牙龈蛋白酶通过C5a途径调控M1型巨噬细胞的极化
发布时间：2019-06-20 17:46
【摘要】：背景:牙周炎是以牙周结缔组织破坏和牙槽骨吸收为特点的一种慢性感染性疾病。巨噬细胞是牙周组织中重要的免疫防御细胞,在不同的免疫微环境下可分化为不同的表型,即:M1型巨噬细胞(M1)和M2型巨噬细胞(M2)。M1高表达细胞表面共刺激分子CD86,抑菌细胞因子IL-12、IL-23和iNOS,低表达CD206和抑炎因子IL-10等,发挥清除病原菌的作用。此外M1还可表达促炎因子TNF-ɑ、IL-1β和IL-6,导致牙周组织免疫病理损伤。牙龈卟啉单胞菌(Porphyromonas gingivlis,Pg)是牙周炎的主要致病菌,可释放多种毒力因子如牙龈蛋白酶(gingipain)、脂多糖(lipopolysaccharide,LPS)和菌毛(fimbriae)等,其中牙龈蛋白酶是一种半胱氨酸蛋白酶,具有C5转化酶样的活性,可分解补体C5(complement 5,C5)产生大量C5a,C5a与LPS等可共同激活巨噬细胞表面的C5a受体和Toll样受体(toll-like receptor,TLR),激活细胞内信号串扰,抑制TLR依赖性途径M1极化,并释放促炎和骨吸收细胞因子如:TNF-α、IL-1β和IL-6等,因此牙龈蛋白酶可通过C5a途径抑制M1型巨噬细胞对Pg免疫清除作用的同时破坏牙周组织。方法:1.牙龈蛋白酶的制备采用sheets改良法从Pg ATCC 33277培养物上清中制备牙龈蛋白酶,应用质谱法鉴定牙龈蛋白酶,底物发色法测定酶活性。2.牙龈蛋白酶对巨噬细胞极化的影响使用活性0、0.25、1、4和8U/L及有/无活性牙龈蛋白酶分别与小鼠RAW264.7巨噬细胞系共培养24h后,采用q RT-PCR检测M1型巨噬细胞相关细胞因子基因表达的水平。3.牙龈蛋白酶对Ec-LPS和Pg-LPS诱导的M1型巨噬细胞极化的影响实验分组为:阴性对照组、LPS组和LPS+牙龈蛋白酶组、LPS+牙龈蛋白酶组+PMX-53(C5a R拮抗剂),共培养24h,采用q RT-PCR、ELISA及流式细胞术法检测M1相关细胞因子表达的水平。结果:1.制备的牙龈蛋白酶主要成分为RgpA,其中Rgps活性为20U/L。2.不同活性牙龈蛋白酶刺激RAW264.7细胞24h后,促炎细胞因子IL-12、IL-23、iNOS、TNF-α、IL-1β和IL-6基因表达在4U/L时达到最高(P0.05);相比与无活性的牙龈蛋白酶,有活性牙龈蛋白酶(4U/L)组可弱的促进上述促炎细胞因子基因的表达(P0.05)。PMX-53可促进有活性牙龈蛋白酶组IL-12、IL-23、iNOS基因的表达(P0.05),而抑制TNF-α、IL-1β和IL-6基因的表达,但PMX-53对无活性牙龈蛋白酶组无此影响(P0.05)。3.牙龈蛋白酶可抑制Ec-LPS诱导的CD86及促炎细胞因子IL-12、IL-23、iNOS、TNF-α、IL-1β和IL-6基因和蛋白的的表达水平(P0.01),PMX-53促进牙龈蛋白酶+Ec-LPS组促炎细胞因子IL-12、IL-23、iNOS基因和蛋白的表达(P0.01),抑制TNF-α、IL-1β和IL-6基因和蛋白的表达,但对抑炎细胞因子IL-10及CD206基因和蛋白的表达无影响(P0.01)。4.牙龈蛋白酶可弱的促进Pg-LPS诱导的细胞因子IL-12、IL-23基因和蛋白的表达(P0.01),却抑制CD86及细胞因子iNOS、TNF-α、IL-1β和IL-6基因和蛋白的表达水平(P0.05),对IL-10和CD206的表达无影响。PMX-53可促进牙龈蛋白酶+Pg-LPS组促炎细胞因子IL-12、IL-23、iNOS基因和蛋白的表达(P0.05),抑制TNF-α、IL-1β和IL-6基因和蛋白的表达(P0.05),但对抑炎细胞因子IL-10及CD206基因和蛋白的表达无明显影响。结论:1.牙龈蛋白酶可弱的促进M1型巨噬细胞极化;2.牙龈蛋白酶可通过C5a途径选择性调控LPS诱导的M1型巨噬细胞抑菌因子IL-12、IL-23、iNOS的表达水平;3.PMX-53可作为潜在免疫调节制剂,在牙周炎的治疗中具有重要应用前景。
[Abstract]:Background: Periodontitis is a chronic infectious disease characterized by periodontal connective tissue destruction and alveolar bone resorption. Macrophages are important immune defense cells in the periodontal tissues, and can be differentiated into different phenotypes under different immune microenvironment, namely, the M1-type macrophages (M1) and the M2-type macrophages (M2). The high-expression cell surface of the M1 has the costimulatory molecules CD86, the antibacterial cytokines IL-12, IL-23 and iNOS, The low-expression CD206 and the anti-inflammatory factor IL-10 play a role in removing pathogenic bacteria. In addition, the pro-inflammatory factor, TNF-1, IL-1, and IL-6, can be expressed as a pro-inflammatory factor, leading to the immune and pathological injury of the periodontal tissue. P. gingiva (Pg) is the main pathogenic bacteria of periodontitis, and can release various virulence factors such as gingival protease, lipopolysaccharides (LPS) and fimbriae, etc., wherein the gingival protease is a cysteine protease, and has the activity of C5 convertase. The decomposable complement C5 (C5) produces a large amount of C5a, C5a and LPS, which can co-activate the C5a receptor and the Toll-like receptor (TLR) on the surface of the macrophage, activate intracellular signal cross-talk, inhibit the polarization of the TLR-dependent pathway M1, and release the pro-inflammatory and bone-absorbing cytokines such as TNF-1, IL-1 and IL-6 and so on, therefore, the gingival protease can inhibit the role of the M1-type macrophage on the Pg immune clearance by the C5a pathway and destroy the periodontal tissue. Method:1. Gingival protease was prepared from Pg ATCC 33277 culture by the method of sheet modification, and the gingival protease was identified by mass spectrometry, and the enzyme activity was determined by the substrate chromophoric method. The effect of gingival protease on the polarization of macrophage was used to detect the level of the expression of the related cytokine gene of the M1-type macrophage by using the q-RT-PCR after 24 h co-culture with the mouse RAW264.7 macrophage system by using the active 0, 0.25,1,4 and 8 U/ L and the/ inactive gingival protease. The effect of gingival protease on the polarization of the M1-type macrophages induced by Ec-LPS and Pg-LPS was as follows: negative control group, LPS group and LPS + gingival protease group, LPS + gingival protease group + PMX-53 (C5a R antagonist), co-culture for 24 h, and q RT-PCR. The expression level of M1-associated cytokines was detected by ELISA and flow cytometry. Results:1. The main component of the prepared gingival protease is RgpA, wherein the activity of the Rgps is 20U/ L.2. The expression of pro-inflammatory cytokines IL-12, IL-23, iNOS, TNF-1, IL-1 and IL-6 was the highest at the time of 4 U/ L (P0.05). The expression of IL-12, IL-23 and iNOS gene in the active gingival protease group (P0.05) can be promoted by the active gingival protease (4U/ L), and the expression of the TNF-1, IL-1 and IL-6 genes can be inhibited. However, PMX-53 did not have the effect on the non-active gingival protease group (P0.05). The expression of IL-12, IL-23, iNOS, TNF-1, IL-1 and IL-6, and the expression of IL-12, IL-23, iNOS gene and protein were inhibited by gingival protease (P0.01). The expression of IL-1 and IL-6 gene and protein was not affected by the expression of IL-10 and CD206 gene and protein (P0.01). The expression of IL-12 and IL-23 and the expression of IL-6 and IL-6 in the expression of IL-12, IL-23 and the expression of IL-6 and the expression of IL-6 and the expression of IL-6 and the expression of IL-6 and the expression of IL-6, and the expression of IL-6 and IL-6 in the expression of IL-10 and CD206 were not affected. PMX-53 could promote the expression of pro-inflammatory cytokines IL-12, IL-23, iNOS gene and protein in the gingival protease + Pg-LPS group (P0.05), and inhibit the expression of the TNF-1, IL-1 and IL-6 genes and proteins (P0.05), but have no significant effect on the expression of the anti-inflammatory cytokine IL-10 and the CD206 gene and the protein. Conclusion:1. Gingival protease is weak to promote the polarization of M1-type macrophages;2. The expression level of IL-12, IL-23, and iNOS induced by LPS can be selectively regulated by the C5a pathway, and the PMX-53 can be used as a potential immunomodulating preparation, and has an important application prospect in the treatment of periodontitis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.42
本文编号：2503406
[Abstract]:Background: Periodontitis is a chronic infectious disease characterized by periodontal connective tissue destruction and alveolar bone resorption. Macrophages are important immune defense cells in the periodontal tissues, and can be differentiated into different phenotypes under different immune microenvironment, namely, the M1-type macrophages (M1) and the M2-type macrophages (M2). The high-expression cell surface of the M1 has the costimulatory molecules CD86, the antibacterial cytokines IL-12, IL-23 and iNOS, The low-expression CD206 and the anti-inflammatory factor IL-10 play a role in removing pathogenic bacteria. In addition, the pro-inflammatory factor, TNF-1, IL-1, and IL-6, can be expressed as a pro-inflammatory factor, leading to the immune and pathological injury of the periodontal tissue. P. gingiva (Pg) is the main pathogenic bacteria of periodontitis, and can release various virulence factors such as gingival protease, lipopolysaccharides (LPS) and fimbriae, etc., wherein the gingival protease is a cysteine protease, and has the activity of C5 convertase. The decomposable complement C5 (C5) produces a large amount of C5a, C5a and LPS, which can co-activate the C5a receptor and the Toll-like receptor (TLR) on the surface of the macrophage, activate intracellular signal cross-talk, inhibit the polarization of the TLR-dependent pathway M1, and release the pro-inflammatory and bone-absorbing cytokines such as TNF-1, IL-1 and IL-6 and so on, therefore, the gingival protease can inhibit the role of the M1-type macrophage on the Pg immune clearance by the C5a pathway and destroy the periodontal tissue. Method:1. Gingival protease was prepared from Pg ATCC 33277 culture by the method of sheet modification, and the gingival protease was identified by mass spectrometry, and the enzyme activity was determined by the substrate chromophoric method. The effect of gingival protease on the polarization of macrophage was used to detect the level of the expression of the related cytokine gene of the M1-type macrophage by using the q-RT-PCR after 24 h co-culture with the mouse RAW264.7 macrophage system by using the active 0, 0.25,1,4 and 8 U/ L and the/ inactive gingival protease. The effect of gingival protease on the polarization of the M1-type macrophages induced by Ec-LPS and Pg-LPS was as follows: negative control group, LPS group and LPS + gingival protease group, LPS + gingival protease group + PMX-53 (C5a R antagonist), co-culture for 24 h, and q RT-PCR. The expression level of M1-associated cytokines was detected by ELISA and flow cytometry. Results:1. The main component of the prepared gingival protease is RgpA, wherein the activity of the Rgps is 20U/ L.2. The expression of pro-inflammatory cytokines IL-12, IL-23, iNOS, TNF-1, IL-1 and IL-6 was the highest at the time of 4 U/ L (P0.05). The expression of IL-12, IL-23 and iNOS gene in the active gingival protease group (P0.05) can be promoted by the active gingival protease (4U/ L), and the expression of the TNF-1, IL-1 and IL-6 genes can be inhibited. However, PMX-53 did not have the effect on the non-active gingival protease group (P0.05). The expression of IL-12, IL-23, iNOS, TNF-1, IL-1 and IL-6, and the expression of IL-12, IL-23, iNOS gene and protein were inhibited by gingival protease (P0.01). The expression of IL-1 and IL-6 gene and protein was not affected by the expression of IL-10 and CD206 gene and protein (P0.01). The expression of IL-12 and IL-23 and the expression of IL-6 and IL-6 in the expression of IL-12, IL-23 and the expression of IL-6 and the expression of IL-6 and the expression of IL-6 and the expression of IL-6 and the expression of IL-6, and the expression of IL-6 and IL-6 in the expression of IL-10 and CD206 were not affected. PMX-53 could promote the expression of pro-inflammatory cytokines IL-12, IL-23, iNOS gene and protein in the gingival protease + Pg-LPS group (P0.05), and inhibit the expression of the TNF-1, IL-1 and IL-6 genes and proteins (P0.05), but have no significant effect on the expression of the anti-inflammatory cytokine IL-10 and the CD206 gene and the protein. Conclusion:1. Gingival protease is weak to promote the polarization of M1-type macrophages;2. The expression level of IL-12, IL-23, and iNOS induced by LPS can be selectively regulated by the C5a pathway, and the PMX-53 can be used as a potential immunomodulating preparation, and has an important application prospect in the treatment of periodontitis.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.42
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