TLR4受体介导的壳寡糖对巨噬细胞激活机制的研究

发布时间：2018-11-20 13:16

【摘要】：背景：壳寡糖（COS）是壳聚糖的降解产物，由于具有分子量小、没有毒性、容易吸收以及水溶性好等特点，较壳聚糖能够发挥更重要的生物学功能。壳寡糖在提高免疫力、抗肿瘤、抗氧化、抗菌、促进伤口愈合等方面具有重要的应用价值。近年来壳寡糖在提高免疫力、抗肿瘤方面的研究受到很大关注。前期研究表明壳寡糖能够调节机体的免疫功能，抑制肿瘤的生长和转移，但是壳寡糖与巨噬细胞相互作用的机制尚未见详细报道。方法：本项目通过壳聚糖酶水解法水解壳聚糖制备壳寡糖，高效液相色谱法（HPLC）对壳寡糖产物的成分进行鉴定。在体外研究了不同浓度壳寡糖对小鼠腹腔巨噬细胞增殖、细胞吞噬中性红能力以及分泌α-肿瘤坏死因子（TNF-α）能力的影响。对小鼠灌胃不同剂量的壳寡糖，在体内研究了壳寡糖对小鼠血清中免疫球蛋白G（IgG）和免疫球蛋白M（IgM）含量以及小鼠胸腺、脾脏指数的影响。用异硫氰酸酯荧光素（FITC）将壳寡糖进行荧光标记，制备FITC-COS；以巨噬细胞系RAW264.7为对象，研究巨噬细胞对壳寡糖的吞噬作用以及探究TLR4受体在吞噬过程中扮演的关键角色，研究壳寡糖对巨噬细胞分泌NO能力的影响以及TLR4受体沉默对巨噬细胞分泌功能的影响，初步比较壳寡糖混合物、壳五糖和壳六糖功能的异同，并用RT-PCR法检测壳寡糖对RAW264.7细胞TNF-α，iNOS，TLR4等基因转录的影响。结果：HPLC分析结果显示壳聚糖酶水解法制备的壳寡糖主要为3~7单糖聚合度的寡糖。壳寡糖具有较好的免疫调节功能：体外研究表明壳寡糖被巨噬细胞吞噬后可显著增强巨噬细胞的增殖功能，刺激巨噬细胞分泌TNF-α；体内研究结果表明小鼠灌胃壳寡糖能够显著增加小鼠的脾脏指数，增加血清中IgG的含量，对胸腺指数和血清中IgM的含量没有显著影响。将FITC-COS作用于巨噬细胞不同时间后，荧光显微镜观察结果表明巨噬细胞能够吞噬壳寡糖，随着时间的延长细胞吞噬壳寡糖的量不断增加，在15min左右达到吞噬高峰；TLR4单克隆抗体预处理巨噬细胞1h后再加入FITC-COS，巨噬细胞对壳寡糖的吞噬作用几乎完全被抑制。壳寡糖能促进RAW264.7细胞的增殖，提高其吞噬中性红的能力，以及分泌NO的能力，各类寡糖糖样中以壳六糖的效果最好。TLR4单克隆抗体预处理细胞后再加入COS，RAW264.7细胞分泌NO的能力急剧降低。壳寡糖能够诱导巨噬细胞内TNF，iNOS，，TLR4等基因转录，但结果仍需进一步探讨。结论：壳寡糖（3~7聚合度）能够被巨噬细胞吞噬，进而激活巨噬细胞，具有较好的体外、体内免疫调节功能，壳寡糖对巨噬细胞的激活是通过细胞表面TLR4受体参与介导的。
[Abstract]:Background: chitosan oligosaccharide (COS) is the degradation product of chitosan. Because of its small molecular weight, no toxicity, easy absorption and good water solubility, chitosan can play a more important biological function than chitosan. Chitosan oligosaccharide has important application value in improving immunity, anti-tumor, anti-oxidation, anti-bacterial and promoting wound healing. In recent years, the research of chitosan oligosaccharide in improving immunity and anti-tumor has received great attention. Previous studies have shown that chitosan can regulate immune function and inhibit tumor growth and metastasis, but the mechanism of interaction between chitosan oligosaccharide and macrophage has not been reported in detail. Methods: chitosan was hydrolyzed by enzymatic hydrolysis of chitosan. The components of chitosan oligosaccharides were identified by (HPLC). The effects of different concentrations of chitosan oligosaccharides on the proliferation, phagocytosis of neutrophil and the secretion of 伪-tumor necrosis factor (TNF- 伪) of mouse peritoneal macrophages were studied in vitro. The effects of chitosan oligosaccharides on the contents of immunoglobulin G (IgG) and immunoglobulin M (IgM) in serum, thymus and spleen index of mice were studied in vivo. Chitosan oligosaccharides were labeled with fluorescein isothiocyanate (FITC) to prepare FITC-COS;. The phagocytosis of macrophages to chitosan oligosaccharide and the key role of TLR4 receptor in phagocytosis were studied in the macrophage cell line RAW264.7. To study the effect of chitosan oligosaccharide on the secretion of NO by macrophages and the effect of TLR4 receptor silencing on the secretion function of macrophages, and to compare the functions of chitosan oligosaccharide mixture, chitosan pentaccharide and chitosan hexose. The effect of chitosan oligosaccharide on the transcription of TNF- 伪, iNOS,TLR4 and other genes in RAW264.7 cells was detected by RT-PCR assay. Results: the results of HPLC analysis showed that the main chitosan oligosaccharides prepared by enzymatic hydrolysis of chitosan were 3 / 7 monosaccharide oligosaccharides with degree of polymerization. Chitosan oligosaccharide has good immunomodulatory function: in vitro studies showed that chitosan oligosaccharide phagocytosis significantly enhanced the proliferation of macrophages and stimulated macrophages to secrete TNF- 伪; The results in vivo showed that chitosan oligosaccharide could significantly increase the spleen index and the content of IgG in serum, but had no significant effect on the thymus index and the content of IgM in serum. After treated with FITC-COS for different time, the results of fluorescence microscope showed that macrophages could swallow chitosan oligosaccharide, and the amount of chitosan oligosaccharide was increased with time, and reached the phagocytosis peak around 15min. The phagocytosis of oligosaccharide was almost completely inhibited by TLR4 monoclonal antibody pretreating macrophage for 1 h and then adding FITC-COS, macrophage. Chitosan oligosaccharide can promote the proliferation of RAW264.7 cells, improve its ability of phagocytosis of neutral red, as well as the ability to secrete NO. Chitosan has the best effect in all kinds of oligosaccharides. TLR4 monoclonal antibody pretreated the cells and then added COS,. The ability of RAW264.7 cells to secrete NO decreased sharply. Chitosan oligosaccharides can induce transcription of TNF,iNOS,TLR4 and other genes in macrophages, but the results need further study. Conclusion: chitosan can be phagocytized by macrophages and then activate macrophages. It has good immunomodulatory function in vitro. The activation of chitosan oligosaccharides on macrophages is mediated by TLR4 receptor on the surface of macrophages.
【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392.1

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