【作者】 严虎；

【导师】 杨毅；

【作者基本信息】 复旦大学 ， 儿科学， 2010， 博士

【摘要】 [目的]星形胶质细胞作为神经发生所必须的小生境中数量最丰富的细胞,可通过细胞间接触和分泌可溶性因子调控神经干细胞的增殖和分化。然而,目前对星形胶质细胞产生哪些因子参与调控以及何种因子在其中起重要作用知之甚少。本课题拟通过运用体外细胞培养与干预、定量蛋白组学技术和生物信息学方法等,筛选在神经干细胞增殖和分化中发挥主要作用的关键分子并加以验证。以期通过对关键分子的干预,探索促进未成熟脑损伤修复的有效途径,为临床早产儿脑损伤的治疗提供新的思路。[方法]首先建立新生1日龄大鼠脑纹状体星形胶质细胞和神经干细胞体外共培养体系,分别采用适度的氧糖剥夺、适宜浓度的布雷福德菌素和氟代柠檬酸抑制星形胶质细胞的分泌功能。通过采用BrdU/Nestin和Tuj1/Nestin免疫荧光双染,观察共培养下的神经干细胞增殖、分化的改变,判断星形胶质细胞的功能状态。在成功建立细胞培养和干预模型的基础上,收集各组星形胶质细胞培养上清,超滤浓缩后采用iTRAQ标记定量蛋白组学方法和生物信息学分析方法,鉴定不同处理组与对照组上清中差异表达的分泌蛋白,并从中筛选出可能发挥主要作用的关键分子。对这些关键分子的作用通过已建立的细胞培养体系进一步加以验证,探讨其对神经干细胞的作用及其机制。[结果]通过星形胶质细胞和神经干细胞非接触状态下共培养,我们证实了星形胶质细胞可以通过分泌可溶性因子调控神经干细胞的增殖和分化。分别经适度的氧糖剥夺、一定浓度的布雷福德菌素和氟代柠檬酸处理后的星形胶质细胞分泌功能被抑制,不再具有促进神经干细胞增殖和分化的能力,体外细胞共培养和干预的实验模型成功建立。采用iTRAQ标记定量蛋白组学技术在星形胶质细胞培养上清中共鉴定出375个蛋白,通过三个相互互补的分泌蛋白预测方法,确认其中有130个分泌蛋白,并且有44个蛋白系首次发现由星形胶质细胞分泌产生的。通过Gene ontology分析,发现在这130个蛋白中,分别有29个蛋白参与调控细胞的分化、增殖和凋亡,18个蛋白参与调控免疫、炎症防御、神经生理过程,29个蛋白属于细胞外基质蛋白。运用Proteinpilot软件计算,显示在氧糖剥夺、布雷福德菌素和氟代柠檬酸干预后分别有27、20、25个蛋白上调和33个、48个、27个蛋白下调；其中三个干预组共同上调的蛋白有4个,共同下调的蛋白有10个在共同变化蛋白中我们选取了4个变化最为显著的(P<0.001)蛋白：Postn(下调)、Psap(下调)、Tptl(上调)和Ppia(上调)进行初步的功能验证。免疫印迹法检测结果显示这4种候选蛋白在星形胶质细胞培养上清中的浓度变化与iTRAQ蛋白组学检测的结果一致。体外细胞培养实验证实Postn蛋白在加入神经干细胞培养体系3天起可显著的促进神经干细胞的增殖(P<0.05)。[结论]1.星形胶质细胞可以通过分泌可溶性因子影响神经干细胞的增殖和分化；2.星形胶质细胞分泌的130种蛋白主要参与调控细胞增殖、分化、凋亡、神经发育、免疫反应、以及细胞外基质构成和营养支持功能；3.Postn蛋白在体外环境可以促进神经干细胞的增殖,可能有助于未成熟脑损伤后的神经修复。更多还原

【Abstract】 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.更多还原