牙齦蛋白酶通過(guò)C5a途徑調(diào)控M1型巨噬細(xì)胞的極化
發(fā)布時(shí)間：2019-06-20 17:46
【摘要】：背景:牙周炎是以牙周結(jié)締組織破壞和牙槽骨吸收為特點(diǎn)的一種慢性感染性疾病。巨噬細(xì)胞是牙周組織中重要的免疫防御細(xì)胞,在不同的免疫微環(huán)境下可分化為不同的表型,即:M1型巨噬細(xì)胞(M1)和M2型巨噬細(xì)胞(M2)。M1高表達(dá)細(xì)胞表面共刺激分子CD86,抑菌細(xì)胞因子IL-12、IL-23和iNOS,低表達(dá)CD206和抑炎因子IL-10等,發(fā)揮清除病原菌的作用。此外M1還可表達(dá)促炎因子TNF-ɑ、IL-1β和IL-6,導(dǎo)致牙周組織免疫病理?yè)p傷。牙齦卟啉單胞菌(Porphyromonas gingivlis,Pg)是牙周炎的主要致病菌,可釋放多種毒力因子如牙齦蛋白酶(gingipain)、脂多糖(lipopolysaccharide,LPS)和菌毛(fimbriae)等,其中牙齦蛋白酶是一種半胱氨酸蛋白酶,具有C5轉(zhuǎn)化酶樣的活性,可分解補(bǔ)體C5(complement 5,C5)產(chǎn)生大量C5a,C5a與LPS等可共同激活巨噬細(xì)胞表面的C5a受體和Toll樣受體(toll-like receptor,TLR),激活細(xì)胞內(nèi)信號(hào)串?dāng)_,抑制TLR依賴性途徑M1極化,并釋放促炎和骨吸收細(xì)胞因子如:TNF-α、IL-1β和IL-6等,因此牙齦蛋白酶可通過(guò)C5a途徑抑制M1型巨噬細(xì)胞對(duì)Pg免疫清除作用的同時(shí)破壞牙周組織。方法:1.牙齦蛋白酶的制備采用sheets改良法從Pg ATCC 33277培養(yǎng)物上清中制備牙齦蛋白酶,應(yīng)用質(zhì)譜法鑒定牙齦蛋白酶,底物發(fā)色法測(cè)定酶活性。2.牙齦蛋白酶對(duì)巨噬細(xì)胞極化的影響使用活性0、0.25、1、4和8U/L及有/無(wú)活性牙齦蛋白酶分別與小鼠RAW264.7巨噬細(xì)胞系共培養(yǎng)24h后,采用q RT-PCR檢測(cè)M1型巨噬細(xì)胞相關(guān)細(xì)胞因子基因表達(dá)的水平。3.牙齦蛋白酶對(duì)Ec-LPS和Pg-LPS誘導(dǎo)的M1型巨噬細(xì)胞極化的影響實(shí)驗(yàn)分組為:陰性對(duì)照組、LPS組和LPS+牙齦蛋白酶組、LPS+牙齦蛋白酶組+PMX-53(C5a R拮抗劑),共培養(yǎng)24h,采用q RT-PCR、ELISA及流式細(xì)胞術(shù)法檢測(cè)M1相關(guān)細(xì)胞因子表達(dá)的水平。結(jié)果:1.制備的牙齦蛋白酶主要成分為RgpA,其中Rgps活性為20U/L。2.不同活性牙齦蛋白酶刺激RAW264.7細(xì)胞24h后,促炎細(xì)胞因子IL-12、IL-23、iNOS、TNF-α、IL-1β和IL-6基因表達(dá)在4U/L時(shí)達(dá)到最高(P0.05);相比與無(wú)活性的牙齦蛋白酶,有活性牙齦蛋白酶(4U/L)組可弱的促進(jìn)上述促炎細(xì)胞因子基因的表達(dá)(P0.05)。PMX-53可促進(jìn)有活性牙齦蛋白酶組IL-12、IL-23、iNOS基因的表達(dá)(P0.05),而抑制TNF-α、IL-1β和IL-6基因的表達(dá),但PMX-53對(duì)無(wú)活性牙齦蛋白酶組無(wú)此影響(P0.05)。3.牙齦蛋白酶可抑制Ec-LPS誘導(dǎo)的CD86及促炎細(xì)胞因子IL-12、IL-23、iNOS、TNF-α、IL-1β和IL-6基因和蛋白的的表達(dá)水平(P0.01),PMX-53促進(jìn)牙齦蛋白酶+Ec-LPS組促炎細(xì)胞因子IL-12、IL-23、iNOS基因和蛋白的表達(dá)(P0.01),抑制TNF-α、IL-1β和IL-6基因和蛋白的表達(dá),但對(duì)抑炎細(xì)胞因子IL-10及CD206基因和蛋白的表達(dá)無(wú)影響(P0.01)。4.牙齦蛋白酶可弱的促進(jìn)Pg-LPS誘導(dǎo)的細(xì)胞因子IL-12、IL-23基因和蛋白的表達(dá)(P0.01),卻抑制CD86及細(xì)胞因子iNOS、TNF-α、IL-1β和IL-6基因和蛋白的表達(dá)水平(P0.05),對(duì)IL-10和CD206的表達(dá)無(wú)影響。PMX-53可促進(jìn)牙齦蛋白酶+Pg-LPS組促炎細(xì)胞因子IL-12、IL-23、iNOS基因和蛋白的表達(dá)(P0.05),抑制TNF-α、IL-1β和IL-6基因和蛋白的表達(dá)(P0.05),但對(duì)抑炎細(xì)胞因子IL-10及CD206基因和蛋白的表達(dá)無(wú)明顯影響。結(jié)論:1.牙齦蛋白酶可弱的促進(jìn)M1型巨噬細(xì)胞極化;2.牙齦蛋白酶可通過(guò)C5a途徑選擇性調(diào)控LPS誘導(dǎo)的M1型巨噬細(xì)胞抑菌因子IL-12、IL-23、iNOS的表達(dá)水平;3.PMX-53可作為潛在免疫調(diào)節(jié)制劑,在牙周炎的治療中具有重要應(yīng)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R781.42
本文編號(hào)：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R781.42
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