生物活性透明质酸对LPS诱导的人树突状细胞和巨噬细胞炎症应答的作用研究

发布时间：2018-01-05 00:00

本文关键词：生物活性透明质酸对LPS诱导的人树突状细胞和巨噬细胞炎症应答的作用研究　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：机体发生脓毒血症时,Toll样受体4(TLR4)信号通路参与固有免疫和急性炎症的过程。因此,通过TLR4信号通路调节炎症反应,被越来越多的研究者看作是临床上治疗脓毒血症的新方向。小分子量的透明质酸(hyaluronic acid,HA)具有多种生物学功能,可刺激内皮细胞迁移、增殖和新生血管的形成,促进肿瘤细胞的生长和转移,也可使损伤部位炎症细胞聚集、释放各种炎症介质和细胞因子。然而,近年有研究者发现小分子量的透明质酸可发挥抗炎作用。目前,小分子量的HA在炎症效应中的作用尚无一致的定论,且其在炎症反应中的作用机制也并不十分明确。在本实验中,我们应用经透明质酸酶PH20特殊处理的分子量为10~50 k Da的重组透明质酸(既生物活性透明质酸,bioactive hyaluronic acid,B-HA),观察其对LPS刺激的人树突状细胞(Dendritic cell,DC)和巨噬细胞炎症应答的影响,并探究其在炎症应答中的作用机制。研究方法1.人外周血分离培养的成熟树突状细胞和THP-1细胞分化来源的巨噬细胞,经不同浓度的B-HA预先孵育2小时,加终浓度为100ng/ml的LPS刺激4小时,收集细胞,提取细胞总RNA,实时荧光定量PCR方法检测细胞因子TNF-α、IL-1、IL-6、IL-8、IL-10和IFN-βm RNA表达水平。确定B-HA的最适浓度。2.THP-1分化来源的巨噬细胞用最适浓度的B-HA预先保护2小时,LPS刺激不同时间(分别为2小时、4小时、8小时),提取细胞总RNA,实时荧光定量PCR方法检测细胞因子TNF-α、IL-1、IL-6、IL-8、IL-10和IFN-βm RNA表达水平。3.最适浓度的B-HA预先保护THP-1细胞分化来源的巨噬细胞2小时,LPS刺激8小时后,收集细胞培养上清,酶联免疫吸附测定法(ELISA)检测细胞因子蛋白表达水平。4.B-HA预先保护THP-1细胞分化来源的巨噬细胞2小时,LPS刺激不同时间(分别为15 min、30 min、60 min),提取细胞蛋白,western blot方法测定TLR4下游信号通路NF-κB、MAPKs、和IRF-3中蛋白分子的磷酸化水平改变情况。研究结果1.在THP-1细胞分化来源的巨噬细胞实验中,与LPS组相比,20 mg/ml B-HA不仅能抑制炎症因子TNF-α、IL-1、IL-6、IL-8和IFN-β的m RNA和蛋白表达水平,而且可增强抗炎因子IL-10的m RNA和蛋白表达水平。B-HA对TLR4信号通路下游的NF-κB(IKK、IκB、p65)、MAPKs(JNK、ERK、p38)和IRF-3蛋白分子的磷酸化水平均有不同程度的抑制。2.在人外周血分离培养的DCs细胞实验中,不同浓度的B-HA对LPS刺激的促炎因子(TNF-α、IL-6、IL-8)和抗炎因子(IL-10)均无明显影响。结论小分子量的B-HA能通过TLR4信号通路有效地调节人巨噬细胞中促炎因子和抗炎因子的表达,抑制其炎症反应;对人DC细胞的炎症反应无明显影响。本实验提示B-HA对人炎性免疫细胞的炎症反应的作用具有选择性。同时,可为临床上治疗脓毒血症提供一种新的方法。
[Abstract]:Toll-like receptor (TLR4) signaling pathway is involved in the process of innate immunity and acute inflammation during sepsis. Therefore, the inflammatory response is regulated by TLR4 signaling pathway. More and more researchers consider it a new direction in clinical treatment of sepsis. Hyaluronic acido (HA) with low molecular weight has a variety of biological functions. It can stimulate endothelial cell migration, proliferation and angiogenesis, promote the growth and metastasis of tumor cells, but also can make inflammatory cells in the injured site to aggregate, release a variety of inflammatory mediators and cytokines. In recent years, some researchers have found that low molecular weight hyaluronic acid can play an anti-inflammatory effect. At present, there is no consistent conclusion on the role of small molecular weight HA in inflammatory effects. And the mechanism of its role in inflammatory response is not very clear. In this experiment. We used recombinant hyaluronic acid with a molecular weight of 10 ~ 50 kDa (i.e. bioactive hyaluronic acid) specially treated by hyaluronidase PH20. Bioactive hyaluronic acidine B-HAN was used to observe LPS stimulated human dendritic cells (LPS). Effect of DC) and macrophage inflammatory response. Methods 1. Mature dendritic cells isolated from human peripheral blood and macrophages derived from differentiation of THP-1 cells. 2. The cells were preincubated with different concentrations of B-HA for 2 hours and stimulated with 100ng / ml LPS for 4 hours. The cells were collected and the total RNA was extracted. The cytokines, TNF- 伪, IL-1and IL-6, were detected by real-time fluorescence quantitative PCR. Expression of IL-10 and IFN- 尾 m RNA. The optimal concentration of B-HA was determined. 2. Macrophages derived from THP-1 differentiation were pre-protected with the optimal concentration of B-HA for 2 hours. LPS was stimulated at different time (2 hours, 4 hours and 8 hours, respectively). The total RNAs were extracted, and the cytokines TNF- 伪 and IL-1- 6 were detected by real-time fluorescence quantitative PCR. IL-8 IL-10 and IFN- 尾 m RNA expression levels 路3.The optimal concentration of B-HA pre-protected macrophages derived from differentiation of THP-1 cells for 2 hours. After LPS was stimulated for 8 hours, the supernatant of cell culture was collected. Enzyme linked immunosorbent assay (Elisa) was used to detect cytokine protein expression. 4. B-HA pre-protected macrophages derived from differentiation of THP-1 cells for 2 hours. Cell protein was extracted by LPS stimulation at different time (15 min ~ 30 min ~ 60 min ~ 60 min, respectively). The downstream signal pathway NF- 魏 B mapks of TLR4 were measured by western blot method. Changes of phosphorylation level of protein molecules in IRF-3. Results 1. Compared with LPS group, macrophages derived from the differentiation of THP-1 cells. 2. 20 mg/ml B-HA could not only inhibit the expression of m RNA and protein of IL-8 and IFN- 尾 in the inflammatory cytokines TNF- 伪, IL-1, IL-6 and IL-6. It also enhanced the expression level of m RNA and protein of anti-inflammatory factor IL-10. B-HA on NF- 魏 B downstream of TLR4 signaling pathway. The phosphorylation levels of IRF-3 and MAPKs were inhibited in different degrees. 2. In DCs cells isolated from human peripheral blood. Different concentrations of B-HA stimulated LPS stimulated TNF- 伪 IL-6. Conclusion small molecular weight B-HA can effectively regulate the expression of proinflammatory factor and anti-inflammatory factor in human macrophages through TLR4 signaling pathway. Inhibition of its inflammatory response; This study showed that B-HA had a selective effect on the inflammatory response of human inflammatory immune cells. It can provide a new method for the treatment of sepsis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

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