两种旋毛虫丝氨酸蛋白酶抑制剂对巨噬细胞免疫功能的影响
发布时间：2018-11-13 11:17
【摘要】：旋毛虫病(Trichinellosis)是一种非常重要的人兽共患寄生虫病,旋毛虫能在宿主体内完成寄生过程,与其入侵宿主、免疫逃避等密不可分。已有研究结果表明丝氨酸蛋白酶抑制剂(Serine Proteinase Inhibitor,SPI)在寄生虫入侵宿主、免疫逃避等的过程中发挥着关键作用,有研究发现埃及血吸虫通过虫体表面丝氨酸蛋白酶抑制因子与人的胰蛋白酶相结合来降低自身的免疫原性,逃脱宿主的免疫攻击。目前SPI已从蛔虫、血吸虫、马来丝虫、捻转血毛线虫及旋毛虫等寄生蠕虫中分离鉴定出来。为了摸清旋毛虫SPI在宿主免疫调控中所发挥的作用,本研究以J774A.1小鼠巨噬细胞为模型,研究旋毛虫成虫Serpin型丝氨酸蛋白酶抑制剂(TsAd SPI)和旋毛虫Kazal型丝氨酸蛋白酶抑制剂(TsKa SPI)对巨噬细胞免疫功能的调节作用。利用实验室已经通过原核表达成功构建,并能表现良好活性的TsAd SPI和TsKa SPI的阳性表达载体,进行IPTG诱导表达,经电洗脱对表达蛋白进行纯化,进行透析复性后获得重组蛋白TsAd SPI和Ts Ka SPI。利用CCK-8检测不同浓度的两种SPI对J774A.1增殖活力的影响。用两种SPI分别单独刺激或经LPS预刺激后再用两种SPI刺激J774A.1,分别对炎性细胞因子TNF-α、IL-1β、IL-6、IL-10、IL-12和TGF-βmRNA表达水平和分泌情况进行定量检测,并应用Westen-blot检测两种SPIs对JAK2/STAT3信号通路的影响。根据Gen Bank中发表的TNF-α、IL-1β、IL-6、IL-10、IL-12、TGF-β和GAPDH基因序列,设计并分别合成荧光引物,建立q PCR检测方法。TsAd SPI和TsKa SPI的CCK-8检测结果基本一致,与PBS空白对照组相比,两种SPI对J774A.1增殖活力的影响表现出一定的浓度依赖性,低浓度的两种SPI均未对J774A.1增殖活力产生影响;但当浓度达到10μg/mL后,J774A.1的增殖活力明显受到损害,并且随着两种SPI浓度的增大,J774A.1的细胞增殖活力受损程度明显增强。故本实验选择TsAd SPI和TsKa SPI的工作浓度均为5μg/mL。SYBR Green I实时荧光定量PCR检测结果表明,Ts Ad SPI和TsKa SPI均可不同程度的抑制LPS活化的J774A.1小鼠巨噬细胞促炎性细胞因子TNF-α、IL-1β、IL-6和IL-12 m RNA的表达水平。并且两种SPI均可不同程度的促进J774A.1小鼠巨噬细胞抗炎性细胞因子IL-10和TGF-β的表达。虽然TsAd SPI和TsKa SPI对巨噬细胞各炎性细胞因子表达与分泌的调节程度各有差异,但均可对以上炎性细胞因子起到抑制或促进的调控,表明这两种SPI对巨噬细胞的免疫功能均具有一定程度的调节功能。ELISA对巨噬细胞促炎性细胞因子和抗炎性细胞因子的分泌情况进行检测,结果与q PCR检测结果相一致,印证了两种SPI对巨噬细胞各炎性细胞因子的产生具有调节能力的论断。同时,Westen-blot检测到TsAd SPI和TsKa SPI均可诱导JAK2和STAT3受体发生磷酸化,表明两种SPIs都可以通过作用于JAK2/STAT3信号通路影响巨噬细胞信号传导。因此推测这两种SPI均能够在旋毛虫感染宿主时作为影响机体免疫功能的效应分子,对宿主巨噬细胞所发挥的免疫功能进行调节,进而实现免疫逃避,来达到抵御宿主机体排虫反应和在宿主体内长期寄生的目的。
[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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：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.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.72
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