【摘要】： 蛋白酶激活受體(proteinase activated receptors或protease activated receptors，PARs)，白細胞介素(interleukin，IL)-4，IL-6參與過敏反應的發(fā)生，IL-12參與后天獲得性免疫反應。然而IL-12對酶引起的肥大細胞分泌細胞因子的影響，細胞因子對肥大細胞PAR表達的影響，Th1類細胞因子IL-12對肥大細胞Th2類細胞因子分泌的影響及其信號轉(zhuǎn)導通路還不甚清楚。因此本研究探討IL-12對肥大細胞PARs表達和IL-4，IL-6分泌以及組胺釋放的影響，并探討可能的信號轉(zhuǎn)導通路。 肥大細胞激發(fā)后，收集各時間點的細胞和上清。細胞處理后采用流式細胞術(shù)和實時定量PCR的方法在蛋白水平和mRNA水平檢測PAR-1，PAR-2，PAR-3和PAR-4表達情況，用細胞激發(fā)信號ELISA(eellular activation of signaling ELISA，CASE)方法檢測信號轉(zhuǎn)導通路蛋白Akt，，ERK，p38和STAT3磷酸化情況；上清用ELISA方法檢測IL-4，IL-6和組胺水平。 結(jié)果顯示：肥大細胞P815在蛋白水平和mRNA水平表達PAR-1，PAR-2，PAR-3和PAR-4。IL-12下調(diào)PAR-2蛋白和mRNA在肥大細胞P815上的表達；IL-12上調(diào)PAR-4蛋白和mRNA在肥大細胞P815上的表達；IL-12上調(diào)PAR-1和PAR-3 mRNA在肥大細胞P815的表達；以低濃度IL-12(1 ng／ml)預處理肥大細胞60分鐘后，胰蛋白酶和類胰蛋白酶引起的PAR-2蛋白和mRNA在肥大細胞P815上的表達增強；IL-12抗體的應用可以阻斷IL-12對PAR表達的上調(diào)作用。IL-12以濃度依賴的方式促進肥大細胞P815分泌IL-4；低濃度IL-12(1ng／ml)預處理P815細胞60分鐘后，胰蛋白酶和類胰蛋白酶引起的IL-4分泌被抑制；PAR-2拮抗肽FSLLRY-NH_2的應用能阻斷胰蛋白酶和類胰蛋白酶引起的IL-4分泌，但對IL-12引起的IL-4分泌無影響；IL-12對肥大細胞IL-6分泌和組胺釋放無明顯影響。應用PD98059，U0126和LY294002可以阻斷IL-12引起的肥大細胞IL-4分泌并抑制IL-12引起的ERK和Akt磷酸化。 總之，IL-12能夠調(diào)節(jié)肥大細胞PARs表達并刺激肥大細胞分泌IL-4。IL-12引起肥大細胞P815分泌IL-4很可能是通過激活Akt和ERK信號轉(zhuǎn)導通路實現(xiàn)的，而胰蛋白酶和類胰蛋白酶引起肥大細胞分泌IL-4似乎是有賴于激活PAR-2途徑實現(xiàn)的。這些發(fā)現(xiàn)提示在過敏性炎癥反應中，IL-12可作為一種調(diào)節(jié)因子，通過調(diào)節(jié)PAR-2在肥大細胞表達和調(diào)節(jié)肥大細胞分泌IL-4，發(fā)揮其維持Th1細胞因子和Th2細胞因子分泌平衡的功能。同時，胰蛋白酶和類胰蛋白酶刺激肥大細胞分泌IL-4的發(fā)現(xiàn)為肥大細胞激活的自我放大機制提供了一個新的理論依據(jù)。
[Abstract]:Protease activated receptors (proteinase activated receptors or protease activated receptor PARs) and interleukin-4- (IL-6) were involved in the development of allergic reaction. IL-12 was involved in acquired immune response. However, the effect of IL-12 on the secretion of cytokines from mast cells induced by enzymes and the effect of cytokines on the expression of PAR in mast cells are unclear. The effect of Th1 cytokine IL-12 on the secretion of Th2 cytokines of mast cells and its signal transduction pathway are not clear. The present study was designed to investigate the effects of IL-12 on the expression of PARs, IL-4 and IL-6 secretion and histamine release in mast cells, and to explore the possible signal transduction pathways. After mast cells were stimulated, cells and supernatants were collected at each time point. Flow cytometry and real-time quantitative PCR were used to detect the expression of PAR-1, PAR-2, PAR-3 and PAR-4 at the protein level and mRNA level, and the phosphorylation of the signal transduction pathway proteins AktnERKP38 and STAT3 were detected by ELISA (eellular activation of signaling elisa case. The levels of IL-4, IL-6 and histamine in supernatant were detected by ELISA. The results showed that P815 and PAR-2PAR-3 and PAR-4.IL-12 down-regulated the expression of PAR-2 and mRNA in P815 and P815, respectively. IL-12 up-regulated the expression of PAR-4 and mRNA on P815. IL-12 up-regulated the expression of PAR-1 and PAR-3 mRNA in mast cell P815, and the expression of PAR-2 protein and mRNA on mast cell P815 was enhanced by trypsin and trypsin induced by low concentration IL-12 (1 ng/ml) for 60 minutes. The application of IL-12 antibody could block the up-regulation of PAR expression by IL-12. IL-12 promoted the secretion of IL-4 by mast cells in a concentration-dependent manner, and the secretion of IL-4 induced by trypsin and trypsin was inhibited after pretreatment of P815 cells with low concentration of IL-12 (1ng/ml) for 60 minutes. PAR-2 antagonistic peptide FSLLRY-NH_2 could block IL-4 secretion induced by trypsin and trypsin, but had no effect on IL-4 secretion induced by IL-12. IL-12 had no effect on IL-6 secretion and histamine release of mast cells. PD98059 U0126 and LY294002 could block IL-4 secretion induced by IL-12 and inhibit ERK and Akt phosphorylation induced by IL-12. In conclusion, IL-12 can regulate the expression of PARs in mast cells and stimulate the secretion of IL-4.IL-12 by mast cells. It is possible that IL-12 can activate the signal transduction pathways of Akt and ERK by activating the IL-4 secretion of mast cells P815. The secretion of IL-4 by mast cells induced by trypsin and trypsin seems to depend on the activation of PAR-2 pathway. These findings suggest that IL-12 may act as a regulatory factor in allergic inflammatory response by regulating the expression of PAR-2 in mast cells and regulating the secretion of IL-4 by mast cells, thus exerting its function of maintaining the balance between Th1 cytokines and Th2 cytokines. At the same time, the discovery of trypsin and trypsin stimulated mast cells to secrete IL-4 provides a new theoretical basis for the self-amplifying mechanism of mast cell activation.
【學位授予單位】：汕頭大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R392

【引證文獻】

中國碩士學位論文全文數(shù)據(jù)庫 前1條

1 張旭;針刺對EAE小鼠模型IL-12和MIP-1α的實驗研究[D];北京中醫(yī)藥大學;2010年

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