本文選題：金屬硫蛋白 + 鉛　； 參考：《新疆醫(yī)科大學》2013年碩士論文

【摘要】：目的：探討一種從新疆地產(chǎn)藥食兼用植物鷹嘴豆中提取純化金屬硫蛋白用于鉛防治的方法，并進一步觀察不同濃度外源性植物金屬硫蛋白對鉛染毒淋巴細胞和海馬神經(jīng)元細胞作用不同時間的拮抗效應，為預防和治療鉛中毒提供理論依據(jù)。方法：采用紫外掃描對鷹嘴豆金屬硫蛋白提取液進行定性鑒定，使用鎘-血紅蛋白飽和法以及火焰原子吸收（AAS）法對鷹嘴豆中金屬硫蛋白的含量進行測定，采用凝膠過濾層析技術初步純化鷹嘴豆中的金屬硫蛋白。將純化的鷹嘴豆-金屬硫蛋白配制成不同濃度溶液對染毒鉛的Wistar大鼠淋巴細胞和海馬神經(jīng)元細胞進行體外干預實驗，觀察外源性鷹嘴豆金屬硫蛋白拮抗鉛毒性的效果。實驗中用50μmol/L的醋酸鉛對淋巴細胞和海馬神經(jīng)元細胞進行染毒，將金屬硫蛋白分成三個濃度組：高劑量組100μmol/L、中劑量組1μmol/L、低劑量組0.01μmol/L進行干預，同時設陰性對照與陽性對照組，陰性對照組細胞不做任何處理，陽性對照組細胞只染毒鉛。采用MTT法測定不同作用時間（24h、48h、72h）各組淋巴細胞和海馬神經(jīng)元細胞存活率；通過流式細胞技術觀察細胞凋亡情況；應用酶聯(lián)免疫吸附法檢測細胞外液中乳酸脫氫酶（LDH）活性、金屬硫蛋白及總蛋白含量；并用原子吸收光譜法測定細胞中鉀離子及鉛離子含量。結(jié)果：1）鷹嘴豆中金屬硫蛋白含量為0.16mg/g；鎘-金屬硫蛋白在255nm處有一吸收峰；純化的金屬硫蛋白含量達91.30%。2）與醋酸鉛染毒組相比72h金屬硫蛋白高劑量干預組細胞存活率增高（P0.05）；金屬硫蛋白24h高劑量干預組，，48h中、高劑量干預組與72h各劑量干預組海馬神經(jīng)元細胞存活率較醋酸鉛染毒組增高（P0.05），不同劑量金屬硫蛋白干預24h后，海馬神經(jīng)元細胞凋亡率均有所下降。3）與醋酸鉛染毒組相比，高、中劑量金屬硫蛋白作用72h后，淋巴細胞上清液中LDH活性降低（P0.05），金屬硫蛋白24h中劑量干預組、48h高劑量與中劑量干預組、72h各劑量干預組鉀離子含量降低（P0.05）；與醋酸鉛染毒組相比，海馬神經(jīng)元細胞金屬硫蛋白24h各劑量干預組、48h與72h中、高干預組細胞上清液中LDH活性降低（P0.05），金屬硫蛋白干預24h高、中劑量組，48h與72h各劑量組海馬神經(jīng)元細胞鉀離子含量降低（P0.05）。4）與醋酸鉛染毒組相比，淋巴細胞金屬硫蛋白干預各劑量組不同時間金屬硫蛋白含量均增加（P0.05），金屬硫蛋白24h、48h高、中劑量干預組和72h高劑量干預組淋巴細胞總蛋白含量升高（P0.05）；金屬硫蛋白干預各劑量組不同時間海馬神經(jīng)元細胞金屬硫蛋白含量均增加（P0.05），與醋酸鉛染毒組相比，金屬硫蛋白干預24h各劑量組、48h中、高劑量組與72h中、高劑量組海馬神經(jīng)元細胞總蛋白含量升高（P0.05）。5）與醋酸鉛染毒組相比，金屬硫蛋白干預24h高、中組，48h與72h各劑量組淋巴細胞中鉛離子含量降低（P0.05）；金屬硫蛋白干預24h高劑量組、48h與72h各劑量組海馬神經(jīng)元細胞鉛離子含量降低（P0.05）。結(jié)論：一定劑量范圍內(nèi)金屬硫蛋白可促進淋巴細胞和海馬神經(jīng)元細胞的體外存活，抑制淋巴細胞和海馬神經(jīng)元細胞的凋亡，降低細胞上清中LDH活性和細胞中鉀離子含量，增加兩種細胞中金屬硫蛋白和總蛋白的水平，同時降低細胞中的鉛含量。
[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.

【學位授予單位】：新疆醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R135.11

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