氧糖剥夺诱导的胶质疤痕离体模型的构建
发布时间：2018-11-13 10:45
【摘要】：背景和目的：脑缺血后星形胶质细胞激活成为反应性星形胶质细胞,并进一步形成胶质疤痕,抑制轴突再生,影响神经功能恢复。目前尚无模拟脑缺血引起的离体胶质疤痕模型,本实验目的是建立缺糖缺氧(Oxygen-Glucose Deprivation, OGD)诱导的离体胶质疤痕模型,为更好的研究脑缺血后胶质疤痕的形成机制及为寻找促进神经功能恢复的靶点或相关药物提供便利。 方法：体外培养的大鼠原代星形胶质细胞经OGD处理6 h,分别在再灌后0 h、24 h和48 h检测各类星形胶质激活及疤痕形成的指标,包括利用MTT法和5-溴脱氧尿核苷(5-Bromodeoxyuridine, BrdU)标记新生细胞数量法观察细胞增殖情况,利用细胞免疫化学或免疫印记的方法考察胶质原纤维酸性蛋白(Glial Fibrillary Acidic Protein, GFAP)的表达和硫酸软骨素类蛋白聚糖neurocan、phosphacan的表达。并将经OGD处理的星形胶质细胞与大鼠胚胎皮层神经元共培养24 h,观察对神经元突起生长的影响。 结果：在再灌24h,OGD处理的星形胶质细胞增殖明显,GFAP表达显著上调,BrdU标记的新生细胞数明显增加,胶质疤痕特异性标记蛋白neurocan表达呈显著上调,而phosphacan的表达在再灌注后呈先下降后恢复的动态变化。同时,在共培养模型中,将OGD处理的星形胶质细胞在再灌0h时与神经元共培养,神经元突起长度比对照组明显增长；在再灌24 h时进行共培养,两组间无明显变化；而在再灌48 h后将两者共培养,则发现OGD处理组的神经元突起生长明显受到抑制。 结论：本实验成功构建了一个OGD诱导的离体胶质疤痕模型,该模型将可用于缺血后胶质疤痕的形成机制研究及神经功能保护策略的研究。
[Abstract]:Background & AIM: after cerebral ischemia, astrocytes are activated into reactive astrocytes, and glial scars are formed, axon regeneration is inhibited, and neural function recovery is affected. At present, there is no model of glial scar induced by cerebral ischemia in vitro. The aim of this experiment is to establish an isolated glial scar model induced by glucose deficiency and hypoxia (Oxygen-Glucose Deprivation, OGD). In order to better study the formation mechanism of glial scar after cerebral ischemia and to find the target or related drugs to promote the recovery of neural function. Methods: cultured rat astrocytes were treated with OGD for 6 h. The activation of astrocytes and scar formation were detected at 0 h and 48 h after reperfusion respectively. MTT method and 5-Bromodeoxyuridine (BrdU) labeling method were used to observe cell proliferation and glial fibrillary acidic protein (Glial Fibrillary Acidic Protein,) was detected by immunocytochemistry or immunological imprinting. GFAP and neurocan,phosphacan expression of chondroitin sulfate proteoglycan. The astrocytes treated with OGD were co-cultured with rat embryonic cortical neurons for 24 h to observe the effect on neurite growth. Results: the proliferation of astrocytes, the expression of GFAP, the number of new cells labeled with BrdU and the expression of glial scar specific marker protein (neurocan) were significantly increased after 24 h reperfusion. The expression of phosphacan decreased first and then recovered after reperfusion. At the same time, in co-culture model, the neurite length of astrocytes treated with OGD was significantly longer than that of control group at 0 h after reperfusion, but there was no obvious change between the two groups at 24 h after reperfusion. However, after 48 h of reperfusion, the neurite growth of OGD treated group was obviously inhibited. Conclusion: this experiment successfully constructed an isolated glial scar model induced by OGD, which can be used to study the mechanism of glial scar formation and the neuroprotective strategy after ischemia.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332
本文编号：2328863
[Abstract]:Background & AIM: after cerebral ischemia, astrocytes are activated into reactive astrocytes, and glial scars are formed, axon regeneration is inhibited, and neural function recovery is affected. At present, there is no model of glial scar induced by cerebral ischemia in vitro. The aim of this experiment is to establish an isolated glial scar model induced by glucose deficiency and hypoxia (Oxygen-Glucose Deprivation, OGD). In order to better study the formation mechanism of glial scar after cerebral ischemia and to find the target or related drugs to promote the recovery of neural function. Methods: cultured rat astrocytes were treated with OGD for 6 h. The activation of astrocytes and scar formation were detected at 0 h and 48 h after reperfusion respectively. MTT method and 5-Bromodeoxyuridine (BrdU) labeling method were used to observe cell proliferation and glial fibrillary acidic protein (Glial Fibrillary Acidic Protein,) was detected by immunocytochemistry or immunological imprinting. GFAP and neurocan,phosphacan expression of chondroitin sulfate proteoglycan. The astrocytes treated with OGD were co-cultured with rat embryonic cortical neurons for 24 h to observe the effect on neurite growth. Results: the proliferation of astrocytes, the expression of GFAP, the number of new cells labeled with BrdU and the expression of glial scar specific marker protein (neurocan) were significantly increased after 24 h reperfusion. The expression of phosphacan decreased first and then recovered after reperfusion. At the same time, in co-culture model, the neurite length of astrocytes treated with OGD was significantly longer than that of control group at 0 h after reperfusion, but there was no obvious change between the two groups at 24 h after reperfusion. However, after 48 h of reperfusion, the neurite growth of OGD treated group was obviously inhibited. Conclusion: this experiment successfully constructed an isolated glial scar model induced by OGD, which can be used to study the mechanism of glial scar formation and the neuroprotective strategy after ischemia.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332
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