异丙威影响神经发育主要细胞的迁移分化及突起生长的体外研究
发布时间：2019-05-30 00:30
【摘要】：目的以体外培养的小鼠胚胎神经干细胞(mNSC)、大鼠C6神经胶质瘤细胞和人神经母细胞瘤SH-SY5Y细胞作为神经干细胞、神经胶质细胞和神经元研究模型,研究异丙威对分化中的神经干细胞(NSC)、C6和SH-SY5Y细胞的毒性作用以及细胞迁移、分化和突起生长的影响,初步探讨异丙威的神经发育毒性及可能的细胞机制。 方法以5、25、50、100 mg/L异丙威对诱导分化中的mNSC、C6和SH-SY5Y神经细胞染毒,同时设立培养液空白对照和溶剂对照,通过四噻氮唑盐(MTT)比色法和细胞活性/毒性染色法(LIVE/DEAD)测定染毒24 h和/或48 h后的细胞毒性,并采用免疫荧光细胞化学的方法分析mNSC的迁移与分化,考马斯亮蓝染色法分析C6和SH-SY5Y细胞突起生长状况。 统计学处理:SPSS11.0 for windows软件包建立数据库并进行统计分析结果MTT法测分化中mNSC的细胞毒性:在50 mg/L异丙威作用24h和48h后,分化中mNSC表现出明显的细胞毒性,随着异丙威浓度的升高,细胞存活率降低,且呈剂量依赖性(24 h,rs=-0.820,48h,rs=-0.950;均P0.01);MTT和LIVE/DEAD两种方法测分化中的C6和SH-SY5Y细胞的细胞毒性:结果一致,处理48 h后,50 mg/L异丙威对分化中的SH-SY5Y细胞引起明显的细胞毒性,而在100 mg/L剂量下,分化中的C6细胞存活率的下降才有显著性差异。随着异丙威浓度的升高,这两种细胞的细胞毒性增大,且呈剂量效应关系(C6: MTT,rs=-0.862;LIVE/DEAD,rs=0.818;SH-SY5Y:MTT,rs=-0.796 ;LIVE/DEAD,rs=0.906;均P0.05)。 免疫荧光细胞化学结果显示,25 mg/L异丙威已明显抑制神经干细胞的迁移(P0.01),随着异丙威浓度浓度的升高,mNSC迁移面积和迁移距离均下降,且呈剂量依赖性(Aa/Ab,rs=-0.998;Dm/Db,rs=-0.995;均P0.01);随着异丙威浓度的升高,mNSC分化出的GFAP和Tuj阳性细胞率下降,呈剂量依赖性(rs=-0.900,rs=-0.984,均P0.05),在50 mg/L剂量时GFAP和Tuj阳性细胞率分别下降了8.34%±1.78%和1.97%±0.35%,且存在明显的差异(P0.05),实验还观察到25mg/L剂量下,GFAP阳性细胞突起减少,Tuj阳性细胞突起缩短,至100mg/L,这种现象更加明显。 考马斯亮蓝染色结果显示,50 mg/L异丙威已抑制C6突起延伸细胞率,而25 mg/L剂量就抑制了SH-SY5Y突起延伸细胞率。随着异丙威浓度的升高,这两种细胞的突起延伸细胞率均下降,且呈剂量效应关系(C6,rs=-0.806;SH-SY5Y,rs=-0.975;均P0.01)。 结论在不引起明显细胞毒性的前提条件下,异丙威能抑制mNSC的迁移,同时也能抑制其向星形胶质细胞和神经元分化,且向星形胶质细胞分化的抑制作用尤为明显;异丙威还能抑制胶质细胞(C6)和神经元(SH-SY5Y)的突起生长。异丙威干扰了神经系统发育的关键事件(细胞的迁移、分化及突起生长),这提示异丙威可能具有一定的神经发育毒性。
[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% 卤1.78% and 1.97% 卤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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2488356
[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% 卤1.78% and 1.97% 卤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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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