PTEN调控神经元轴突再生和凋亡的实验研究

发布时间：2018-03-24 08:41

本文选题：PTEN　切入点：神经元　出处：《天津医科大学》2017年硕士论文

【摘要】：【目的】PI3K-AKT信号通路参与细胞的生长代谢、增殖分化及凋亡等多种生物过程。沉默PTEN基因能够有效的促进神经元轴突再生及脊髓损伤小鼠功能恢复,其可能作用于PI3K-AKT信号通路从而发挥作用。本课题通过采用具有高抑制效率的PTEN特异性抑制剂Bpv(pic),特异性干扰PTEN蛋白的表达,同时分别选用PI3K及mTOR特异性抑制剂阻断PI3K-AKT-mTOR通路,然后观察特异性抑制剂对神经元轴突再生以及凋亡的作用。探讨PTEN蛋白被抑制时,PI3K-AKT-mTOR信号通路在神经元轴突生长和凋亡作用中的关键机制。【方法】1.神经元原代培养:本实验首先对出生后0天(P0)的Wistar新生大鼠脑皮层神经元细胞进行原代分离、培养与纯化后进行体外培养和形态学观察,应用细胞免疫荧光染色以鉴定神经元细胞。2.抑制剂效用:将分离获得的P0 Wistar新生大鼠大脑皮层神经元接种于预先包被的10cm培养皿中,以进行神经元原代培养,并将神经元随机分为四组:(1)对照组;(2)LY294002+bpv(pic)组;(3)Ridaforolimus+bpv(pic)组;(4)bpv(pic)组,应用蛋白印迹(western blotting)技术对培养7天后的神经元细胞PI3K-AKT-mTOR通路相关蛋白表达情况进行观察。3.神经元再生:将分离获得的P0 Wistar新生大鼠大脑皮层神经元接种于预先包被的六孔板中,并将神经元随机分为四组:(1)对照组;(2)LY294002+bpv(pic)组;(3)Ridaforolimus+bpv(pic)组;(4)bpv(pic)组,利用细胞免疫荧光染色的方法对神经元轴突及CSPGs进行染色,并利用Image-Pro Plus 6.0软件测量六孔板中接种于CSPGs基质上神经元轴突长度、数量以及接种于CSPGs基质外神经元轴突穿越CSPGs的百分比,探索特异性抑制剂干扰PTEN蛋白表达后神经元轴突的再生及穿越能力,并研究PI3K-AKT-mTOR信号通路在PTEN蛋白表达调控对神经元细胞产生生理效应中的作用机制。4.神经元凋亡:将分离获得的P0 Wistar新生大鼠大脑皮层神经元接种于预先包被的九十六孔板及培养皿中,并将神经元随机分为四组:(1)对照组;(2)LY294002+bpv(pic)组;(3)Ridaforolimus+bpv(pic)组;(4)bpv(pic)组,建立双氧水细胞凋亡模型,通过Western-blot技术检测细胞凋亡相关蛋白的表达,同时进行细胞Tunel化学染色标记凋亡神经元,计算神经元的存活率,探索干扰PTEN对神经元细胞的抗凋亡能力的影响。【结果】1.实验成功分离纯化P0 Wistar新生鼠大脑皮层神经元细胞,通过形态学观察、β-tubulin III免疫学染色鉴定,证实其具有神经元特性。2.利用PI3K-AKT-m TOR信号通路的特异性抑制剂LY294002与Ridaforolimus,证实m TOR作为PI3K-AKT信号通路的关键分子,参与抑制PTEN后细胞通路作用。3.在CSPGs基质上,bpv(pic)组神经元轴突长度明显长于对照组、LY294002+bpv(pic)组及Ridaforolimus+bpv(pic)组神经元轴突长度,差异具有统计学意义(p0.001);同时,bpv(pic)组CSPGs基质外神经元轴突穿越CSPGs能力明显强于其他三组,差异具有统计学意义(p0.001)。4.双氧水细胞凋亡模型基础上,Ridaforolimus+bpv(pic)组及bpv(pic)组神经元细胞存活率明显高于对照组,而LY294002+bpv(pic)组神经元存活率明显降低(p0.05)。【结论】1.本实验证实抑制PTEN具有显著有效减轻CSPGs的轴突抑制作用;PI3K-AKT-mTOR信号通路在抑制PTEN对神经元细胞轴突增生的调控作用中发挥关键性作用。2.抑制PTEN能够通过PI3K/AKT信号通路,有效增强神经元在脊髓损伤条件下存活能力。3.本课题为抑制PTEN促进中枢神经系统的再生修复提供实验依据,并为进一步研究抑制PTEN治疗中枢神经系统损伤提供理论基础。
[Abstract]:[Objective] the growth and metabolism of PI3K-AKT signaling pathway involved in cell proliferation, differentiation and apoptosis of a variety of biological processes. PTEN gene silencing can promote axonal regeneration and functional recovery of spinal cord injury in mice effectively, which may play a role in the PI3K-AKT signaling pathway and play an important role. PTEN specific inhibitor Bpv, this research uses high inhibition efficiency (PIC), expression of specific interference of PTEN protein, and PI3K were selected and mTOR specific inhibitor of PI3K-AKT-mTOR signaling pathway, and then observe the specific inhibitor of neuronal axon regeneration and apoptosis. To study the expression of PTEN was inhibited, the key mechanism of PI3K-AKT-mTOR signal pathway on growth and apoptosis of neuron axons. [Methods] 1. primary culture of neurons: the first 0 days after birth (P0) Wistar in neonatal rat cortical neurons in primary cell Isolated, cultured in vitro and the morphological observation and purification, immunofluorescence staining to identify neurons:.2. inhibitor utility P0 isolated Wistar neurons were inoculated on 10cm pre coated Petri dishes, for primary culture of neurons, and the neurons were randomly divided into the four group: (1) control group; (2) LY294002+bpv (PIC) group; (3) Ridaforolimus+bpv (PIC) group; (4) BPV (PIC) group, using Western blot (Western blotting) were observed in.3. neurons regeneration expression in neurons of PI3K-AKT-mTOR pathway related proteins of cultured for 7 days: the isolated P0 Wistar neurons were inoculated on the pre coated 6-wel plate, and the neurons were randomly divided into four groups: (1) control group; (2) LY294002+bpv (PIC) group; (3) Ridaforolimus+bpv (PIC) group; (4) BPV (PIC) group, Method of staining by immunofluorescence staining of axons and CSPGs, and using Image-Pro Plus 6 software measurement of six well plates were inoculated in the CSPGs matrix on the length of axons, the number and percentage of inoculated in CSPGs matrix axons through CSPGs, to explore the expression of specific inhibitors of PTEN protein interference after regeneration of axon and crossing ability, and PI3K-AKT-mTOR pathway on regulation of apoptosis mechanism of.4. neurons in the physiological effects of neuronal cells in the expression of PTEN protein: ninety-six hole plate will be isolated P0 Wistar of new born neurons in the cerebral cortex of rats inoculated and coated Petri dish, and the neurons were randomly divided into four groups: (1) the control group; (2) LY294002+bpv (PIC) group; (3) Ridaforolimus+bpv (PIC) group; (4) BPV (PIC) group, a model of cell apoptosis by hydrogen peroxide, Western-bl The expression of apoptosis related protein ot detection technology, cell Tunel chemical staining of apoptotic neurons at the same time, to calculate the survival rate of neurons, explore the effect of PTEN interference on the anti apoptosis ability of neuron cells. [results] 1. successful experiments for separation and purification of P0 Wistar in neonatal rat cortex neurons, by morphological observation, -tubulin beta III immunology staining confirmed that it has the characteristics of neurons by.2. specific inhibitor LY294002 and Ridaforolimus PI3K-AKT-m of the TOR signaling pathway, and confirmed that the m TOR as the key molecules in PI3K-AKT signaling pathway, PTEN pathway involved in the inhibition of cell.3. in CSPGs matrix, BPV (PIC) group of axonal length was significantly longer than that of the control group, LY294002+bpv group (PIC) Ridaforolimus+bpv (PIC) group and the length of axons, the difference was statistically significant (p0.001); at the same time, BPV (PIC) CSPGs matrix Outside the axons through CSPGs was stronger than the other three groups, the difference was statistically significant (p0.001) based on cell model of apoptosis of.4. hydrogen peroxide (PIC), Ridaforolimus+bpv group and BPV group (PIC) neuronal cell survival rate was significantly higher than the control group, and LY294002+bpv group (PIC) neuron survival rate was significantly lower (P0.05). [conclusion 1.] this experiment confirmed that inhibition of PTEN is significantly effective in reducing CSPGs axon inhibition; PI3K-AKT-mTOR signaling pathway play a key role in the inhibition of.2. PTEN through PI3K/AKT signaling pathway in the regulation of inhibitory effect of PTEN on neuronal axon hyperplasia, enhance neuronal survival ability of.3. in spinal cord injury under this project to provide the experimental basis for the inhibition PTEN promote the regeneration of the central nervous system, and to further study the inhibition of PTEN for treatment of central nervous system injury and provide theoretical basis.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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