【作者】 韩璐；

【导师】 潘风光；

【作者基本信息】 吉林大学 ， 食品科学， 2016， 硕士

【摘要】 本课题依托于国家“十二五”科技支撑计划项目——“食源性功能肽生物制备技术研究”,项目编号为2012BAD33B03,主要从事蛋清肽消化代谢的研究。本文以蛋清源活性多肽中活性较强的肽段RVPSL为原料,通过体外实验建立模拟胃肠道消化代谢模型和细胞共同培养模型,应用分子生物学相关技术对细胞共同培养模型进行评价,并结合超高效液相色谱分析技术分析细胞共同培养模型的代谢产物,最终获得最佳的评价寡肽代谢的胃肠道消化模型。首先建立体外模拟胃肠液的模型,利用体外模拟胃肠道的方法研究RVPSL蛋清寡肽在人体吸收代谢的过程,通过高效液相色谱仪测得RVPSL蛋清寡肽消化代谢量,为进一步研究寡肽的消化代谢研究奠定基础。试验结果表明,在模拟胃环境中,当蛋清寡肽RVPSL代谢量为初始含量的61.40%±3.02%,且其在胃环境中代谢60~90min时,RVPSL代谢率最高;而在模拟肠环境中,蛋清寡肽RVPSL代谢量为初始含量的30.44%±5.82%,且其在肠环境中代谢60~90min时,RVPSL代谢率最高。为了进一步研究蛋清寡肽RVPSL在小肠中吸收代谢的情况,本研究初步建立了Caco-2/HT29细胞共同培养模型。为了优化共培养细胞的接种比例,本研究检测了细胞单层膜的跨上皮电阻值(TEER),并用FITC-Phalloidin将细胞单层膜染色后,用激光共聚焦显微镜观察细胞成膜状态,同时用阿尔新蓝对共培养细胞中的粘液进行染色,最后提取细胞总RNA,用荧光定量Real-time PCR法对不同比例共培养模型中细胞的代表性基因表达量进行分析。综上所述,我们比较系统地评价了不同接种比例的Caco-2/HT29共培养细胞模型,确定了Caco-2:HT29为9:1为共培养细胞的最优接种比例,为后续蛋清寡肽的吸收代谢试验奠定基础。本研究还利用建立的最优细胞共同培养模型研究蛋清寡肽RVPSL的代谢。MTS试验证明蛋清寡肽RVPSL对Caco-2、HT29细胞无生殖毒性,随后在细胞模型的AP侧(嵌入小室的顶端)加入蛋清寡肽RVPSL,利用超高效液相色谱仪在不同时间点检测RVPSL的分解率与代谢率。结果表明在Caco-2/HT29细胞共同培养模型中,RVPSL在0~30min时AP侧测得的分解率最高,在30~60min时BL侧(嵌入小室的基底侧)测得的代谢率最高。以Caco-2/HT29细胞共同培养模型为依托,研究蛋清寡肽RVPSL对细胞模型中细胞因子IL-6和IL-8分泌的影响。在加入不同浓度的RVPSL后2h测定其IL-6、IL-8细胞因子的浓度,测定结果显示浓度大于3m M时能促进细胞因子IL-6的产生,但RVPSL对IL-8细胞因子的产生无促进作用。在加入不同浓度的RVPSL后12h测定其IL-6、IL-8细胞因子浓度,当RVPSL浓度大于5m M时能促进细胞因子IL-6的产生;而RVPSL对IL-8细胞因子均有明显的抑制作用。更多还原

【Abstract】 The study supported by Project of National Key Technology Research and Development Program for the 12 th Five-year Plan——‘Biological preparation technology research of food-derived bioactive peptides’, the project number is 2012BAD33B03, mainly engaged in digestion and metabolism research of peptides from egg white. This study took the activity peptitde RVPSL which from egg white as raw material, established gastrointestinal digestion and metabolism model and cell co-culture model by in vitro test. Application of molecular biology related technology to evaluate the cell model, and analysed the metabolites of cell co-culture model through ultra high performance liquid chromatography(HPLC). Finally, we got the optimal digestion model of gastrointestinal digestion to evaluate the metabolism of oligopeptide.First of all, this study established the gastrointestinal digestion and metabolism model. By using the method of simulated in vitro gastrointestinal tract, researched the absorption and metabolism of RVPSL which from egg white. We measured the quantity of digestion and metabolism of RVPSL by high performance liquid chromatograph, which can lay the foundation for further study of oligopeptide digestion and metabolism study. The result of simulated gastric environment shows that, when the metabolism of RVPSL reached 61.40% ± 3.02% of the initial content and the metabolic time is 0~30min in the gastric environment, we can get the highest metabolic rate. At the same time, the result of simulated intestinal environment shows that, when the metabolism of RVPSL reached 30.44%±5.82% of the initial content and the metabolic time is 60~90min in the intestinal metabolic, we can get the highest metabolic rate.In order to further study absorption and metabolism of egg white oligopeptides RVPSL in the small intestine, this study established Caco-2/HT29 co-culture cell model preliminarily. With the purpose of optimized the inoculation proportion of co-culture cell model, this study tested the transepithelial electrical resistance, and observed the cellular morphology with laser confocal microscope after stained the cell monolayer with FITC-Phalloidin. And then, we stained the mucous with alcian blue dyeing on the co-culture cell\model. Finally we analysed the representative gene expressions of different inoculation proportion of co-culture cell model with the methods of total RNA extraction, Real-time PCR and so on. Through the above research, we systematic evaluated different inoculation proportion of Caco-2/HT29 co-culture cell model from various aspects, and determined the ratio of 9:1(Caco-2:HT29) was the optimal proportion of co-culture cell model. This result will lay the foundation for subsequent absorption metabolism studies of egg white oligopeptide.We studied the metabolism of RVPSL using the optimized co-culture cell model which established in previous studies. The MTS test showed RVPSL had no reproductive toxicity on Caco-2 and HT29 cells. RVPSL were added on the AP side of the transwell insert, and than we detected the decomposition rate and metabolic rate of RVPSL in different time node using ultra high performance liquid chromatograph. The results showed that, in Caco-2/HT29 co-culture cell model, the decomposition rate of RVPSL was highest at 0~30 min(AP side), and the metabolic rate was highest at 30~60 min(BL side).Basing on the established Caco-2/HT29 co-culture cell model, we studied the influence of RVPSL to cytokine IL-6 and IL-8. After 2h of added different concentrations of RVPSL, we detected the concentration of cytokine IL-6 and IL-8. The results showed that, it can promote the production of IL-6 when the concentrations of RVPSL were higher than 3m M. However various concentrations of RVPSL almost had no effect on IL-8. In the results of detected the concentration of cytokine IL-6 and IL-8 after 12 h of added different concentrations of RVPSL, we had known that, it can promote the production of IL-8 when the concentrations of RVPSL were higher than 5m M. However various concentrations of RVPSL almost had obvious inhibitory effect on the IL-8.更多还原