酪蛋白酶解肽谱对树突状细胞成熟的影响

发布时间：2018-01-03 00:39

本文关键词：酪蛋白酶解肽谱对树突状细胞成熟的影响　出处：《天津商业大学》2010年硕士论文　论文类型：学位论文

【摘要】： 酪蛋白经水解制得的多肽是重要的生物活性肽,具有免疫调节能力。本研究分别利用胰蛋白酶及胃蛋白酶的酶解酪蛋白,经乙醇、超滤以及Sephadex G-25两次分离纯化后,制备两种不同的酪蛋白水解多肽。一方面以Balb/c小鼠为实验动物,分别将同一浓度的胰蛋白酶水解的酪蛋白多肽通过口服,腹腔注射和静脉注射给药。利用悬浮液体芯片系统,检测小鼠外周血血清中24种细胞因子的浓度变化,研究胰蛋白酶酪蛋白水解多肽对小鼠细胞因子网络的影响并推测产生的先天免疫应答。结果灌胃组中,大部分细胞因子的水平是升高的,抗炎细胞因子IL-5,IL-9,IL-10,MIP-1α是降低的,说明酪蛋白水解多肽促进发炎细胞因子的合成,具有刺激免疫升高的作用。腹腔注射组与此类似。静脉注射的酪蛋白水解多肽对细胞因子水平的影响与灌胃组、腹腔注射组相比明显不同。因为多肽不经过肠粘膜免疫系统直接进入血液,推测通过激活多种免疫细胞内部不同的信号途径影响了细胞因子的基因表达。另一方面诱导培养C57BL/6雄性小鼠骨髓源的树突状细胞(Dendritic cells),用不同浓度的水解多肽刺激DCs,在48h后进行流式细胞仪检测,鉴定DCs的成熟程度,并将成熟后的DCs与T细胞共孵育96h,CCK-8检测T细胞增殖。结果表明:两种酶水解酪蛋白多肽均能够刺激DCs的成熟,但刺激程度低于阳性对照LPS。而且成熟的DCs能够刺激T细胞增殖,说明DCs具有对这些多肽的抗原递呈能力。其上清液中的细胞因子IL-1α、IL-β、IL-2、IL-6、IL-12的分泌提高, TNF-α、IFN-γ浓度降低,促使幼稚T细胞向Th2极化。说明酪蛋白水解多肽对获得性免疫具有信号传递作用。
[Abstract]:The peptides obtained by hydrolysis of casein are important bioactive peptides with immunomodulatory ability. In this study, casein was hydrolyzed by trypsin and pepsin respectively, and ethanol was used to hydrolyze casein. Two different casein hydrolysates were prepared after ultrafiltration and Sephadex G-25 purification. On the one hand, Balb/c mice were used as experimental animals. Casein peptides hydrolyzed with the same concentration of trypsin were administered orally, intraperitoneally and intravenously. To study the effect of trypsin casein hydrolysate polypeptide on cytokine network and to speculate the innate immune response in mice peripheral blood serum 24 cytokines were detected. Results in the gastric perfusion group the effect of trypsin casein hydrolysate polypeptide on the cytokine network of mice was studied. The levels of most cytokines were increased, and the anti-inflammatory cytokines IL-5, IL-9, IL-10 and MIP-1 伪 were decreased, indicating that casein hydrolyzed polypeptides promoted the synthesis of inflammatory cytokines. It has the effect of stimulating immune elevation. The intraperitoneal injection group is similar. The effect of intravenously injected casein hydrolysate polypeptide on cytokine level is similar to that in the gavage group. The intraperitoneal injection group was significantly different because peptides entered the blood without going through the intestinal mucosal immune system. It is speculated that the gene expression of cytokines is affected by activating different signaling pathways within immune cells. On the other hand, dendritic cells derived from bone marrow of C57BL / 6 male mice are induced. Dendritic cells. DCs were stimulated with different concentrations of hydrolyzed polypeptides. Flow cytometry was performed after 48 hours to identify the maturity of DCs. The mature DCs was co-incubated with T cells for 96 h. CCK-8 was used to detect the proliferation of T cells. The results showed that casein peptides hydrolyzed by two enzymes could stimulate the maturation of DCs. Moreover, mature DCs can stimulate T cell proliferation, indicating that DCs has the ability to present antigen to these polypeptides. The cytokine IL-1 伪 in the supernatant of DCs. The secretion of IL-12 was increased, and the concentration of TNF- 伪 and IFN- 纬 was decreased. The results showed that casein hydrolysate polypeptide had signal transduction effect on acquired immunity.
【学位授予单位】：天津商业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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