本文選題：α-甘草次酸 + 成體神經(jīng)干細(xì)胞��； 參考：《中國(guó)病理生理雜志》2017年10期

【摘要】：目的:明確18α-甘草次酸(18α-GA)對(duì)核因子E2相關(guān)因子2(Nrf2)的激活作用,觀察上調(diào)Nrf2對(duì)成體神經(jīng)干細(xì)胞(a NSCs)增殖和分化能力的影響,探討維持a NSCs活力的有效途徑。方法:體外培養(yǎng)新生、成年和老年小鼠的側(cè)腦室室膜下區(qū)神經(jīng)干細(xì)胞(NSCs),觀察隨年齡增長(zhǎng)NSCs中Nrf2的表達(dá)變化。以18α-GA作用a NSCs,用real-time PCR和Western blot實(shí)驗(yàn)比較18α-GA組與DMSO對(duì)照組a NSCs的Nrf2表達(dá)變化。構(gòu)建攜帶綠色熒光蛋白(GFP)基因的Nrf2-shRNA慢病毒載體(LV)感染a NSCs,用real-time PCR和Western blot實(shí)驗(yàn)檢測(cè)shRNA對(duì)Nrf2的沉默效率。將a NSCs按照干預(yù)條件不同分為DMSO組、18α-GA組、LV-GFP組和LV-Nrf2-shRNA組,運(yùn)用Brd U摻入實(shí)驗(yàn)、Tuj1免疫熒光染色、CCK-8實(shí)驗(yàn)、Hoechst 33342/PI雙染色和活性氧簇(ROS)水平測(cè)定等方法評(píng)價(jià)18α-GA是否通過激活Nrf2對(duì)a NSCs的增殖、分化、細(xì)胞活力、細(xì)胞凋亡以及氧化應(yīng)激水平產(chǎn)生影響。結(jié)果:隨年齡增長(zhǎng),成年和老年小鼠a NSCs的Nrf2 mRNA表達(dá)水平較新生小鼠NSCs顯著降低(P0.01),但a NSCs的ROS水平顯著高于新生小鼠NSCs(P0.05)。應(yīng)用18α-GA后,a NSCs的Nrf2 mRNA與蛋白表達(dá)水平較DMSO組明顯升高(P0.01),并且a NSCs的Brd U陽性率也顯著增加(P0.01)。2 mg/L 18α-GA作用后,a NSCs的Tuj1陽性率和細(xì)胞活力較DMSO組顯著升高(P0.05),而凋亡率和ROS水平顯著下降(P0.05和P0.01)。但是,敲減a NSCs的Nrf2并應(yīng)用18α-GA干預(yù)后,LV-Nrf2-shRNA組Brd U陽性率、Tuj1陽性率以及細(xì)胞活力均較LV-GFP組顯著下降(P0.05),ROS水平卻顯著上升(P0.05)。結(jié)論:18α-GA可能通過激活Nrf2提高a NSCs的抗氧化能力,促進(jìn)a NSCs增殖和分化,維持a NSCs潛能。
[Abstract]:Aim: to investigate the activation of 18 偽 -glycyrrhetinic acid (18 偽 -GA) on the nuclear factor E2 related factor 2 (Nrf2), to observe the effect of Nrf2 on the proliferation and differentiation of adult neural stem cells (a NSCs), and to explore an effective way to maintain the activity of a NSCs. Methods: neural stem cells (NSCs) in the submembranous region of lateral ventricle of newborn, adult and aged mice were cultured in vitro, and the expression of Nrf2 in NSCs was observed with age. The expression of nrf2 in 18 偽 -GA group was compared with that in DMSO control group by real-time PCR and Western blot. Nrf2-shRNA lentivirus vector (LV) carrying green fluorescent protein (GFP) gene was constructed to infect a NSCs.The silencing efficiency of shRNA to Nrf2 was detected by real-time PCR and Western blot assay. A NSCs were divided into LV-GFP group and LV-Nrf2-shRNA group in DMSO group according to different intervention conditions. The proliferation of a NSCs was evaluated by Brd U incorporation assay (Brd U incorporation) and Hoechst 33342% Pi double staining and reactive oxygen species cluster (Ros) level. Differentiation, cell viability, apoptosis and oxidative stress levels have an effect. Results: the expression of Nrf2 mRNA in adult and aged mice was significantly lower than that in newborn NSCs (P0.01), but the Ros level of a NSCs was significantly higher than that of NSCs in newborn mice (P0.05). The expression of Nrf2 mRNA and protein in NSCs was significantly higher than that in DMSO group (P0.01), and the positive rate of BrdU in a-NSCs was also significantly increased (P0.01). 2 mg / L 18 偽 -GA treatment, the Tuj1 positive rate and cell viability of NSCs were significantly higher than those of DMSO group (P0.05), while the apoptosis rate and apoptosis rate of NSCs were significantly higher than those of DMSO group (P0.05). Ros level decreased significantly (P0.05 and P0.01). However, the positive rate of Brd-U and cell viability in LV-Nrf2-shRNA group were significantly lower than those in LV-GFP group (P0.05) after Nrf2 knockout and 18 偽 -GA intervention (P0.05). Conclusion it is suggested that the activation of Nrf2 may increase the antioxidant capacity of a NSCs, promote the proliferation and differentiation of a NSCs, and maintain the potential of a NSCs.
【作者單位】： 山西醫(yī)科大學(xué)人體解剖學(xué)教研室;山西醫(yī)科大學(xué)生物制藥系;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(No.81571381) 山西省自然科學(xué)基金資助項(xiàng)目(No.2015011132) 山西醫(yī)科大學(xué)校級(jí)博士啟動(dòng)基金(No.03201604)
【分類號(hào)】：R329.2

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