本文選題：賴氨酸 + 免疫球蛋白G�。� 參考：《揚州大學(xué)》2017年碩士論文

【摘要】：目的:我國目前尚未制定中國人群膳食必需氨基酸推薦攝入量,有關(guān)蛋白質(zhì)、氨基酸代謝動力學(xué)研究的方法也很少。本課題組前期實驗得出中國健康青年男性膳食賴氨酸的需要量,為進(jìn)一步驗證此結(jié)果是否滿足該人群的實際生理需要,本論文通過“賴氨酸攝入量對大鼠血漿IgG代謝動力學(xué)的影響”這一方面深入探討。研究賴氨酸攝入量過低或過高,對體內(nèi)IgG合成率的影響,與正常賴氨酸攝入量相比的差別,為進(jìn)一步分析本課題組前期的人體賴氨酸需要量研究結(jié)果是否真正滿足人體生理需要提供依據(jù)。方法:通過用重水腹腔注射大鼠來標(biāo)記樣品,90min后取血并觀察血液中IgG是否結(jié)合氘。梯度洗脫法確定IgG最佳純化方案,并SDS-PAGE進(jìn)行鑒定。酸水解IgG,衍生水解物中丙氨酸,最后GC/MS檢測。檢測飼料中氨基酸組分,分析大鼠體重和進(jìn)食量。用在日常飲食狀態(tài)下,較長時間段內(nèi)大鼠攝入高劑量(1.6%)、正常劑量(0.9%)、低劑量(0.2%)三種賴氨酸水平的飼料。通過利用2H20作為示蹤劑,腹腔注射生理鹽水(2H20配制)和日常飲水中添加4%的2H20兩種方法,對大鼠體內(nèi)的蛋白進(jìn)行同位素氘標(biāo)記。探究三組不同賴氨酸攝入量對大鼠血液IgG的代謝動力學(xué)和IgG的分?jǐn)?shù)合成率的影響,剖析血漿IgG代謝動力學(xué)參數(shù)對賴氨酸攝入量的敏感程度,為進(jìn)一步研究對賴氨酸攝入量較敏感的檢測參數(shù)構(gòu)建方法體系,從而為確定人體賴氨酸需要量的研究奠基。結(jié)果:梯度洗脫法確定IgG純化最佳方案(緩沖液濃度、pH、流速、時間)并用SDS-PAGE鑒定表明此方案IgG純化量多、純度高。GC/MS結(jié)果顯示,重水可以在大鼠腹腔注射90 min時,短時間內(nèi)完成對血漿IgG水解物中丙氨酸的標(biāo)記,即重水標(biāo)記丙氨酸檢測方法建立成功。第三章實驗表明:三組的大鼠體重和進(jìn)食量都有顯著差異,從大到小依次:高劑量組、正常劑量組、低劑量組;低劑量組的IgG、TP含量與高劑量組比,存在顯著性差異(P0.05),高劑量組IgG和TP含量最高,然后依次為正常劑量組,低劑量組;隨著賴氨酸攝入量的增加,血漿IgG的分?jǐn)?shù)合成率呈增加趨勢;IgG合成動力學(xué)方程:高劑量組ft = 0.076027×(1—e-0.2231t),正常劑量組ft=0 084139×(1—e.0.1483t),低劑量組ft=0.10620×(1-e-0.1051t)。由合成動力學(xué)曲線可知,隨賴氨酸攝入量增加,大鼠血漿IgG中2H-丙氨酸豐度達(dá)到穩(wěn)態(tài)的時間縮短,穩(wěn)態(tài)豐度呈下降趨勢。結(jié)論:低劑量賴氨酸能顯著減少大鼠的體重和進(jìn)食量,且短期內(nèi)就能體現(xiàn)出對大鼠體重的影響效果。低劑量賴氨酸組血漿IgG含量和TP含量都顯著低于高劑量賴氨酸組。隨著賴氨酸攝入量的增加,血漿IgG的分?jǐn)?shù)合成率呈增加趨勢,穩(wěn)態(tài)豐度均呈下降趨勢。綜上,大鼠血漿IgG的分?jǐn)?shù)合成率、合成動力學(xué)方程均對賴氨酸攝入量具有較高的敏感性。
[Abstract]:Objective: at present, the recommended intake of essential amino acids in Chinese diet has not been formulated, and there are few methods to study the metabolism kinetics of protein and amino acids. In order to further verify whether this result can meet the actual physiological needs of healthy young men in China, the previous experiments of our group have obtained the dietary lysine requirements of healthy young men in China. In this paper, the effect of lysine intake on the metabolic kinetics of plasma IgG in rats was studied. To study the effect of low or high lysine intake on IgG synthesis in vivo, and the difference between lysine intake and normal lysine intake. It provides a basis for further analysis on whether the results of the study on lysine requirement in the early stage of our group really meet the physiological needs of the human body. Methods: blood samples were labeled by intraperitoneal injection of heavy water for 90 minutes and blood IgG binding deuterium was observed. Gradient elution method was used to determine the best purification method of IgG and SDS-PAGE was used to identify it. Acid hydrolysis of IgG, derivative hydrolysate of alanine, the final GC/MS detection. The content of amino acids in the diet was determined, and the body weight and food intake of the rats were analyzed. Under the condition of daily diet, the rats were fed with three kinds of lysine levels: high dose (1.6%), normal dose (0.9%) and low dose (0.2%) for a long period of time. By using 2H20 as tracer and intraperitoneal injection of normal saline 2H20) and adding 4% 2H20 to daily drinking water, the protein in rats were labeled with deuterium isotope. To explore the effects of different lysine intake on the metabolic kinetics of blood IgG and the fraction synthesis rate of IgG in three groups, and to analyze the sensitivity of plasma IgG metabolic kinetic parameters to lysine intake. In order to further study the more sensitive lysine intake detection parameters to build a system, so as to determine the human lysine requirements laid a foundation for the study. Results: gradient elution method was used to determine the best method of IgG purification (buffer concentration, pH, flow rate, time). The results of SDS-PAGE identification showed that the IgG purified by gradient elution method had high purity. The results showed that the heavy water could be injected intraperitoneally into rats for 90 min. In a short time, the method for the detection of alanine in plasma IgG hydrolysates was established successfully. Chapter 3 experiment showed that there were significant differences in body weight and food intake among the three groups: high dose group, normal dose group, low dose group, high dose group, and low dose group, and the content of IgGN TP in the low dose group was higher than that in the high dose group. There was significant difference (P 0.05). The contents of IgG and TP in high dose group were the highest, then were normal dose group and low dose group, with the increase of lysine intake. The fractional synthesis rate of IgG in plasma showed an increasing trend: FT = 0.076027 脳 10 ~ (-1) -0.2231 t ~ (-1) for high dose group, 084139 脳 1 -e.0.1483 t ~ (-1) for ft=0 in normal dose group, and ft=0.10620 脳 1 -e -0.1051 t ~ (-1) for low dose group. According to the kinetic curve of synthesis, the time of 2H-alanine abundance in plasma IgG of rats was shortened and the steady-state abundance decreased with the increase of lysine intake. Conclusion: low dose lysine can significantly reduce the weight and food intake of rats, and can reflect the effect on the weight of rats in the short term. The levels of plasma IgG and TP in low dose lysine group were significantly lower than those in high dose lysine group. With the increase of lysine intake, the fraction synthesis rate of plasma IgG increased and the steady-state abundance decreased. In conclusion, the fractional synthesis rate and synthesis kinetics of plasma IgG in rats were highly sensitive to lysine intake.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R151

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