本文選題：共培養(yǎng) + 丙烯酰胺�。� 參考：《江蘇大學》2017年碩士論文

【摘要】：海馬神經元/星形膠質細胞體外共培養(yǎng)體系,已被廣泛用于預防神經退行性疾病的天然活性成分篩選及其機制研究,但用于神經毒性早期篩選模型的報道甚少。鑒于此,本論文利用已知神經毒性的丙烯酰胺(Acrylamide,ACR)為陽性藥物,作用于共培養(yǎng)細胞,建立早期神經毒性體外評價方法。在此基礎上,利用該法對檳榔和虎杖的水煎液進行早期安全性體外神經毒性評價。主要研究內容和結果如下:1.建立海馬神經元和星形膠質細胞體外共培養(yǎng)模型建立海馬神經元和星形膠質細胞直接混合共培養(yǎng)和transwell小室非接觸式共培養(yǎng)兩種細胞模型。結果發(fā)現(xiàn),兩種共培養(yǎng)方式,星形膠質細胞都可以很好地促進神經元的生長,并促進神經元突起數(shù)目的增加,混合共培養(yǎng)第3、5、7 d的多突起神經元的數(shù)目和突起長度均高于transwell小室共培養(yǎng)。表明混合共培養(yǎng)能更好的模擬體內狀態(tài),維持神經元的生長,后續(xù)實驗以直接混合共培養(yǎng)為實驗模型。2.建立體外神經毒性早期評價方法(1)2-10 mmol/L濃度的ACR處理共培養(yǎng)細胞24 h、48 h和72 h,對共培養(yǎng)細胞已產生一般毒性(均為P0.01)。后續(xù)實驗選擇ACR處理共培養(yǎng)細胞的時間為24 h,濃度為3、7.26和10 mmol/L。(2)7.26和10 mmol/L ACR處理細胞后,細胞釋放LDH增加的量分別是對照組的1.19倍和2.53倍,Ca2+濃度升高的量分別是對照組的0.5倍和0.71倍;ACR各組線粒體膜電位下降的量是對照組的0.26-0.56倍(均為P0.01)。表明ACR在該濃度范圍內對共培養(yǎng)細胞產生了一般毒性作用。(3)經7.26和10 mmol/L ACR處理共培養(yǎng)細胞導致多突起神經元的數(shù)目降低、突起變短,突觸可塑性相關蛋白Arc、SYN和β-ΙΙΙ-Tubulin表達均下降;神經遞質NO和谷氨酸的含量均增加,ACh含量下降,均具有統(tǒng)計學意義(均為P0.05)。表明ACR引起突觸可塑性產生變化,影響了神經遞質的釋放,最終產生神經毒性。3.檳榔水煎液和虎杖水煎液早期安全性體外神經毒性評價檳榔和虎杖的飲片分別煎煮后得水煎液,45℃下減壓濃縮,冷凍干燥分別得到檳榔和虎杖水煎液的干燥粉末,進行早期神經毒性評價:(1)檳榔水煎液的神經毒性評價共培養(yǎng)細胞經25-1000μg/m L檳榔水煎液處理6-96 h,其中150μg/mL檳榔水煎液處理細胞96 h時,細胞存活率為(77±4.5)%(P0.01);經25-200μg/m L的檳榔水煎液處理細胞24 h,其中150μg/m L組已使細胞LDH釋放量增加、胞內Ca2+濃度升高、線粒體膜電位水平下降(均為P0.01)。表明該濃度對共培養(yǎng)細胞產生一般毒性作用。經25-200μg/m L的檳榔水煎液作用共培養(yǎng)細胞1 h和3 h,突觸可塑性相關蛋白Arc、SYN和β-ΙΙΙ-Tubulin表達均無顯著變化(均為P0.05);當作用6 h時,50μg/mL組細胞SYN蛋白表達顯著降低,神經遞質NO和谷氨酸的含量升高,ACh含量下降,均具有統(tǒng)計學意義(均為P0.05)。表明50μg/mL檳榔水煎液作用共培養(yǎng)細胞6 h時,已對共培養(yǎng)細胞產生神經毒性,SYN可作為快速檢測檳榔水煎液神經毒性的敏感蛋白。(2)虎杖水煎液的神經毒性評價共培養(yǎng)細胞經25-1000μg/m L虎杖水煎液處理6-96 h,其中300μg/mL組作用細胞48 h時,細胞存活率為(79±6.5)%(P0.01)。經25-400μg/mL虎杖水煎液處理細胞24 h,其中300μg/mL組已使細胞LDH釋放量增加、胞內Ca2+濃度升高、線粒體膜電位水平下降(均為P0.01)。表明該濃度對共培養(yǎng)細胞有一般毒性作用。50-400μg/mL虎杖水煎液處理共培養(yǎng)細胞1 h和3 h,突觸可塑性相關蛋白Arc、SYN和β-ΙΙΙ-Tubulin表達均無顯著變化(均為P0.05);當作用6 h后,400μg/mL組已使Arc蛋白表達顯著降低,神經遞質NO和谷氨酸的含量顯著上升,ACh含量下降(均為P0.01)。表明400μg/mL虎杖水煎液作用細胞6 h時,已對細胞產生神經毒性,Arc可作為快速檢測虎杖水煎液神經毒性的敏感蛋白。
[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.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.1

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