早期神经毒性体外评价方法的建立及槟榔和虎杖的神经毒性评价

发布时间：2018-06-19 06:44

本文选题：共培养 + 丙烯酰胺　；参考：《江苏大学》2017年硕士论文

【摘要】：海马神经元/星形胶质细胞体外共培养体系,已被广泛用于预防神经退行性疾病的天然活性成分筛选及其机制研究,但用于神经毒性早期筛选模型的报道甚少。鉴于此,本论文利用已知神经毒性的丙烯酰胺(Acrylamide,ACR)为阳性药物,作用于共培养细胞,建立早期神经毒性体外评价方法。在此基础上,利用该法对槟榔和虎杖的水煎液进行早期安全性体外神经毒性评价。主要研究内容和结果如下:1.建立海马神经元和星形胶质细胞体外共培养模型建立海马神经元和星形胶质细胞直接混合共培养和transwell小室非接触式共培养两种细胞模型。结果发现,两种共培养方式,星形胶质细胞都可以很好地促进神经元的生长,并促进神经元突起数目的增加,混合共培养第3、5、7 d的多突起神经元的数目和突起长度均高于transwell小室共培养。表明混合共培养能更好的模拟体内状态,维持神经元的生长,后续实验以直接混合共培养为实验模型。2.建立体外神经毒性早期评价方法(1)2-10 mmol/L浓度的ACR处理共培养细胞24 h、48 h和72 h,对共培养细胞已产生一般毒性(均为P0.01)。后续实验选择ACR处理共培养细胞的时间为24 h,浓度为3、7.26和10 mmol/L。(2)7.26和10 mmol/L ACR处理细胞后,细胞释放LDH增加的量分别是对照组的1.19倍和2.53倍,Ca2+浓度升高的量分别是对照组的0.5倍和0.71倍;ACR各组线粒体膜电位下降的量是对照组的0.26-0.56倍(均为P0.01)。表明ACR在该浓度范围内对共培养细胞产生了一般毒性作用。(3)经7.26和10 mmol/L ACR处理共培养细胞导致多突起神经元的数目降低、突起变短,突触可塑性相关蛋白Arc、SYN和β-ΙΙΙ-Tubulin表达均下降;神经递质NO和谷氨酸的含量均增加,ACh含量下降,均具有统计学意义(均为P0.05)。表明ACR引起突触可塑性产生变化,影响了神经递质的释放,最终产生神经毒性。3.槟榔水煎液和虎杖水煎液早期安全性体外神经毒性评价槟榔和虎杖的饮片分别煎煮后得水煎液,45℃下减压浓缩,冷冻干燥分别得到槟榔和虎杖水煎液的干燥粉末,进行早期神经毒性评价:(1)槟榔水煎液的神经毒性评价共培养细胞经25-1000μg/m L槟榔水煎液处理6-96 h,其中150μg/mL槟榔水煎液处理细胞96 h时,细胞存活率为(77±4.5)%(P0.01);经25-200μg/m L的槟榔水煎液处理细胞24 h,其中150μg/m L组已使细胞LDH释放量增加、胞内Ca2+浓度升高、线粒体膜电位水平下降(均为P0.01)。表明该浓度对共培养细胞产生一般毒性作用。经25-200μg/m L的槟榔水煎液作用共培养细胞1 h和3 h,突触可塑性相关蛋白Arc、SYN和β-ΙΙΙ-Tubulin表达均无显著变化(均为P0.05);当作用6 h时,50μg/mL组细胞SYN蛋白表达显著降低,神经递质NO和谷氨酸的含量升高,ACh含量下降,均具有统计学意义(均为P0.05)。表明50μg/mL槟榔水煎液作用共培养细胞6 h时,已对共培养细胞产生神经毒性,SYN可作为快速检测槟榔水煎液神经毒性的敏感蛋白。(2)虎杖水煎液的神经毒性评价共培养细胞经25-1000μg/m L虎杖水煎液处理6-96 h,其中300μg/mL组作用细胞48 h时,细胞存活率为(79±6.5)%(P0.01)。经25-400μg/mL虎杖水煎液处理细胞24 h,其中300μg/mL组已使细胞LDH释放量增加、胞内Ca2+浓度升高、线粒体膜电位水平下降(均为P0.01)。表明该浓度对共培养细胞有一般毒性作用。50-400μg/mL虎杖水煎液处理共培养细胞1 h和3 h,突触可塑性相关蛋白Arc、SYN和β-ΙΙΙ-Tubulin表达均无显著变化(均为P0.05);当作用6 h后,400μg/mL组已使Arc蛋白表达显著降低,神经递质NO和谷氨酸的含量显著上升,ACh含量下降(均为P0.01)。表明400μg/mL虎杖水煎液作用细胞6 h时,已对细胞产生神经毒性,Arc可作为快速检测虎杖水煎液神经毒性的敏感蛋白。
[Abstract]:The in vitro co culture system of hippocampal neurons / astrocytes has been widely used to screen the natural active components of neurodegenerative diseases and their mechanisms. However, there are few reports on early screening models for neurotoxicity. In this paper, the neurotoxic acrylamide (Acrylamide, ACR) is used as a positive drug in this paper. On the basis of this method, the early safety in vitro neurotoxicity of areca and Polygonum cuspidatum was evaluated by using this method. The main contents and results were as follows: 1. the model of hippocampal neurons and astrocytes was established to establish hippocampal neurons and astrocytes. Two kinds of cell models were cultured directly with the co culture of the stromal cells and the non contact co culture of the Transwell compartment. The results showed that the two co culture methods, astrocytes could promote the growth of neurons well, and increase the number of neurites, and the number and the protuberance length of the mixed Co culture of the 3,5,7 D were both. The results showed that the mixed co culture could better simulate the state of the body and maintain the growth of the neurons. After the subsequent experiment, a direct mixed co culture was used as the experimental model.2. to establish an early evaluation method of neurotoxicity in vitro (1) the 2-10 mmol/L concentration of ACR treatment co cultured cells 24 h, 48 h and 72 h, and the co culture cells had produced one. The time for the treatment of co cultured cells by ACR was 24 h, and the concentration of 3,7.26 and 10 mmol/L. (2) 7.26 and 10 mmol/L ACR in the subsequent experiment was 1.19 and 2.53 times as much as that of the control group, and the increase of Ca2+ concentration was 0.5 and 0.71 times as high as that of the control group; the mitochondrial membrane electricity of ACR groups was different from that of the control group. The amount of the decrease was 0.26-0.56 times (P0.01) of the control group. It showed that ACR produced general toxic effects on co culture cells in the concentration range. (3) the number of multiple neurites was reduced, the protuberance was shortened, the synaptic plasticity related protein Arc, SYN and beta -Tubulin expression were all expressed in the co cultured cells treated with 7.26 and 10 mmol/L ACR. Decrease, the content of neurotransmitter NO and glutamic acid increased, and the content of ACh decreased, which were all statistically significant (P0.05). It indicated that ACR caused changes in synaptic plasticity, influenced the release of neurotransmitters, and finally produced neurotoxic.3. areca water decoction and the early safety of the decoction of Polygonum cuspidatum in vitro neurotoxicity evaluation of areca and Polygonum cuspidatum The tablets were decocted after decocting respectively. The dry powder of areca and Polygonum cuspidatum was obtained by decompression and concentration at 45 C, and the early neurotoxicity was evaluated. (1) the neurotoxicity of areca water decoction was evaluated by 25-1000 mu g/m L areca Decoction and 6-96 h, of which 150 mu g/mL areca water decoction was used to treat cells 96 h. The cell survival rate was (77 + 4.5)% (P0.01); the areca water decoction of 25-200 mu g/m was used to treat 24 h cells, of which 150 mu g/m L group had made the cell LDH release increase, the intracellular Ca2+ concentration increased and the mitochondrial membrane potential level decreased (all P0.01). It showed that the concentration had general toxic effect on the co cultured cells. The effect of this concentration on the co cultured cells was performed by 25-200 mu g/m L. The co cultured cells were 1 h and 3 h. There were no significant changes in the expression of synaptic plasticity related protein Arc, SYN and beta -Tubulin. When the effect of 6 h, the expression of SYN protein in the 50 mu g/mL group decreased significantly, the content of NO and glutamic acid in the neurotransmitter increased, and the ACh content decreased, all of which were P0.05). It showed 50 micron areca water. When the decoction was co cultured with 6 h, the co cultured cells had neurotoxicity. SYN could be used as a sensitive protein for rapid detection of neurotoxicity in areca decoction. (2) the neurotoxicity of the decoction of Polygonum cuspidatum was evaluated by 25-1000 mu g/m L Polygonum cuspidatum Decoction for 6-96 h, and the cell survival rate was 79 when the 300 mu g/mL group was 48 h. (+ 6.5)%)% (P0.01). Treatment of cells 24 h through 25-400 mu g/mL Decoction of Polygonum cuspidatum, among which 300 g/mL group had increased the release of LDH cells, increased intracellular Ca2+ concentration, and decreased mitochondrial membrane potential (P0.01). It showed that the concentration of the co cultured cells had general toxicity of.50-400 u g/mL Polygonum cuspidatum Decoction for the treatment of co cultured cells 1 h and 3 h, synapses There was no significant change in the expression of plastic related protein Arc, SYN and beta -Tubulin. When the action of 6 h, the expression of Arc protein was significantly reduced, the content of NO and glutamic acid in neurotransmitters increased significantly, and the content of ACh decreased (all P0.01). It showed that the cells produced nerve to the cells when the 400 mu g /mL Polygonum cuspidatum water decoction was 6. Toxicity, Arc can be used as a sensitive protein for rapid detection of neurotoxicity in water extract of Polygonum cuspidatum.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.1

【参考文献】