本文選題：PTEN　切入點(diǎn)：神經(jīng)元　出處：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：【目的】PI3K-AKT信號(hào)通路參與細(xì)胞的生長(zhǎng)代謝、增殖分化及凋亡等多種生物過(guò)程。沉默PTEN基因能夠有效的促進(jìn)神經(jīng)元軸突再生及脊髓損傷小鼠功能恢復(fù),其可能作用于PI3K-AKT信號(hào)通路從而發(fā)揮作用。本課題通過(guò)采用具有高抑制效率的PTEN特異性抑制劑Bpv(pic),特異性干擾PTEN蛋白的表達(dá),同時(shí)分別選用PI3K及mTOR特異性抑制劑阻斷PI3K-AKT-mTOR通路,然后觀察特異性抑制劑對(duì)神經(jīng)元軸突再生以及凋亡的作用。探討PTEN蛋白被抑制時(shí),PI3K-AKT-mTOR信號(hào)通路在神經(jīng)元軸突生長(zhǎng)和凋亡作用中的關(guān)鍵機(jī)制。【方法】1.神經(jīng)元原代培養(yǎng):本實(shí)驗(yàn)首先對(duì)出生后0天(P0)的Wistar新生大鼠腦皮層神經(jīng)元細(xì)胞進(jìn)行原代分離、培養(yǎng)與純化后進(jìn)行體外培養(yǎng)和形態(tài)學(xué)觀察,應(yīng)用細(xì)胞免疫熒光染色以鑒定神經(jīng)元細(xì)胞。2.抑制劑效用:將分離獲得的P0 Wistar新生大鼠大腦皮層神經(jīng)元接種于預(yù)先包被的10cm培養(yǎng)皿中,以進(jìn)行神經(jīng)元原代培養(yǎng),并將神經(jīng)元隨機(jī)分為四組:(1)對(duì)照組;(2)LY294002+bpv(pic)組;(3)Ridaforolimus+bpv(pic)組;(4)bpv(pic)組,應(yīng)用蛋白印跡(western blotting)技術(shù)對(duì)培養(yǎng)7天后的神經(jīng)元細(xì)胞PI3K-AKT-mTOR通路相關(guān)蛋白表達(dá)情況進(jìn)行觀察。3.神經(jīng)元再生:將分離獲得的P0 Wistar新生大鼠大腦皮層神經(jīng)元接種于預(yù)先包被的六孔板中,并將神經(jīng)元隨機(jī)分為四組:(1)對(duì)照組;(2)LY294002+bpv(pic)組;(3)Ridaforolimus+bpv(pic)組;(4)bpv(pic)組,利用細(xì)胞免疫熒光染色的方法對(duì)神經(jīng)元軸突及CSPGs進(jìn)行染色,并利用Image-Pro Plus 6.0軟件測(cè)量六孔板中接種于CSPGs基質(zhì)上神經(jīng)元軸突長(zhǎng)度、數(shù)量以及接種于CSPGs基質(zhì)外神經(jīng)元軸突穿越CSPGs的百分比,探索特異性抑制劑干擾PTEN蛋白表達(dá)后神經(jīng)元軸突的再生及穿越能力,并研究PI3K-AKT-mTOR信號(hào)通路在PTEN蛋白表達(dá)調(diào)控對(duì)神經(jīng)元細(xì)胞產(chǎn)生生理效應(yīng)中的作用機(jī)制。4.神經(jīng)元凋亡:將分離獲得的P0 Wistar新生大鼠大腦皮層神經(jīng)元接種于預(yù)先包被的九十六孔板及培養(yǎng)皿中,并將神經(jīng)元隨機(jī)分為四組:(1)對(duì)照組;(2)LY294002+bpv(pic)組;(3)Ridaforolimus+bpv(pic)組;(4)bpv(pic)組,建立雙氧水細(xì)胞凋亡模型,通過(guò)Western-blot技術(shù)檢測(cè)細(xì)胞凋亡相關(guān)蛋白的表達(dá),同時(shí)進(jìn)行細(xì)胞Tunel化學(xué)染色標(biāo)記凋亡神經(jīng)元,計(jì)算神經(jīng)元的存活率,探索干擾PTEN對(duì)神經(jīng)元細(xì)胞的抗凋亡能力的影響。【結(jié)果】1.實(shí)驗(yàn)成功分離純化P0 Wistar新生鼠大腦皮層神經(jīng)元細(xì)胞,通過(guò)形態(tài)學(xué)觀察、β-tubulin III免疫學(xué)染色鑒定,證實(shí)其具有神經(jīng)元特性。2.利用PI3K-AKT-m TOR信號(hào)通路的特異性抑制劑LY294002與Ridaforolimus,證實(shí)m TOR作為PI3K-AKT信號(hào)通路的關(guān)鍵分子,參與抑制PTEN后細(xì)胞通路作用。3.在CSPGs基質(zhì)上,bpv(pic)組神經(jīng)元軸突長(zhǎng)度明顯長(zhǎng)于對(duì)照組、LY294002+bpv(pic)組及Ridaforolimus+bpv(pic)組神經(jīng)元軸突長(zhǎng)度,差異具有統(tǒng)計(jì)學(xué)意義(p0.001);同時(shí),bpv(pic)組CSPGs基質(zhì)外神經(jīng)元軸突穿越CSPGs能力明顯強(qiáng)于其他三組,差異具有統(tǒng)計(jì)學(xué)意義(p0.001)。4.雙氧水細(xì)胞凋亡模型基礎(chǔ)上,Ridaforolimus+bpv(pic)組及bpv(pic)組神經(jīng)元細(xì)胞存活率明顯高于對(duì)照組,而LY294002+bpv(pic)組神經(jīng)元存活率明顯降低(p0.05)�！窘Y(jié)論】1.本實(shí)驗(yàn)證實(shí)抑制PTEN具有顯著有效減輕CSPGs的軸突抑制作用;PI3K-AKT-mTOR信號(hào)通路在抑制PTEN對(duì)神經(jīng)元細(xì)胞軸突增生的調(diào)控作用中發(fā)揮關(guān)鍵性作用。2.抑制PTEN能夠通過(guò)PI3K/AKT信號(hào)通路,有效增強(qiáng)神經(jīng)元在脊髓損傷條件下存活能力。3.本課題為抑制PTEN促進(jìn)中樞神經(jīng)系統(tǒng)的再生修復(fù)提供實(shí)驗(yàn)依據(jù),并為進(jìn)一步研究抑制PTEN治療中樞神經(jīng)系統(tǒng)損傷提供理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R651.2

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本文編號(hào)：1657545