【摘要】：目的以體外培養(yǎng)的小鼠胚胎神經(jīng)干細(xì)胞(mNSC)、大鼠C6神經(jīng)膠質(zhì)瘤細(xì)胞和人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞作為神經(jīng)干細(xì)胞、神經(jīng)膠質(zhì)細(xì)胞和神經(jīng)元研究模型,研究異丙威對(duì)分化中的神經(jīng)干細(xì)胞(NSC)、C6和SH-SY5Y細(xì)胞的毒性作用以及細(xì)胞遷移、分化和突起生長(zhǎng)的影響,初步探討異丙威的神經(jīng)發(fā)育毒性及可能的細(xì)胞機(jī)制。 方法以5、25、50、100 mg/L異丙威對(duì)誘導(dǎo)分化中的mNSC、C6和SH-SY5Y神經(jīng)細(xì)胞染毒,同時(shí)設(shè)立培養(yǎng)液空白對(duì)照和溶劑對(duì)照,通過(guò)四噻氮唑鹽(MTT)比色法和細(xì)胞活性/毒性染色法(LIVE/DEAD)測(cè)定染毒24 h和/或48 h后的細(xì)胞毒性,并采用免疫熒光細(xì)胞化學(xué)的方法分析mNSC的遷移與分化,考馬斯亮藍(lán)染色法分析C6和SH-SY5Y細(xì)胞突起生長(zhǎng)狀況。 統(tǒng)計(jì)學(xué)處理:SPSS11.0 for windows軟件包建立數(shù)據(jù)庫(kù)并進(jìn)行統(tǒng)計(jì)分析結(jié)果MTT法測(cè)分化中mNSC的細(xì)胞毒性:在50 mg/L異丙威作用24h和48h后,分化中mNSC表現(xiàn)出明顯的細(xì)胞毒性,隨著異丙威濃度的升高,細(xì)胞存活率降低,且呈劑量依賴(lài)性(24 h,rs=-0.820,48h,rs=-0.950;均P0.01);MTT和LIVE/DEAD兩種方法測(cè)分化中的C6和SH-SY5Y細(xì)胞的細(xì)胞毒性:結(jié)果一致,處理48 h后,50 mg/L異丙威對(duì)分化中的SH-SY5Y細(xì)胞引起明顯的細(xì)胞毒性,而在100 mg/L劑量下,分化中的C6細(xì)胞存活率的下降才有顯著性差異。隨著異丙威濃度的升高,這兩種細(xì)胞的細(xì)胞毒性增大,且呈劑量效應(yīng)關(guān)系(C6: MTT,rs=-0.862;LIVE/DEAD,rs=0.818;SH-SY5Y:MTT,rs=-0.796 ;LIVE/DEAD,rs=0.906;均P0.05)。 免疫熒光細(xì)胞化學(xué)結(jié)果顯示,25 mg/L異丙威已明顯抑制神經(jīng)干細(xì)胞的遷移(P0.01),隨著異丙威濃度濃度的升高,mNSC遷移面積和遷移距離均下降,且呈劑量依賴(lài)性(Aa/Ab,rs=-0.998;Dm/Db,rs=-0.995;均P0.01);隨著異丙威濃度的升高,mNSC分化出的GFAP和Tuj陽(yáng)性細(xì)胞率下降,呈劑量依賴(lài)性(rs=-0.900,rs=-0.984,均P0.05),在50 mg/L劑量時(shí)GFAP和Tuj陽(yáng)性細(xì)胞率分別下降了8.34%±1.78%和1.97%±0.35%,且存在明顯的差異(P0.05),實(shí)驗(yàn)還觀察到25mg/L劑量下,GFAP陽(yáng)性細(xì)胞突起減少,Tuj陽(yáng)性細(xì)胞突起縮短,至100mg/L,這種現(xiàn)象更加明顯。 考馬斯亮藍(lán)染色結(jié)果顯示,50 mg/L異丙威已抑制C6突起延伸細(xì)胞率,而25 mg/L劑量就抑制了SH-SY5Y突起延伸細(xì)胞率。隨著異丙威濃度的升高,這兩種細(xì)胞的突起延伸細(xì)胞率均下降,且呈劑量效應(yīng)關(guān)系(C6,rs=-0.806;SH-SY5Y,rs=-0.975;均P0.01)。 結(jié)論在不引起明顯細(xì)胞毒性的前提條件下,異丙威能抑制mNSC的遷移,同時(shí)也能抑制其向星形膠質(zhì)細(xì)胞和神經(jīng)元分化,且向星形膠質(zhì)細(xì)胞分化的抑制作用尤為明顯;異丙威還能抑制膠質(zhì)細(xì)胞(C6)和神經(jīng)元(SH-SY5Y)的突起生長(zhǎng)。異丙威干擾了神經(jīng)系統(tǒng)發(fā)育的關(guān)鍵事件(細(xì)胞的遷移、分化及突起生長(zhǎng)),這提示異丙威可能具有一定的神經(jīng)發(fā)育毒性。
[Abstract]:Objective to study the neural stem cells, glial cells and neurons of mouse embryonic neural stem cells (mNSC), rat C6 glioma cells and human neuroblastoma SH-SY5Y cells cultured in vitro. To study the toxic effect of propofol on the differentiation of neural stem cells (NSC), C 6 and SH-SY5Y cells and the effects of cell migration, differentiation and protruding growth, and to explore the neurodevelopmental toxicity and possible cellular mechanism of propofol. Methods mNSC,C6 and SH-SY5Y nerve cells were exposed to 5, 25 and 50 mg/L isoproxide, and blank culture medium control and solvent control were set up at the same time. The cytotoxicity of mNSC was determined by (MTT) colorimetric assay and cytotoxicity / cytotoxicity staining (LIVE/DEAD) after 24 h and / or 48 h exposure, and the migration and differentiation of mNSC were analyzed by immunofluorescence cytochemistry. Coomassie brilliant blue staining was used to analyze the protrusive growth of C6 and SH-SY5Y cells. Statistical analysis: SPSS11.0 for windows software package established a database and statistical analysis showed that the cytotoxicity of mNSC in differentiation was measured by MTT method. After treated with 50 mg/L isoproxide for 24 h and 48 h, mNSC showed obvious cytotoxicity in differentiation. With the increase of isopropyl concentration, the cell survival rate decreased in a dose-dependent manner (24 h, rs0.820, 48h, rs0.950), and the survival rate of cells decreased in a dose-dependent manner (24 h, rs0.820, 48h, rs0.950). All of them were P 0.01). The cytotoxicity of C6 and SH-SY5Y cells in differentiation was measured by MTT and LIVE/DEAD. The results were consistent. After 48 hours of treatment, 50 mg/L isoproxide induced obvious cytotoxicity to differentiated SH-SY5Y cells, but at the dose of 100 mg/L, The survival rate of C6 cells in differentiation decreased significantly. With the increase of propofol concentration, the cytotoxicity of the two cells increased in a dose-dependent manner (C 6: MTT,rs=-0.862;LIVE/DEAD,rs=0.818;SH-SY5Y:MTT,rs=-0.796; LIVE/DEAD,rs=0.906; P 0.05). The results of immunofluorescence cytochemistry showed that 25 mg/L propofol significantly inhibited the migration of neural stem cells (P01). With the increase of propofol concentration, the migration area and migration distance of mNSC decreased. In a dose-dependent manner (Aa/Ab,rs=-0.998;) Dm/Db,rs=-0.995; were all P01). With the increase of propofol concentration, the rate of GFAP and Tuj positive cells differentiated from mNSC decreased in a dose-dependent manner (rs=-0.900,rs=-0.984, P 0.05). At the dose of 50 mg/L, the positive cell rates of GFAP and Tuj decreased by 8.34% 鹵1.78% and 1.97% 鹵0.35%, respectively, and there was significant difference (P 0.05). It was also observed that the protrusions of GFAP positive cells decreased at the dose of 25mg/L. The protrusions of Tuj positive cells were shortened to 100 mg / L, which was more obvious. The results of Coomassie brilliant blue staining showed that 50 mg/L isopropyl could inhibit the cell rate of C 6 process extension, while the dose of 25 mg/L could inhibit the rate of SH-SY5Y process extension cell. With the increase of isopropyl concentration, the rate of protruding extension cells in both cells decreased in a dose-dependent manner (C6, rs0.806 SHSY5Y, rs0.975; both P01). The percentage of protruding cells in these two kinds of cells decreased in a dose-dependent manner (C6, rs0.806 SHSY5Y, rs0.975; both P 0.01). Conclusion propofol can inhibit the migration of mNSC and inhibit the differentiation of mNSC into astrocytes and neurons without causing obvious cytotoxicity, and the inhibitory effect of isopropyl on the differentiation into astrocytes and neurons is especially obvious. Isopropyl can also inhibit the growth of glial cells (C 6) and neurons (SH-SY5Y). Propofol interferes with the key events of nervous system development (cell migration, differentiation and protruding growth), which suggests that isopropyl may have certain neurodevelopmental toxicity.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

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