鹰嘴豆金属硫蛋白对铅细胞毒性的干预研究

发布时间：2018-05-13 02:38

本文选题：金属硫蛋白 + 铅　；参考：《新疆医科大学》2013年硕士论文

【摘要】：目的：探讨一种从新疆地产药食兼用植物鹰嘴豆中提取纯化金属硫蛋白用于铅防治的方法，并进一步观察不同浓度外源性植物金属硫蛋白对铅染毒淋巴细胞和海马神经元细胞作用不同时间的拮抗效应，为预防和治疗铅中毒提供理论依据。方法：采用紫外扫描对鹰嘴豆金属硫蛋白提取液进行定性鉴定，使用镉-血红蛋白饱和法以及火焰原子吸收（AAS）法对鹰嘴豆中金属硫蛋白的含量进行测定，采用凝胶过滤层析技术初步纯化鹰嘴豆中的金属硫蛋白。将纯化的鹰嘴豆-金属硫蛋白配制成不同浓度溶液对染毒铅的Wistar大鼠淋巴细胞和海马神经元细胞进行体外干预实验，观察外源性鹰嘴豆金属硫蛋白拮抗铅毒性的效果。实验中用50μmol/L的醋酸铅对淋巴细胞和海马神经元细胞进行染毒，将金属硫蛋白分成三个浓度组：高剂量组100μmol/L、中剂量组1μmol/L、低剂量组0.01μmol/L进行干预，同时设阴性对照与阳性对照组，阴性对照组细胞不做任何处理，阳性对照组细胞只染毒铅。采用MTT法测定不同作用时间（24h、48h、72h）各组淋巴细胞和海马神经元细胞存活率；通过流式细胞技术观察细胞凋亡情况；应用酶联免疫吸附法检测细胞外液中乳酸脱氢酶（LDH）活性、金属硫蛋白及总蛋白含量；并用原子吸收光谱法测定细胞中钾离子及铅离子含量。结果：1）鹰嘴豆中金属硫蛋白含量为0.16mg/g；镉-金属硫蛋白在255nm处有一吸收峰；纯化的金属硫蛋白含量达91.30%。2）与醋酸铅染毒组相比72h金属硫蛋白高剂量干预组细胞存活率增高（P0.05）；金属硫蛋白24h高剂量干预组，，48h中、高剂量干预组与72h各剂量干预组海马神经元细胞存活率较醋酸铅染毒组增高（P0.05），不同剂量金属硫蛋白干预24h后，海马神经元细胞凋亡率均有所下降。3）与醋酸铅染毒组相比，高、中剂量金属硫蛋白作用72h后，淋巴细胞上清液中LDH活性降低（P0.05），金属硫蛋白24h中剂量干预组、48h高剂量与中剂量干预组、72h各剂量干预组钾离子含量降低（P0.05）；与醋酸铅染毒组相比，海马神经元细胞金属硫蛋白24h各剂量干预组、48h与72h中、高干预组细胞上清液中LDH活性降低（P0.05），金属硫蛋白干预24h高、中剂量组，48h与72h各剂量组海马神经元细胞钾离子含量降低（P0.05）。4）与醋酸铅染毒组相比，淋巴细胞金属硫蛋白干预各剂量组不同时间金属硫蛋白含量均增加（P0.05），金属硫蛋白24h、48h高、中剂量干预组和72h高剂量干预组淋巴细胞总蛋白含量升高（P0.05）；金属硫蛋白干预各剂量组不同时间海马神经元细胞金属硫蛋白含量均增加（P0.05），与醋酸铅染毒组相比，金属硫蛋白干预24h各剂量组、48h中、高剂量组与72h中、高剂量组海马神经元细胞总蛋白含量升高（P0.05）。5）与醋酸铅染毒组相比，金属硫蛋白干预24h高、中组，48h与72h各剂量组淋巴细胞中铅离子含量降低（P0.05）；金属硫蛋白干预24h高剂量组、48h与72h各剂量组海马神经元细胞铅离子含量降低（P0.05）。结论：一定剂量范围内金属硫蛋白可促进淋巴细胞和海马神经元细胞的体外存活，抑制淋巴细胞和海马神经元细胞的凋亡，降低细胞上清中LDH活性和细胞中钾离子含量，增加两种细胞中金属硫蛋白和总蛋白的水平，同时降低细胞中的铅含量。
[Abstract]:Objective: To explore a method to extract and purify metallothionein from the Xinjiang real estate drug and food concurrently plant chickpea, and to further observe the antagonistic effects of different concentrations of exogenous metallothionein on the cells of lead infected lymphocytes and hippocampal neurons in different time, and provide a theory for the prevention and treatment of lead poisoning. Methods: UV scanning was used to identify the extract of metallothionein in chickpea. The content of metallothionein in chickpea was determined by the method of cadmium hemoglobin saturation and flame atomic absorption (AAS). The metallothionein in chickpea was purified by gel filtration chromatography. The purified chickpeas were purified by the gel filtration chromatography. Metallothionein was prepared into different concentration solutions to interfere with the lymphocyte and hippocampal neurons of Wistar rats infected with lead in vitro. The effect of exogenous olecranon metallothionein on the toxicity of lead was observed. In the experiment, the lead acetate was used to dye the lymphocyte and hippocampal neurons by 50 mol/L lead acetate, and the metallothionein was used. Three concentration groups were divided into three groups: high dose group 100 mu, medium dose group 1 mu mol/L, low dose group 0.01 micron mol/L, negative control and positive control group, negative control group cells do not do any treatment, positive control group cells only dye lead. MTT method was used to determine the different use time (24h, 48h, 72h) each group of lymphocytes and hippocampus God Cell apoptosis was observed by flow cytometry; enzyme linked immunosorbent assay (ELISA) was used to detect the activity of lactate dehydrogenase (LDH), metallothionein and total protein content, and the content of potassium and lead in cells was determined by atomic absorption spectrometry. Results: 1) the content of metallothionein in chickpea was contained. The amount was 0.16mg/g; the cadmium metallothionein had an absorption peak at 255nm; the purified metallothionein content reached 91.30%.2) and the cell survival rate of the 72h metallothionein high dose intervention group was higher than that of the lead acetate exposure group (P0.05); the high dose group of metallothionein 24h, 48h, the high dose intervention group and the hippocampus nerve of each dose of 72h in the intervention group of 72h. The cell survival rate of the cells was higher than that of the lead acetate (P0.05), and the apoptosis rate of hippocampal neurons decreased by.3 after the intervention of 24h with different doses of metallothionein. Compared with the lead acetate exposure group, the LDH activity in the lymphocyte supernatant was lower (P0.05), the dose intervention group in the metallothionein 24h, 48h, 48h, 48h, 48h, 48h, 48h, 48h, 48H In the high dose and middle dose intervention group, the potassium ion content in the 72h intervention group decreased (P0.05), compared with the lead acetate exposure group, the LDH activity in the high dry pregroup cell supernatant was reduced (P0.05) in each dose of metallothionein 24h dose intervention group, 48h and 72h, and the metallothionein interfered 24h high, middle dose group, and 72h dose group. Compared with lead acetate (P0.05).4), the content of metallothionein in each dose group increased (P0.05), metallothionein 24h, 48h high, middle dose intervention group and 72h high dose dry pregroup lymphocyte total protein content increased (P0.05) and metal sulphur eggs. The content of metallothionein in hippocampal neurons in different doses of white dry predose group increased (P0.05). Compared with lead acetate exposure group, metallothionein interfered 24h dose group, 48h, high dose group and 72h, the total protein content of hippocampus neuron increased (P0.05.5) in high dose group (P0.05).5), compared with lead acetate exposure group, metallothionein intervention 24h high, middle group, 48h and 72h lymphocyte lead content decreased (P0.05); metallothionin intervention 24h high dose group, 48h and 72h each dose group of hippocampal neurons lead ion content decreased (P0.05). Conclusion: metallothionin can promote the survival of lymphocyte and hippocampal neurons in a certain dose range, inhibit the survival of cells in vitro. The apoptosis of lymphocyte and hippocampal neurons reduces the LDH activity and the content of potassium ion in the cell supernatant, increases the level of metallothionein and total protein in the two cells, and reduces the lead content in the cells.

【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R135.11

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