發(fā)布時間：2018-09-03 08:37

【摘要】：【目的】神經(jīng)干細(xì)胞(Neural stem cells,NSCs)具有多向分化能力,可代償損傷后的神經(jīng)細(xì)胞并重建被破壞的神經(jīng)系統(tǒng)環(huán)路,被廣泛認(rèn)為是可用于治療中樞神經(jīng)系統(tǒng)(Central nervous system,CNS)損傷,如脊髓損傷(Spinal cord injury,SCI),最具潛力的種子細(xì)胞。然而,如何調(diào)控神經(jīng)干細(xì)胞的分化及分化方向,促進(jìn)其向神經(jīng)元方向分化,進(jìn)而最大化修復(fù)受損神經(jīng)組織,仍是當(dāng)前亟待解決的重大問題。近年的研究發(fā)現(xiàn),曾被認(rèn)為是僅參與生理性和病理性血管形成的血管生成素2(Angiopoietin 2,Ang2),在中樞神經(jīng)系統(tǒng)的發(fā)育及再生修復(fù)的過程中亦發(fā)揮著重要的促進(jìn)性作用。本課題通過分離培養(yǎng)E12.5天的胎鼠腦皮層神經(jīng)干細(xì)胞,研究Ang2對胎鼠腦皮層神經(jīng)干細(xì)胞的誘導(dǎo)分化作用及分化方向;并進(jìn)一步研究其誘導(dǎo)分化作用的分子機(jī)制,應(yīng)用PI3K-AKT-m TOR信號通路特異性抑制劑,觀察Ang2對胎鼠腦皮層神經(jīng)干細(xì)胞誘導(dǎo)分化作用的改變,探討PI3K-AKT-m TOR信號通路在Ang2誘導(dǎo)神經(jīng)干細(xì)胞分化作用中的關(guān)鍵機(jī)制�！痉椒ā勘緦�(shí)驗(yàn)首先對C57BL/6J胎鼠(E12.5天)腦皮層神經(jīng)干細(xì)胞進(jìn)行原代分離、培養(yǎng)與純化,通過體外培養(yǎng)形態(tài)學(xué)觀察、Nestin免疫細(xì)胞化學(xué)染色鑒定、以及神經(jīng)干細(xì)胞分化能力的鑒定;改良凍存與復(fù)蘇神經(jīng)干細(xì)胞的配方與方法,并對經(jīng)過不同凍存時間的神經(jīng)干細(xì)胞(0,1,6,12月)進(jìn)行形態(tài)學(xué)觀察及分化能力的鑒定。實(shí)驗(yàn)使用經(jīng)過兩代無血清培養(yǎng)純化后的神經(jīng)干細(xì)胞,將其接種于包被有多聚賴氨酸(Poly-L-lysine,PLL)的培養(yǎng)孔板及培養(yǎng)皿中,使用含有Ang2的分化培養(yǎng)基進(jìn)行分化培養(yǎng),利用逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)、免疫細(xì)胞化學(xué)染色及免疫印跡試驗(yàn)(Western blot)等技術(shù),觀察Ang2對胎鼠腦皮層神經(jīng)干細(xì)胞的誘導(dǎo)分化方向及分化效率,并使用Image-Pro Plus 6.0軟件及Quantity One軟件量化、比較神經(jīng)干細(xì)胞向神經(jīng)元、星形膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞的分化比例。同時通過免疫細(xì)胞化學(xué)染色、Western blot、流式細(xì)胞術(shù)(Flow cytomery)等技術(shù),利用PI3K-AKT-m TOR信號通路的特異性抑制劑LY294002與Rapamycin,觀察Ang2對神經(jīng)干細(xì)胞向神經(jīng)元方向誘導(dǎo)分化作用的改變,研究PI3K-AKT-mTOR信號通路在Ang2誘導(dǎo)神經(jīng)干細(xì)胞分化作用中的機(jī)制�！窘Y(jié)果】實(shí)驗(yàn)成功分離純化胎鼠(E12.5天)腦皮層神經(jīng)干細(xì)胞,通過形態(tài)學(xué)觀察、Nestin與神經(jīng)干細(xì)胞分化能力的免疫細(xì)胞化學(xué)染色鑒定,證實(shí)其具有神經(jīng)干細(xì)胞特性;改良后的凍存與復(fù)蘇神經(jīng)干細(xì)胞配方與方法有效,經(jīng)過不同凍存時間的神經(jīng)干細(xì)胞(0,1,6,12月)形態(tài)一致,神經(jīng)干細(xì)胞向神經(jīng)元、星形膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞的分化能力無統(tǒng)計(jì)學(xué)差異(p0.05)。通過使用針對神經(jīng)元、星形膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞的特異性細(xì)胞標(biāo)志物(βⅢ-tubulin,MAP2;GFAP;CNPase)進(jìn)行細(xì)胞免疫熒光染色及Western blot檢測,證實(shí)經(jīng)過Ang2誘導(dǎo)處理的神經(jīng)干細(xì)胞向神經(jīng)元分化的細(xì)胞比例及蛋白表達(dá)均顯著升高,差異具有統(tǒng)計(jì)學(xué)意義(p0.001;p0.001),而神經(jīng)干細(xì)胞向星形膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞分化的細(xì)胞比例與蛋白表達(dá)均無統(tǒng)計(jì)學(xué)意義上的改變(p0.05;p0.05)。利用PI3K-AKT-m TOR信號通路的特異性抑制劑LY294002與Rapamycin,證實(shí)m TOR作為PI3K-AKT信號通路的關(guān)鍵分子,介導(dǎo)Ang2對神經(jīng)干細(xì)胞向神經(jīng)元方向的誘導(dǎo)分化�！窘Y(jié)論】本實(shí)驗(yàn)證實(shí)Ang2具有顯著促進(jìn)E12.5天的胎鼠腦皮層神經(jīng)干細(xì)胞向神經(jīng)元方向分化的作用,同時不影響其向星形膠質(zhì)細(xì)胞及少突膠質(zhì)細(xì)胞方向的分化;PI3K-AKT-m TOR信號通路在Ang2對神經(jīng)干細(xì)胞分化方向的調(diào)控作用中發(fā)揮重要作用,m TOR作為PI3K-AKT信號通路的關(guān)鍵分子,介導(dǎo)Ang2對神經(jīng)干細(xì)胞向神經(jīng)元方向的誘導(dǎo)分化作用。課題證實(shí)Ang2調(diào)控神經(jīng)干細(xì)胞分化及其分化方向,提高神經(jīng)干細(xì)胞向神經(jīng)元方向分化效率,探討Ang2在調(diào)控神經(jīng)干細(xì)胞分化方向及分化效率中PI3K/AKT/m TOR信號傳導(dǎo)通路的作用及分化關(guān)鍵因子,從而為Ang2在神經(jīng)系統(tǒng)的發(fā)育與再生修復(fù)中的促進(jìn)性作用提供實(shí)驗(yàn)依據(jù),并為進(jìn)一步研究Ang2促進(jìn)神經(jīng)干細(xì)胞修復(fù)中樞神經(jīng)系統(tǒng)損傷提供研究基礎(chǔ)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R741

【共引文獻(xiàn)】

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