重水标记法测定日粮赖氨酸摄入量对大鼠血液免疫球蛋白G代谢动力学的影响

发布时间：2018-04-30 22:16

本文选题：赖氨酸 + 免疫球蛋白G　；参考：《扬州大学》2017年硕士论文

【摘要】：目的:我国目前尚未制定中国人群膳食必需氨基酸推荐摄入量,有关蛋白质、氨基酸代谢动力学研究的方法也很少。本课题组前期实验得出中国健康青年男性膳食赖氨酸的需要量,为进一步验证此结果是否满足该人群的实际生理需要,本论文通过“赖氨酸摄入量对大鼠血浆IgG代谢动力学的影响”这一方面深入探讨。研究赖氨酸摄入量过低或过高,对体内IgG合成率的影响,与正常赖氨酸摄入量相比的差别,为进一步分析本课题组前期的人体赖氨酸需要量研究结果是否真正满足人体生理需要提供依据。方法:通过用重水腹腔注射大鼠来标记样品,90min后取血并观察血液中IgG是否结合氘。梯度洗脱法确定IgG最佳纯化方案,并SDS-PAGE进行鉴定。酸水解IgG,衍生水解物中丙氨酸,最后GC/MS检测。检测饲料中氨基酸组分,分析大鼠体重和进食量。用在日常饮食状态下,较长时间段内大鼠摄入高剂量(1.6%)、正常剂量(0.9%)、低剂量(0.2%)三种赖氨酸水平的饲料。通过利用2H20作为示踪剂,腹腔注射生理盐水(2H20配制)和日常饮水中添加4%的2H20两种方法,对大鼠体内的蛋白进行同位素氘标记。探究三组不同赖氨酸摄入量对大鼠血液IgG的代谢动力学和IgG的分数合成率的影响,剖析血浆IgG代谢动力学参数对赖氨酸摄入量的敏感程度,为进一步研究对赖氨酸摄入量较敏感的检测参数构建方法体系,从而为确定人体赖氨酸需要量的研究奠基。结果:梯度洗脱法确定IgG纯化最佳方案(缓冲液浓度、pH、流速、时间)并用SDS-PAGE鉴定表明此方案IgG纯化量多、纯度高。GC/MS结果显示,重水可以在大鼠腹腔注射90 min时,短时间内完成对血浆IgG水解物中丙氨酸的标记,即重水标记丙氨酸检测方法建立成功。第三章实验表明:三组的大鼠体重和进食量都有显著差异,从大到小依次:高剂量组、正常剂量组、低剂量组;低剂量组的IgG、TP含量与高剂量组比,存在显著性差异(P0.05),高剂量组IgG和TP含量最高,然后依次为正常剂量组,低剂量组;随着赖氨酸摄入量的增加,血浆IgG的分数合成率呈增加趋势;IgG合成动力学方程:高剂量组ft = 0.076027×(1—e-0.2231t),正常剂量组ft=0 084139×(1—e.0.1483t),低剂量组ft=0.10620×(1-e-0.1051t)。由合成动力学曲线可知,随赖氨酸摄入量增加,大鼠血浆IgG中2H-丙氨酸丰度达到稳态的时间缩短,稳态丰度呈下降趋势。结论:低剂量赖氨酸能显著减少大鼠的体重和进食量,且短期内就能体现出对大鼠体重的影响效果。低剂量赖氨酸组血浆IgG含量和TP含量都显著低于高剂量赖氨酸组。随着赖氨酸摄入量的增加,血浆IgG的分数合成率呈增加趋势,稳态丰度均呈下降趋势。综上,大鼠血浆IgG的分数合成率、合成动力学方程均对赖氨酸摄入量具有较高的敏感性。
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151

【参考文献】