兩種旋毛蟲絲氨酸蛋白酶抑制劑對(duì)巨噬細(xì)胞免疫功能的影響
發(fā)布時(shí)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(nèi)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(jié)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(guān)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(jié)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(zhuǎn)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(diào)控中所發(fā)揮的作用,本研究以J774A.1小鼠巨噬細(xì)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(duì)巨噬細(xì)胞免疫功能的調(diào)節(jié)作用。利用實(shí)驗(yàn)室已經(jīng)通過原核表達(dá)成功構(gòu)建,并能表現(xiàn)良好活性的TsAd SPI和TsKa SPI的陽性表達(dá)載體,進(jìn)行IPTG誘導(dǎo)表達(dá),經(jīng)電洗脫對(duì)表達(dá)蛋白進(jìn)行純化,進(jìn)行透析復(fù)性后獲得重組蛋白TsAd SPI和Ts Ka SPI。利用CCK-8檢測(cè)不同濃度的兩種SPI對(duì)J774A.1增殖活力的影響。用兩種SPI分別單獨(dú)刺激或經(jīng)LPS預(yù)刺激后再用兩種SPI刺激J774A.1,分別對(duì)炎性細(xì)胞因子TNF-α、IL-1β、IL-6、IL-10、IL-12和TGF-βmRNA表達(dá)水平和分泌情況進(jìn)行定量檢測(cè),并應(yīng)用Westen-blot檢測(cè)兩種SPIs對(duì)JAK2/STAT3信號(hào)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(shè)計(jì)并分別合成熒光引物,建立q PCR檢測(cè)方法。TsAd SPI和TsKa SPI的CCK-8檢測(cè)結(jié)果基本一致,與PBS空白對(duì)照組相比,兩種SPI對(duì)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(duì)J774A.1增殖活力產(chǎn)生影響;但當(dāng)濃度達(dá)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(xì)胞增殖活力受損程度明顯增強(qiáng)。故本實(shí)驗(yàn)選擇TsAd SPI和TsKa SPI的工作濃度均為5μg/mL。SYBR Green I實(shí)時(shí)熒光定量PCR檢測(cè)結(jié)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(xì)胞促炎性細(xì)胞因子TNF-α、IL-1β、IL-6和IL-12 m RNA的表達(dá)水平。并且兩種SPI均可不同程度的促進(jìn)J774A.1小鼠巨噬細(xì)胞抗炎性細(xì)胞因子IL-10和TGF-β的表達(dá)。雖然TsAd SPI和TsKa SPI對(duì)巨噬細(xì)胞各炎性細(xì)胞因子表達(dá)與分泌的調(diào)節(jié)程度各有差異,但均可對(duì)以上炎性細(xì)胞因子起到抑制或促進(jìn)的調(diào)控,表明這兩種SPI對(duì)巨噬細(xì)胞的免疫功能均具有一定程度的調(diào)節(jié)功能。ELISA對(duì)巨噬細(xì)胞促炎性細(xì)胞因子和抗炎性細(xì)胞因子的分泌情況進(jìn)行檢測(cè),結(jié)果與q PCR檢測(cè)結(jié)果相一致,印證了兩種SPI對(duì)巨噬細(xì)胞各炎性細(xì)胞因子的產(chǎn)生具有調(diào)節(jié)能力的論斷。同時(shí),Westen-blot檢測(cè)到TsAd SPI和TsKa SPI均可誘導(dǎo)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(hào)通路影響巨噬細(xì)胞信號(hào)傳導(dǎo)。因此推測(cè)這兩種SPI均能夠在旋毛蟲感染宿主時(shí)作為影響機(jī)體免疫功能的效應(yīng)分子,對(duì)宿主巨噬細(xì)胞所發(fā)揮的免疫功能進(jìn)行調(diào)節(jié),進(jìn)而實(shí)現(xiàn)免疫逃避,來達(dá)到抵御宿主機(jī)體排蟲反應(yīng)和在宿主體內(nèi)長(zhǎng)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(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：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(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.72
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相關(guān)期刊論文 前10條
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