironment and promote the repair and regeneration of injured neurons.In addition,this research provides a new thought and direction for the research of Chinese medicine in CNS related diseases.Part ? Effect of "Jisuikang" on engulfment of neuron debris by microgliaObjective:Observing the effect of herbal compound "Jisuikang" on engulfinent of neuron debris by BV2.Methods:The primary neurons were isolated and identified.BV2 was transfected by lentivirus to express green fluorescent protein.The neurons were labeled by Fluorescent nuclear stain.The phagocytosis of treated BV2 was examined by fluorescence microscope.Results:Low,middle and high dose of "Jisuikang" showed higher phagocytosis index than that of control,Middle and high dose of "Jisuikang" had greater phagocytosis percentage versus that of control.And there were no significance between middle and high dose of "Jisuikang" in both higher phagocytosis index and phagocytosis percentage.Conclusion:"Jisuikang" may enhance the engulfment of neuron debris by BV2,which may be related to its good clinical efficacy on spinal cord injury.Additionally,this study provided the research of Chinese Medicine for central nervous system with new methods.Part ? Effects of Chinese herbal compound "Jisuikang" on phagocytosis of primary microglia in natural hybrid systemObjective:To observe the effects of Chinese herbal compound "Jisuikang" on the phagocytosis of primary microglia cells in natural mixed system.Methods:To prepare drug serum of "Jisuikang",isolate and culture the primary nerve cell,establishing mixed nerve cells system.The neuron cells were induced damage by glutamic acid and the mixed system was disposed by drug serum of "Jisuikang".Mark neurons skeleton and debris by first antibody ?3-tubulin and second antibody Alexa 594.Mark microglia associated membrane protein by first antibody CDllb and second antibody Alexa 488.Then the microglia phagocytosis of neurons fragments was observed in the mixed system after the intervention of "Jisuikang".Results:Low,middle and high dose of "Jisuikang" showed higher phagocytosis index than that of control,and there were no significance between middle and high dose of"Jisuikang" among higher phagocytosis index.Middle and high dose of "Jisuikang"had greater phagocytosis percentage versus that of control.In comparing with LPS group,middle dose of "Jisuikang" was lower in phagocytosis percentage.Conclusion:Traditional Chinese medicine compound "Jisuikang" may improve local microenvironment enhance engulfment of neuron debris by microglia,which promote the repair and regeneration of injured neurons.Part ? Effects of Chinese herbal compound "Jisuikang" on phagocytosis of microglia and regeneration of injured neurons in co-culture systemObjective:To observe the effects of Chinese herbal compound "Jisuikang" on the phagocytosis of microglia and the regeneration of injured neurons in co-culture system.Methods:To prepare drug serum of "Jisuikang" and isolate and identify the primary neuron and microglia.The neuron cells were induced apoptosis by glutamic acid and the microglia cells were predisposed by drug serum of "Jisuikang".Then,the co-culture system of injured neurons and microglia cells was established.24h and 96h after co-culture,engulfment of neuron debris by microglia cells and regeneration of injured neurons were observed by immunofluorescence double labeling method.Results:24h after co-culture,middle and high dose of "Jisuikang" showed greater phagocytic percentage and phagocytic index than that of control.In comparison of LPS,high dose of "Jisuikang" showed no significant difference.96h after co-culture,first grade of neuritis of middle and high dose of "Jisuikang" were more than that of control,and there were no significant difference in comparison of LPS.Neuritis'mean length per cell of middle and high dose of "Jisuikang" were larger than that of control.Neuritis' mean length per cell of high dose of "Jisuikang" showed significant difference in comparison of LPS.Conclusion:Traditional Chinese medicine compound "Jisuikang" may enhance engulfment of neuron debris by microglia to improve local microenvironment,which promote the repair and regeneration of injured neurons.'SUMMARY:1.For the first time,the research is conducted in the aspect of TCM intervention in spinal cord injury from the angle of regulating the microglia phagocytosis.The research makes the new scientific interpretation about why "Jisuikang" can against spinal cord injury.2.We study and establish a variety of methods which can accurately,effectively and dynamically observe the microglial phagocytosis of neuronal debris,verify the regulation effect of Jisuikang on microglial phagocytosis.To a certain extent,we clarify the relationship between the effects of nerve regeneration and repair.3.Considering the theory of holistic intervention of TCM and whole lesion of SCI,we simulate a mixed-culture system with multiple factors and integrity and conduct a preliminary study. |
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