The Research Of Autophagy In The Proliferation And Differentiation Of Rat Neural Stem Cells

With the rapid development of molecular biology techniques, researchersgradually found that variety of diseases of the nervous system-related genes and cellstudies have yielded more in-depth understanding, which makes the treatment ofneurological disorders more possibilities of choice. In recent years, neural stem cells(NSCs) as a hot research topic, has attracted a growing number of medical attention.Therefore, in-depth study of neural stem cells will bring the hope for a number ofneurodegenerative diseases and disorders of the nervous system damage treatment.There were very important influence in neural stem cell microenvironment in thesurvival of proliferation, differentiation and plasticity. Nerve injury andneurodegenerative diseases can be seen autophagy phenomenon. Some studies foundthat cell autophagy activation will occur when compared with normal andpathological conditions. Thereby it enhanced the degradation of organelles andprotein levels. However, if the organelle degradation rate of the protein synthesis andbiological substances can not be re-utilization reaches equilibrium, the pressure willcause the activation of autophagy. Therefore, to ease the pressure we need to takeautophagy compound treatment, not only to control the degree of activation ofautophagy, but also the attention of lysosomal degradation efficiency, so that the levelof autophagy and degradation of damaged neurons qualitative level basic balance. Itshould also stimulate and accelerate the processes within the mitochondria with othersubstances biosynthesis, degradation and re-use the material level of equilibrium.Such treatment is more effective for neurodegenerative diseases.In order to study autophagy on neural stem cell proliferation and differentiation,rat neural stem cells as a model to study the EGF, bFGF withdrawal conditionsautophagy rat neural stem cell proliferation and differentiation of the relationship. Inthe neural stem cell culture system removal EGF and bFGF, some technology wereused to observe the neural stem cells, neural stem cell proliferation, neurite outgrowth and expression of Beclin1situation by transmission electron microscopy, secondarycloning experiments, immunocytochemistry, Western Blot techniques ultrastructural.Methods1. separating fetal rat cerebral cortex tissue cells, primary suspension culturesgrown neurospheres form aggregates.2. Nestin immunohistochemical staining, NF/GFAP immunofluorescencestaining cells obtained as rat neural stem cells.3. Neuronal differentiation marker gene expression, MAP-2and GFAP weredetected by RT-PCR technology.4. Using the second method of counting passaged clones generated by secondaryneurospheres number of neural stem cells were observed bFGF, EGF removal onneural stem cell proliferation.5. Using transmission electron microscopy removal EGF, bFGF factor of neuralstem cells and normal neural stem cells cultured ultrastructural differences.6. Immunological detection of fluorescence removal EGF, LC3expression ofbFGF in cultured neural stem cells and normal neural stem cells.7. Western Blot to detect the removal of EGF, bFGF factor in cultured neuralstem cells and normal neural stem cells Beclin1expression.8. Morphological software was used to measure the removal of EGF, bFGFneural stem cells and neural stem cells cultured normal growth projections.Results1.14days gestation fetal rat cerebral cortex cells, primary suspension culture,after immunohistochemical staining Nestin, NF/GFAP immunofluorescencestaining, successful rat neural stem cells.2. Removal of EGF, bFGF factor in cultured neural stem cells and normalneural stem cells compared to the formation of secondary neurospheres significantlyreduced the number.3. Removal EGF, bFGF factor cultured neural stem cells and normal neuralstem cells compared to, Bax expression was significantly increased.4. Removal EGF, bFGF factor cultured neural stem cells and normal neuralstem cells compared to, Beclin1expression was significantly increased.5. Transmission electron microscopy showed that, in the absence of growthVI factors in the environment appear visible autophagic vesicles.6. In the EGF, bFGF missing three days later, most of the neural stem cellscontinued to show Nestin positive cells, but compared with the control group,significant growth of neural stem cell processes.7. LC3-II expression was significantly increased in EGF/bFGF withdrawal3days group，but in3-MA group, LC3-II expression was decreased.8. Application autophagy inhibitor3-MA were acting on the EGF/bFGFwithdrawal group and the control group was found in the EGF/bFGF withdrawalgroup inhibits neurite growth, while the control group did not affect the growth ofneurites.ConclusionsThe use of fetal rat cerebral cortex cell suspension cultures can successfully get ratneural stem cells. As a model, EGF, bFGF the absence of growth factors, nervegrowth slow ball, but still have the characteristics of neural stem cells, as comparedwith the control group, significant growth of neural stem cell processes. Furtherresearch found that the growth factor is missing rat neural stem cells autophagy, andin neural stem cell culture system short-term removal of EGF/bFGF inducesautophagy, and autophagy occurs promotes neural stem cell neurite growth. This isfor further study of the pathogenesis of central nervous system diseases andtherapeutic tools provide a theoretical basis.