本文關(guān)鍵詞：生物活性透明質(zhì)酸對(duì)LPS誘導(dǎo)的人樹突狀細(xì)胞和巨噬細(xì)胞炎癥應(yīng)答的作用研究　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 生物活性透明質(zhì)酸 LPS 炎癥反應(yīng) 樹突狀細(xì)胞 巨噬細(xì)胞

【摘要】：機(jī)體發(fā)生膿毒血癥時(shí),Toll樣受體4(TLR4)信號(hào)通路參與固有免疫和急性炎癥的過程。因此,通過TLR4信號(hào)通路調(diào)節(jié)炎癥反應(yīng),被越來越多的研究者看作是臨床上治療膿毒血癥的新方向。小分子量的透明質(zhì)酸(hyaluronic acid,HA)具有多種生物學(xué)功能,可刺激內(nèi)皮細(xì)胞遷移、增殖和新生血管的形成,促進(jìn)腫瘤細(xì)胞的生長(zhǎng)和轉(zhuǎn)移,也可使損傷部位炎癥細(xì)胞聚集、釋放各種炎癥介質(zhì)和細(xì)胞因子。然而,近年有研究者發(fā)現(xiàn)小分子量的透明質(zhì)酸可發(fā)揮抗炎作用。目前,小分子量的HA在炎癥效應(yīng)中的作用尚無一致的定論,且其在炎癥反應(yīng)中的作用機(jī)制也并不十分明確。在本實(shí)驗(yàn)中,我們應(yīng)用經(jīng)透明質(zhì)酸酶PH20特殊處理的分子量為10~50 k Da的重組透明質(zhì)酸(既生物活性透明質(zhì)酸,bioactive hyaluronic acid,B-HA),觀察其對(duì)LPS刺激的人樹突狀細(xì)胞(Dendritic cell,DC)和巨噬細(xì)胞炎癥應(yīng)答的影響,并探究其在炎癥應(yīng)答中的作用機(jī)制。研究方法1.人外周血分離培養(yǎng)的成熟樹突狀細(xì)胞和THP-1細(xì)胞分化來源的巨噬細(xì)胞,經(jīng)不同濃度的B-HA預(yù)先孵育2小時(shí),加終濃度為100ng/ml的LPS刺激4小時(shí),收集細(xì)胞,提取細(xì)胞總RNA,實(shí)時(shí)熒光定量PCR方法檢測(cè)細(xì)胞因子TNF-α、IL-1、IL-6、IL-8、IL-10和IFN-βm RNA表達(dá)水平。確定B-HA的最適濃度。2.THP-1分化來源的巨噬細(xì)胞用最適濃度的B-HA預(yù)先保護(hù)2小時(shí),LPS刺激不同時(shí)間(分別為2小時(shí)、4小時(shí)、8小時(shí)),提取細(xì)胞總RNA,實(shí)時(shí)熒光定量PCR方法檢測(cè)細(xì)胞因子TNF-α、IL-1、IL-6、IL-8、IL-10和IFN-βm RNA表達(dá)水平。3.最適濃度的B-HA預(yù)先保護(hù)THP-1細(xì)胞分化來源的巨噬細(xì)胞2小時(shí),LPS刺激8小時(shí)后,收集細(xì)胞培養(yǎng)上清,酶聯(lián)免疫吸附測(cè)定法(ELISA)檢測(cè)細(xì)胞因子蛋白表達(dá)水平。4.B-HA預(yù)先保護(hù)THP-1細(xì)胞分化來源的巨噬細(xì)胞2小時(shí),LPS刺激不同時(shí)間(分別為15 min、30 min、60 min),提取細(xì)胞蛋白,western blot方法測(cè)定TLR4下游信號(hào)通路NF-κB、MAPKs、和IRF-3中蛋白分子的磷酸化水平改變情況。研究結(jié)果1.在THP-1細(xì)胞分化來源的巨噬細(xì)胞實(shí)驗(yàn)中,與LPS組相比,20 mg/ml B-HA不僅能抑制炎癥因子TNF-α、IL-1、IL-6、IL-8和IFN-β的m RNA和蛋白表達(dá)水平,而且可增強(qiáng)抗炎因子IL-10的m RNA和蛋白表達(dá)水平。B-HA對(duì)TLR4信號(hào)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.在人外周血分離培養(yǎng)的DCs細(xì)胞實(shí)驗(yàn)中,不同濃度的B-HA對(duì)LPS刺激的促炎因子(TNF-α、IL-6、IL-8)和抗炎因子(IL-10)均無明顯影響。結(jié)論小分子量的B-HA能通過TLR4信號(hào)通路有效地調(diào)節(jié)人巨噬細(xì)胞中促炎因子和抗炎因子的表達(dá),抑制其炎癥反應(yīng);對(duì)人DC細(xì)胞的炎癥反應(yīng)無明顯影響。本實(shí)驗(yàn)提示B-HA對(duì)人炎性免疫細(xì)胞的炎癥反應(yīng)的作用具有選擇性。同時(shí),可為臨床上治療膿毒血癥提供一種新的方法。
[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- 偽, 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- 偽 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- 偽, 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- 偽 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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R459.7

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