Screening And Preliminary Verify Of The Key Factors From Astrocytes That Regulates Neural Stem Cells In Immature Brain

Objectives:Astrocytes are essential components of neurogenic niches that affect neural stem cell differentiation and proliferation through membrane association and/or the release of soluble factors. However, little is known about which factors produced from astrocytes play critical roles in the regulation of these two progresses. Therefore, by set up an in vitro co-culture system of astrocytes and neural stem cells and using some treatments on astrocytes, we collected the conditional cultured supernatants. Then, quantitative proteomics technology and bioinformatics methods were used to select and verify the critical factors which could regulate the proliferation and differentiation of neural stem cells from the supernatants. To find an effective way for repairing the immature brain damage, this may provide a new therapeutic strategy for premature brain injury.Methods:First, we used Transwell system to co-culture the astrocytes and neural stem cells separated from the brain striatum of one day old Sprague-Dawley rat. Further, we utilize mild oxygen-glucose deprivation, appropriate concentration Brefeldin A and Fluorocitrate to inhibit the secretory function of astrocytes, respectively. In order to identify the function of astrocytes whose secretory capacity had been inhibited by the above method, immunofluorescence double staining for Tujl/Nestin or BrdU/Nestin was performed to assess the differentiation or proliferation of neural stem cells. The supernatants of cultured astrocytes in each interventional group and control group were harvested and concentrated. Then, isobaric tagging for the relative and absolute quantitation (iTRAQ) labeled quantitative proteomic technology and bioinformatics methods were used to identify differentially expressed proteins in supernatants of astrocytes of each group and select the critical regulatory factors which may play an important role among them. Each of candidate key regulatory proteins was added into the neural stem cell culture system and further verified its regulatory ability on neural stem cell, respectively.Results:By using of the Transwell system, astrocytes were co-cultured with neural stem cells and without touching, we verified that astrocytes could release some soluble factors to promote neural stem cells differentiation and proliferation. However, after mild oxygen-glucose deprivation, appropriate concentration Brefeldin A or Fluorocitrate treatment the secretory function of astrocytes was inhibited, and the promotive effect of these astrocytes on neural stem cells proliferation and differentiation was blocked.By using iTRAQ proteomics technology,375 proteins were identified in the supernatants of astrocytes, and 130 proteins were characterized as extracellular by using three complementary secreted protein prediction methods. Among the 130 secreted proteins 44 were firstly reported as expressed by astrocyte. By gene ontology analysis of these 130 proteins, we found that 29 proteins were involved in regulating the differentiation, proliferation and apoptosis of cells,18 proteins were involved in modulating immune, inflammatory response, neurophysiology process, and 29 proteins belong to extracellular matrix protein. Using ProteinPilot software analysis, we found that 27,20,25 proteins were significant up-regulated and 33,48,27 proteins were significant down-regulated after oxygen-glucose deprivation, Brefeldin A, Fluorocitrate treatment, respectively. In the co-changed proteins, we selected the four most significantly changed proteins (P<0.001) for further functional verification. Which are Postn (downregulated), Psap (downregulated), Tptl (upregulated) and Ppia (upregulated). The results of western-blotting detection showed that the contentrations of four candidate proteins in the cultured supernatants of astrocytes were consistent with the results detected by iTRAQ proteomic technology. Cell culture experiments confirmed that the proliferation of neural stem cells increased significantly after 3 days when postn was added into the culture (P<0.05).Conclusions:1. Astrocyte could secret soluble factors to regulate the proliferation and differentiation of neural stem cells; 2. The 130 proteins secreted by astrocyte mostly involved in the regulation of proliferation, differentiation, neuronal development, inflammatory response, extracellular matrix composition and supportive functions.3. Postn could promote the proliferation of neural stem cells in vitro, which maybe helpful for the neural repair after premature brain injury.