血管生成素2对胎鼠脑皮层神经干细胞诱导分化作用及其分子机制的研究
发布时间：2018-09-03 08:37
【摘要】：【目的】神经干细胞(Neural stem cells,NSCs)具有多向分化能力,可代偿损伤后的神经细胞并重建被破坏的神经系统环路,被广泛认为是可用于治疗中枢神经系统(Central nervous system,CNS)损伤,如脊髓损伤(Spinal cord injury,SCI),最具潜力的种子细胞。然而,如何调控神经干细胞的分化及分化方向,促进其向神经元方向分化,进而最大化修复受损神经组织,仍是当前亟待解决的重大问题。近年的研究发现,曾被认为是仅参与生理性和病理性血管形成的血管生成素2(Angiopoietin 2,Ang2),在中枢神经系统的发育及再生修复的过程中亦发挥着重要的促进性作用。本课题通过分离培养E12.5天的胎鼠脑皮层神经干细胞,研究Ang2对胎鼠脑皮层神经干细胞的诱导分化作用及分化方向;并进一步研究其诱导分化作用的分子机制,应用PI3K-AKT-m TOR信号通路特异性抑制剂,观察Ang2对胎鼠脑皮层神经干细胞诱导分化作用的改变,探讨PI3K-AKT-m TOR信号通路在Ang2诱导神经干细胞分化作用中的关键机制。【方法】本实验首先对C57BL/6J胎鼠(E12.5天)脑皮层神经干细胞进行原代分离、培养与纯化,通过体外培养形态学观察、Nestin免疫细胞化学染色鉴定、以及神经干细胞分化能力的鉴定;改良冻存与复苏神经干细胞的配方与方法,并对经过不同冻存时间的神经干细胞(0,1,6,12月)进行形态学观察及分化能力的鉴定。实验使用经过两代无血清培养纯化后的神经干细胞,将其接种于包被有多聚赖氨酸(Poly-L-lysine,PLL)的培养孔板及培养皿中,使用含有Ang2的分化培养基进行分化培养,利用逆转录聚合酶链式反应(RT-PCR)、免疫细胞化学染色及免疫印迹试验(Western blot)等技术,观察Ang2对胎鼠脑皮层神经干细胞的诱导分化方向及分化效率,并使用Image-Pro Plus 6.0软件及Quantity One软件量化、比较神经干细胞向神经元、星形胶质细胞、少突胶质细胞的分化比例。同时通过免疫细胞化学染色、Western blot、流式细胞术(Flow cytomery)等技术,利用PI3K-AKT-m TOR信号通路的特异性抑制剂LY294002与Rapamycin,观察Ang2对神经干细胞向神经元方向诱导分化作用的改变,研究PI3K-AKT-mTOR信号通路在Ang2诱导神经干细胞分化作用中的机制。【结果】实验成功分离纯化胎鼠(E12.5天)脑皮层神经干细胞,通过形态学观察、Nestin与神经干细胞分化能力的免疫细胞化学染色鉴定,证实其具有神经干细胞特性;改良后的冻存与复苏神经干细胞配方与方法有效,经过不同冻存时间的神经干细胞(0,1,6,12月)形态一致,神经干细胞向神经元、星形胶质细胞、少突胶质细胞的分化能力无统计学差异(p0.05)。通过使用针对神经元、星形胶质细胞、少突胶质细胞的特异性细胞标志物(βⅢ-tubulin,MAP2;GFAP;CNPase)进行细胞免疫荧光染色及Western blot检测,证实经过Ang2诱导处理的神经干细胞向神经元分化的细胞比例及蛋白表达均显著升高,差异具有统计学意义(p0.001;p0.001),而神经干细胞向星形胶质细胞、少突胶质细胞分化的细胞比例与蛋白表达均无统计学意义上的改变(p0.05;p0.05)。利用PI3K-AKT-m TOR信号通路的特异性抑制剂LY294002与Rapamycin,证实m TOR作为PI3K-AKT信号通路的关键分子,介导Ang2对神经干细胞向神经元方向的诱导分化。【结论】本实验证实Ang2具有显著促进E12.5天的胎鼠脑皮层神经干细胞向神经元方向分化的作用,同时不影响其向星形胶质细胞及少突胶质细胞方向的分化;PI3K-AKT-m TOR信号通路在Ang2对神经干细胞分化方向的调控作用中发挥重要作用,m TOR作为PI3K-AKT信号通路的关键分子,介导Ang2对神经干细胞向神经元方向的诱导分化作用。课题证实Ang2调控神经干细胞分化及其分化方向,提高神经干细胞向神经元方向分化效率,探讨Ang2在调控神经干细胞分化方向及分化效率中PI3K/AKT/m TOR信号传导通路的作用及分化关键因子,从而为Ang2在神经系统的发育与再生修复中的促进性作用提供实验依据,并为进一步研究Ang2促进神经干细胞修复中枢神经系统损伤提供研究基础。
[Abstract]:[Objective] Neural stem cells (NSCs) have the ability of multidirectional differentiation, which can compensate damaged nerve cells and reconstruct damaged nervous system circuits. They are widely considered to be the most potential seed fines for the treatment of central nervous system (CNS) injury, such as spinal cord injury (SCI). However, how to regulate the differentiation and differentiation of neural stem cells, promote their differentiation toward neurons, and then maximize the repair of damaged nerve tissue is still a major problem to be solved. In this study, we isolated and cultured E12.5-day fetal rat cortical neural stem cells to study the induction and differentiation direction of Ang2 on fetal rat cortical neural stem cells, and further studied the molecular mechanism of its induction and differentiation. PI3K-AKT-m TOR signaling pathway specific inhibitors were used to observe the effect of Ang2 on the induction and differentiation of neural stem cells in fetal rat cerebral cortex, and to explore the key mechanism of PI3K-AKT-m TOR signaling pathway in the differentiation of neural stem cells induced by Ang2. Primary isolation, culture and purification, morphological observation in vitro, Nestin immunocytochemical staining identification, and identification of neural stem cell differentiation ability; improved cryopreservation and resuscitation of neural stem cells formula and method, and after different cryopreservation of neural stem cells (0, 1, 6, 12 months) morphological observation and differentiation ability Identification. Neural stem cells cultured and purified in serum-free medium for two generations were inoculated into poly-L-lysine (PLL) coated culture plates and dishes. Ang2-containing differentiation medium was used for differentiation and culture. Reverse transcription polymerase chain reaction (RT-PCR), immunocytochemical staining and immunoblotting were used. Western blot and other techniques were used to observe the differentiation direction and efficiency of neural stem cells induced by Ang2 in fetal rat cerebral cortex. Image-Pro Plus 6.0 software and Quantity One software were used to quantify the differentiation ratio of neural stem cells to neurons, astrocytes and oligodendrocytes. By using the specific inhibitors of PI3K-AKT-mTOR signaling pathway, LY294002 and Rapamycin, we observed the effect of Ang2 on the differentiation of neural stem cells toward neurons, and studied the mechanism of PI3K-AKT-mTOR signaling pathway in the differentiation of neural stem cells induced by Ang2. Neural stem cells from the cerebral cortex of fetal mice (E12.5 days) were successfully isolated and purified. Morphological observation and immunocytochemical staining of Nestin and neural stem cells showed that the neural stem cells possessed the characteristics of neural stem cells. There was no significant difference in the ability of neural stem cells to differentiate into neurons, astrocytes and oligodendrocytes (p0.05). The results showed that the percentage and protein expression of neural stem cells differentiated into neurons after Ang2 induction treatment were significantly increased (p0.001; p0.001), while the percentage and protein expression of neural stem cells differentiated into astrocytes and oligodendrocytes were not significantly changed (p0.05). Using specific inhibitors of PI3K-AKT-m TOR signaling pathway, LY294002 and Rapamycin, it was confirmed that m TOR, as a key molecule of PI3K-AKT signaling pathway, mediated the induction and differentiation of neural stem cells into neurons by Ang2. PI3K-AKT-m TOR signaling pathway plays an important role in the regulation of the differentiation direction of neural stem cells. As a key molecule of PI3K-AKT signaling pathway, m TOR mediates Ang2-induced differentiation of neural stem cells toward neurons. This study confirms that Ang2 regulates the differentiation and differentiation direction of neural stem cells, improves the differentiation efficiency of neural stem cells toward neurons, and explores the role of Ang2 in regulating the differentiation direction and differentiation efficiency of neural stem cells PI3K/AKT/m TOR signal transduction pathway and key factors of differentiation, so as to promote the development and regeneration of Ang2 in the nervous system. The results provide experimental basis for further study on the effect of Ang2 on the repair of central nervous system injury.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R741
[Abstract]:[Objective] Neural stem cells (NSCs) have the ability of multidirectional differentiation, which can compensate damaged nerve cells and reconstruct damaged nervous system circuits. They are widely considered to be the most potential seed fines for the treatment of central nervous system (CNS) injury, such as spinal cord injury (SCI). However, how to regulate the differentiation and differentiation of neural stem cells, promote their differentiation toward neurons, and then maximize the repair of damaged nerve tissue is still a major problem to be solved. In this study, we isolated and cultured E12.5-day fetal rat cortical neural stem cells to study the induction and differentiation direction of Ang2 on fetal rat cortical neural stem cells, and further studied the molecular mechanism of its induction and differentiation. PI3K-AKT-m TOR signaling pathway specific inhibitors were used to observe the effect of Ang2 on the induction and differentiation of neural stem cells in fetal rat cerebral cortex, and to explore the key mechanism of PI3K-AKT-m TOR signaling pathway in the differentiation of neural stem cells induced by Ang2. Primary isolation, culture and purification, morphological observation in vitro, Nestin immunocytochemical staining identification, and identification of neural stem cell differentiation ability; improved cryopreservation and resuscitation of neural stem cells formula and method, and after different cryopreservation of neural stem cells (0, 1, 6, 12 months) morphological observation and differentiation ability Identification. Neural stem cells cultured and purified in serum-free medium for two generations were inoculated into poly-L-lysine (PLL) coated culture plates and dishes. Ang2-containing differentiation medium was used for differentiation and culture. Reverse transcription polymerase chain reaction (RT-PCR), immunocytochemical staining and immunoblotting were used. Western blot and other techniques were used to observe the differentiation direction and efficiency of neural stem cells induced by Ang2 in fetal rat cerebral cortex. Image-Pro Plus 6.0 software and Quantity One software were used to quantify the differentiation ratio of neural stem cells to neurons, astrocytes and oligodendrocytes. By using the specific inhibitors of PI3K-AKT-mTOR signaling pathway, LY294002 and Rapamycin, we observed the effect of Ang2 on the differentiation of neural stem cells toward neurons, and studied the mechanism of PI3K-AKT-mTOR signaling pathway in the differentiation of neural stem cells induced by Ang2. Neural stem cells from the cerebral cortex of fetal mice (E12.5 days) were successfully isolated and purified. Morphological observation and immunocytochemical staining of Nestin and neural stem cells showed that the neural stem cells possessed the characteristics of neural stem cells. There was no significant difference in the ability of neural stem cells to differentiate into neurons, astrocytes and oligodendrocytes (p0.05). The results showed that the percentage and protein expression of neural stem cells differentiated into neurons after Ang2 induction treatment were significantly increased (p0.001; p0.001), while the percentage and protein expression of neural stem cells differentiated into astrocytes and oligodendrocytes were not significantly changed (p0.05). Using specific inhibitors of PI3K-AKT-m TOR signaling pathway, LY294002 and Rapamycin, it was confirmed that m TOR, as a key molecule of PI3K-AKT signaling pathway, mediated the induction and differentiation of neural stem cells into neurons by Ang2. PI3K-AKT-m TOR signaling pathway plays an important role in the regulation of the differentiation direction of neural stem cells. As a key molecule of PI3K-AKT signaling pathway, m TOR mediates Ang2-induced differentiation of neural stem cells toward neurons. This study confirms that Ang2 regulates the differentiation and differentiation direction of neural stem cells, improves the differentiation efficiency of neural stem cells toward neurons, and explores the role of Ang2 in regulating the differentiation direction and differentiation efficiency of neural stem cells PI3K/AKT/m TOR signal transduction pathway and key factors of differentiation, so as to promote the development and regeneration of Ang2 in the nervous system. The results provide experimental basis for further study on the effect of Ang2 on the repair of central nervous system injury.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R741
【共引文献】
相关期刊论文 前6条
1 黄成锋;周正芳;郭惠明;范瑞新;郑少忆;陈寄梅;庄建;朱平;;转酪氨酸激酶C基因神经干细胞移植对脊髓损伤后肿瘤坏死因子-α的影响[J];广东医学;2014年11期
2 倪剑书;曹乃龙;谷宝军;;干细胞移植治疗脊髓损伤后排尿障碍的可行性[J];临床泌尿外科杂志;2015年03期
3 韩辉;吴丽敏;储丹晨;方淑珍;杨文明;王晓e，
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