兩種旋毛蟲絲氨酸蛋白酶抑制劑對巨噬細胞免疫功能的影響
發(fā)布時間：2018-11-13 11:17
【摘要】：旋毛蟲病(Trichinellosis)是一種非常重要的人獸共患寄生蟲病,旋毛蟲能在宿主體內完成寄生過程,與其入侵宿主、免疫逃避等密不可分。已有研究結果表明絲氨酸蛋白酶抑制劑(Serine Proteinase Inhibitor,SPI)在寄生蟲入侵宿主、免疫逃避等的過程中發(fā)揮著關鍵作用,有研究發(fā)現(xiàn)埃及血吸蟲通過蟲體表面絲氨酸蛋白酶抑制因子與人的胰蛋白酶相結合來降低自身的免疫原性,逃脫宿主的免疫攻擊。目前SPI已從蛔蟲、血吸蟲、馬來絲蟲、捻轉血毛線蟲及旋毛蟲等寄生蠕蟲中分離鑒定出來。為了摸清旋毛蟲SPI在宿主免疫調控中所發(fā)揮的作用,本研究以J774A.1小鼠巨噬細胞為模型,研究旋毛蟲成蟲Serpin型絲氨酸蛋白酶抑制劑(TsAd SPI)和旋毛蟲Kazal型絲氨酸蛋白酶抑制劑(TsKa SPI)對巨噬細胞免疫功能的調節(jié)作用。利用實驗室已經(jīng)通過原核表達成功構建,并能表現(xiàn)良好活性的TsAd SPI和TsKa SPI的陽性表達載體,進行IPTG誘導表達,經(jīng)電洗脫對表達蛋白進行純化,進行透析復性后獲得重組蛋白TsAd SPI和Ts Ka SPI。利用CCK-8檢測不同濃度的兩種SPI對J774A.1增殖活力的影響。用兩種SPI分別單獨刺激或經(jīng)LPS預刺激后再用兩種SPI刺激J774A.1,分別對炎性細胞因子TNF-α、IL-1β、IL-6、IL-10、IL-12和TGF-βmRNA表達水平和分泌情況進行定量檢測,并應用Westen-blot檢測兩種SPIs對JAK2/STAT3信號通路的影響。根據(jù)Gen Bank中發(fā)表的TNF-α、IL-1β、IL-6、IL-10、IL-12、TGF-β和GAPDH基因序列,設計并分別合成熒光引物,建立q PCR檢測方法。TsAd SPI和TsKa SPI的CCK-8檢測結果基本一致,與PBS空白對照組相比,兩種SPI對J774A.1增殖活力的影響表現(xiàn)出一定的濃度依賴性,低濃度的兩種SPI均未對J774A.1增殖活力產(chǎn)生影響;但當濃度達到10μg/mL后,J774A.1的增殖活力明顯受到損害,并且隨著兩種SPI濃度的增大,J774A.1的細胞增殖活力受損程度明顯增強。故本實驗選擇TsAd SPI和TsKa SPI的工作濃度均為5μg/mL。SYBR Green I實時熒光定量PCR檢測結果表明,Ts Ad SPI和TsKa SPI均可不同程度的抑制LPS活化的J774A.1小鼠巨噬細胞促炎性細胞因子TNF-α、IL-1β、IL-6和IL-12 m RNA的表達水平。并且兩種SPI均可不同程度的促進J774A.1小鼠巨噬細胞抗炎性細胞因子IL-10和TGF-β的表達。雖然TsAd SPI和TsKa SPI對巨噬細胞各炎性細胞因子表達與分泌的調節(jié)程度各有差異,但均可對以上炎性細胞因子起到抑制或促進的調控,表明這兩種SPI對巨噬細胞的免疫功能均具有一定程度的調節(jié)功能。ELISA對巨噬細胞促炎性細胞因子和抗炎性細胞因子的分泌情況進行檢測,結果與q PCR檢測結果相一致,印證了兩種SPI對巨噬細胞各炎性細胞因子的產(chǎn)生具有調節(jié)能力的論斷。同時,Westen-blot檢測到TsAd SPI和TsKa SPI均可誘導JAK2和STAT3受體發(fā)生磷酸化,表明兩種SPIs都可以通過作用于JAK2/STAT3信號通路影響巨噬細胞信號傳導。因此推測這兩種SPI均能夠在旋毛蟲感染宿主時作為影響機體免疫功能的效應分子,對宿主巨噬細胞所發(fā)揮的免疫功能進行調節(jié),進而實現(xiàn)免疫逃避,來達到抵御宿主機體排蟲反應和在宿主體內長期寄生的目的。
[Abstract]:Trichinella, a very important human and animal, has a parasitic disease, and the trichinella can complete the parasitic process in the host, and it is closely related to its invasion and host, immune evasion, and so on. It has been shown that Serine Protease Inhibitor (SPI) plays a key role in the process of parasite invasion of host and immune escape. It has been found that the binding of a serine protease inhibitor on the surface of the worm through the surface of the worm is combined with human trypsin to reduce its immunogenicity and escape the immune attack of the host. At present, the SPI has been isolated and identified from the parasitic worms such as ascariasis, schistosome, malaysia, and trichinella spiralis. In order to study the role of Trichinella spiralis in host immune regulation, this study is based on J774A. 1 mouse macrophage. To study the regulation of Serpin-type Serpin-type serine protease inhibitor (TsAd SPI) and Cazal-type serine protease inhibitor (TsKa SPI) of Trichinella spiralis on the immune function of macrophages. The expression of TsAd SPI and TsKa SPI was successfully constructed by the expression of TsAd SPI and TsKa SPI, and the recombinant protein TsAd SPI and Ts Ka SPI were obtained by electroelution. The effect of two SPI on the proliferation of J774A. 1 was detected by CCK-8. The expression level and secretion of the inflammatory cytokines TNF-1, IL-1, IL-6, IL-10, IL-12 and TGF-1 mRNA were detected by two kinds of SPI separately or after LPS pre-stimulation, and the effects of the two SPIs on the JAK2/ STAT3 signal pathway were detected by using the Westen-blot. according to the sequence of TNF-1, IL-1, IL-6, IL-10, IL-12, TGF-1 and GAPDH published in the Gen Bank, and the fluorescent primers are designed and synthesized respectively, and a q-PCR detection method is established. The results of CCK-8 test of TsAd SPI and TsKa SPI are basically the same, and compared with the blank control group of PBS, the effect of two SPI on the proliferation activity of J774A. 1 shows a certain concentration dependence. Both SPI of low concentration have no effect on the proliferation activity of J774A. 1, but when the concentration reaches 10. m u.g/ mL, The proliferation activity of J774A. 1 was obviously damaged, and with the increase of the two SPI concentrations, the degree of cell proliferation of J774A-1 was significantly increased. Therefore, the working concentration of TsAd SPI and TsKa SPI was 5. m u.g/ mL. The results of real-time fluorescence quantitative PCR of SYBR Green I showed that both the TsAd SPI and TsKa SPI could inhibit the expression of TNF-1, IL-1, IL-6 and IL-12 mRNA in the macrophage of the mice. and the expression of the anti-inflammatory cytokine IL-10 and TGF-1 in the macrophage of the mouse macrophage can be promoted to different extent by the two SPI. Although TsAd SPI and TsKa SPI have different levels of regulation of inflammatory cytokine expression and secretion of macrophages, it is possible to inhibit or promote the above inflammatory cytokines, indicating that these two kinds of SPI have a certain degree of regulation on the immune function of macrophages. The secretion of pro-inflammatory cytokines and anti-inflammatory cytokines in macrophages was detected by ELISA, and the results were consistent with the results of q-PCR. At the same time, both the TsAd SPI and the TsKa SPI can induce phosphorylation of the JAK2 and STAT3 receptors, indicating that both SPIs can influence the signaling of the macrophages by acting on the JAK2/ STAT3 signaling pathway. therefore, the two SPI can be used as the effector molecule for influencing the immune function of the organism when the trichinella is infected with the host, the immune function of the host macrophage is regulated, and the immune escape is realized, so as to resist the reaction of the host organism and the purpose of long-term parasitism in the host.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.72
[Abstract]:Trichinella, a very important human and animal, has a parasitic disease, and the trichinella can complete the parasitic process in the host, and it is closely related to its invasion and host, immune evasion, and so on. It has been shown that Serine Protease Inhibitor (SPI) plays a key role in the process of parasite invasion of host and immune escape. It has been found that the binding of a serine protease inhibitor on the surface of the worm through the surface of the worm is combined with human trypsin to reduce its immunogenicity and escape the immune attack of the host. At present, the SPI has been isolated and identified from the parasitic worms such as ascariasis, schistosome, malaysia, and trichinella spiralis. In order to study the role of Trichinella spiralis in host immune regulation, this study is based on J774A. 1 mouse macrophage. To study the regulation of Serpin-type Serpin-type serine protease inhibitor (TsAd SPI) and Cazal-type serine protease inhibitor (TsKa SPI) of Trichinella spiralis on the immune function of macrophages. The expression of TsAd SPI and TsKa SPI was successfully constructed by the expression of TsAd SPI and TsKa SPI, and the recombinant protein TsAd SPI and Ts Ka SPI were obtained by electroelution. The effect of two SPI on the proliferation of J774A. 1 was detected by CCK-8. The expression level and secretion of the inflammatory cytokines TNF-1, IL-1, IL-6, IL-10, IL-12 and TGF-1 mRNA were detected by two kinds of SPI separately or after LPS pre-stimulation, and the effects of the two SPIs on the JAK2/ STAT3 signal pathway were detected by using the Westen-blot. according to the sequence of TNF-1, IL-1, IL-6, IL-10, IL-12, TGF-1 and GAPDH published in the Gen Bank, and the fluorescent primers are designed and synthesized respectively, and a q-PCR detection method is established. The results of CCK-8 test of TsAd SPI and TsKa SPI are basically the same, and compared with the blank control group of PBS, the effect of two SPI on the proliferation activity of J774A. 1 shows a certain concentration dependence. Both SPI of low concentration have no effect on the proliferation activity of J774A. 1, but when the concentration reaches 10. m u.g/ mL, The proliferation activity of J774A. 1 was obviously damaged, and with the increase of the two SPI concentrations, the degree of cell proliferation of J774A-1 was significantly increased. Therefore, the working concentration of TsAd SPI and TsKa SPI was 5. m u.g/ mL. The results of real-time fluorescence quantitative PCR of SYBR Green I showed that both the TsAd SPI and TsKa SPI could inhibit the expression of TNF-1, IL-1, IL-6 and IL-12 mRNA in the macrophage of the mice. and the expression of the anti-inflammatory cytokine IL-10 and TGF-1 in the macrophage of the mouse macrophage can be promoted to different extent by the two SPI. Although TsAd SPI and TsKa SPI have different levels of regulation of inflammatory cytokine expression and secretion of macrophages, it is possible to inhibit or promote the above inflammatory cytokines, indicating that these two kinds of SPI have a certain degree of regulation on the immune function of macrophages. The secretion of pro-inflammatory cytokines and anti-inflammatory cytokines in macrophages was detected by ELISA, and the results were consistent with the results of q-PCR. At the same time, both the TsAd SPI and the TsKa SPI can induce phosphorylation of the JAK2 and STAT3 receptors, indicating that both SPIs can influence the signaling of the macrophages by acting on the JAK2/ STAT3 signaling pathway. therefore, the two SPI can be used as the effector molecule for influencing the immune function of the organism when the trichinella is infected with the host, the immune function of the host macrophage is regulated, and the immune escape is realized, so as to resist the reaction of the host organism and the purpose of long-term parasitism in the host.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.72
【參考文獻】
相關期刊論文 前10條
1 馬靜;李達;孔令杰;劉暢;李爽;毛貽賢;杜娟;路義鑫;;旋毛蟲Kazal型絲氨酸蛋白酶抑制劑的表達及免疫相關性[J];中國獸醫(yī)學報;2015年08期
2 李達;萬悅;馬靜;孔令杰;路佳琦;武文浩;路義鑫;;旋毛蟲成蟲絲氨酸蛋白酶抑制劑的原核表達及免疫原性研究[J];中國獸醫(yī)雜志;2015年03期
3 龐宇;李巍;韓彩霞;伊娜娜;劉暢;俞昭e，
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