重组蛋白P29对细粒棘球蚴感染小鼠肝脏组织TGF-β/Smad信号通路的影响
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摘要
目的观察重组蛋白P29 (r P29)对细粒棘球蚴感染小鼠肝脏组织转化生长因子β(TGF-β)/Smad信号通路的作用。方法36只雌性BALB/c小鼠随机分为r P29免疫组(r P29组)、佐剂组、 PBS组,每组12只。r P29组小鼠皮下注射r P29蛋白(10μg/只,与等体积弗氏佐剂乳化),第2和4周各加强免疫1次;佐剂组、 PBS组仅注射相应的佐剂或PBS。末次免疫后2周, 3组小鼠均腹腔接种细粒棘球蚴原头节(1 500个/只)进行攻击感染。感染后12周,剖杀小鼠,分离肝脏组织,提取肝脏组织mRNA,实时荧光定量PCR检测小鼠肝脏组织中TGF-β1基因的相对表达水平,蛋白质印迹(Western blotting)检测小鼠肝脏组织中TGF-β/Smad信号通路下游相关蛋白TGFβ-RI、 TGFβ-RII、 p-Smad2,3、 Smad4、 Smad7的表达水平。结果实时荧光定量PCR检测结果显示,佐剂组、 PBS组和r P29组小鼠肝脏组织中的TGF-β1 mRNA的相对表达量分别为0.98±0.42、 1.71±0.29和1.24±0.56, r P29组低于PBS组(P <0.01),高于佐剂组,但无统计学意义。Western blotting检测结果显示, r P29组小鼠肝脏组织TGF-β1蛋白表达灰度值为372.04±0.01,低于PBS组(673.02±0.06),高于佐剂组(213.69±0.31)。小鼠肝脏组织TGF-β/Smad信号通路相关蛋白的检测结果显示, r P29组TGF-β-RI、 TGF-β-RII、 p-Smad 2,3、 Smad 4的灰度值分别为357.59±0.83、 289.07±0.21、 204.06±0.19、 396.11±0.01,低于PBS组(496.89±0.09、 378.41±0.01、428.11±0.29、 566.95±0.21),高于佐剂组(171.99±0.82、 185.77±0.09、 128.09±0.08、 205.87±0.59)。r P29免疫组Smad 7蛋白灰度值为278.89±0.12,高于PBS组(142.32±0.07),低于佐剂组(384.17±0.51)。结论r P29能够降低细粒棘球蚴感染小鼠肝脏组织中TGF-β1 mRNA和蛋白的表达水平;下调TGF-β/Smad下游信号通路相关蛋白TGF-β-RⅠ、 TGF-β-RⅡ、 p-Smad 2,3和Smad 4的表达,上调Smad 7的表达。
Objective To evaluate the effect of recombinant protein P29(rP29), a 29 kDa antigen identified in hydatid cyst fluid which induces protective immunity, on the transforming growth factor-β(TGF-β)/Smad signaling pathway in the liver of mice infected with Echinococcus granulosus. Methods A group of 12 female BALB/c mice were subcutaneously immunized with 10 μg of r P29 emulsified with Freund 's adjuvant, then boosted twice with two weeks interval. Another two groups of mice were given with Freund 's adjuvant or PBS only as controls. The three groups of mice were intraperitoneally challenged with 1 500 protoscolex of E. granulosus per mouse two weeks after the last immunization. Twelve weeks after infection, the mice were euthanized and the liver tissues were collected.The TGF-β1 mRNA expression level was detected by RT-PCR in the liver of each infected mouse. The expression levels of TGF-β/Smad signaling pathway regulated downstream proteins such as TGF-RI, TGF-RII, p-Smad2,3, Smad4 and Smad7 were measured by Western blotting in the liver tissues. Results RT-PCR results showed that the relative expression level of TGF-β1 mRNA in the liver tissues of mice immunized with r P29(1.24 ± 0.56) was reduced compared to PBS control mice(1.71 ± 0.29) with significant difference(P < 0.01), however, it was higher than that in adjuvant control mice(0.98 ± 0.42). The changes of the TGF-β1 m RNA expression level in the liver tissues were consistent with the TGF-β1 protein expression level detected by Western blotting. The expression density of TGF-β1 protein in the r P29 immunized group was 372.04 ± 0.01(measured by densitometry) which was lower than that in the PBS group(673.02 ± 0.06), but higher than that in the adjuvant group(213.69 ± 0.31). The TGF-β/Smad signaling pathway-regulated downstream proteins also showed the similar changes except for Smad 7. The expression levels of TGF-β-RI, TGF-β-RII, p-Smad 2,3 and Smad 4 in the liver of r P29 immunized mice(357.59 ± 0.83,289.07 ± 0.21, 204.06 ± 0.19, 396.11 ± 0.01, respectively) were reduced compared with the PBS group(496.89 ± 0.09,378.41 ± 0.01, 428.11 ± 0.29, 566.95 ± 0.21, respectively), but higher than that of the adjuvant control group(171.99 ± 0.82, 185.77 ± 0.09, 128.09 ± 0.08, 205.87 ± 0.59, respectively). However, Smad 7 level expressed in r P29 immunized mice(278.89 ± 0.12) was up-regulated compared with PBS group(142.32 ± 0.07), but significantly lower than that in adjuvant group(384.17 ± 0.51). Conclusion Immunization of recombinant P29 protein significantly reduced the expression of TGF-β1 at both mRNA and protein levels in the liver of mice infected with E. granulosus. Most of the TGF-β/Smad signaling pathway-regulated downstream proteins(TGF-β-RI, TGF-β-RII, pSmad 2,3 and Smad 4) were also reduced except for Smad 7 which was up-regulated.
Objective To evaluate the effect of recombinant protein P29(rP29), a 29 kDa antigen identified in hydatid cyst fluid which induces protective immunity, on the transforming growth factor-β(TGF-β)/Smad signaling pathway in the liver of mice infected with Echinococcus granulosus. Methods A group of 12 female BALB/c mice were subcutaneously immunized with 10 μg of r P29 emulsified with Freund 's adjuvant, then boosted twice with two weeks interval. Another two groups of mice were given with Freund 's adjuvant or PBS only as controls. The three groups of mice were intraperitoneally challenged with 1 500 protoscolex of E. granulosus per mouse two weeks after the last immunization. Twelve weeks after infection, the mice were euthanized and the liver tissues were collected.The TGF-β1 mRNA expression level was detected by RT-PCR in the liver of each infected mouse. The expression levels of TGF-β/Smad signaling pathway regulated downstream proteins such as TGF-RI, TGF-RII, p-Smad2,3, Smad4 and Smad7 were measured by Western blotting in the liver tissues. Results RT-PCR results showed that the relative expression level of TGF-β1 mRNA in the liver tissues of mice immunized with r P29(1.24 ± 0.56) was reduced compared to PBS control mice(1.71 ± 0.29) with significant difference(P < 0.01), however, it was higher than that in adjuvant control mice(0.98 ± 0.42). The changes of the TGF-β1 m RNA expression level in the liver tissues were consistent with the TGF-β1 protein expression level detected by Western blotting. The expression density of TGF-β1 protein in the r P29 immunized group was 372.04 ± 0.01(measured by densitometry) which was lower than that in the PBS group(673.02 ± 0.06), but higher than that in the adjuvant group(213.69 ± 0.31). The TGF-β/Smad signaling pathway-regulated downstream proteins also showed the similar changes except for Smad 7. The expression levels of TGF-β-RI, TGF-β-RII, p-Smad 2,3 and Smad 4 in the liver of r P29 immunized mice(357.59 ± 0.83,289.07 ± 0.21, 204.06 ± 0.19, 396.11 ± 0.01, respectively) were reduced compared with the PBS group(496.89 ± 0.09,378.41 ± 0.01, 428.11 ± 0.29, 566.95 ± 0.21, respectively), but higher than that of the adjuvant control group(171.99 ± 0.82, 185.77 ± 0.09, 128.09 ± 0.08, 205.87 ± 0.59, respectively). However, Smad 7 level expressed in r P29 immunized mice(278.89 ± 0.12) was up-regulated compared with PBS group(142.32 ± 0.07), but significantly lower than that in adjuvant group(384.17 ± 0.51). Conclusion Immunization of recombinant P29 protein significantly reduced the expression of TGF-β1 at both mRNA and protein levels in the liver of mice infected with E. granulosus. Most of the TGF-β/Smad signaling pathway-regulated downstream proteins(TGF-β-RI, TGF-β-RII, pSmad 2,3 and Smad 4) were also reduced except for Smad 7 which was up-regulated.
引文
[1]李润乐,格日力.我国包虫病基础研究现状及存在的问题[J].中国高原医学与生物学杂志,2017,38(4):217-218,234.
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[9]张梦媛,伍卫平,官亚宜,等.我国棘球蚴病疾病负担分析[J].中国寄生虫学与寄生虫病杂志,2018,36(1):15-19,25.
[10] Cadavid Restrepo AM,Yang YR,McManus DP,et al.Spatiotemporal patterns and environmental drivers of human echinococcoses over a twenty-year period in Ningxia Hui Autonomous Region,China[J]. Parasit Vectors,2018,11(1):108.
[11]何叶,尹家祥.棘球蚴病流行因素分析[J].中国热带医学,2017,17(4):418-420.
[12]张文宝,张壮志,郑雪婷,等.棘球蚴(包虫)病预防疫苗的研制与应用[J].中国人兽共患病学报,2018,34(9):834-838.
[13]师志云,李昭宇,卜阳,等.细粒棘球绦虫(中国大陆株)诊断抗原P-29基因的表达、纯化及免疫原性初步分析[J].中国人兽共患病学报,2009,25(11):1065-1067.
[14] Wang H,Li ZH,Gao F,et al. Immunoprotection of recombinant Eg.P29 against Echinococcus granulosus in sheep[J]. Vet Res Commun,2016,40(2):73-79.
[15] Zhao X,Zhang FB,Li ZW,et al. Bioinformatics analysis of EgA31 and EgG1Y162 proteins for designing a multi-epitope vaccine against Echinococcus granulosus[J]. Infect Genet Evol,2019:S1567-S1348(19)30057-7.
[16] Liu FX,Fan XX,Li L,et al. Development of recombinant goatpox virus expressing Echinococcus granulosus EG9 5 vaccine antigen[J]. J Virol Methods,2018,261:28-33.
[17]张静,曾静,李亮,等. Smad蛋白抑制剂SIS3对细粒棘球蚴原头节的作用研究[J].中国病原生物学杂志,2017,12(5):389-393.
[18] Hu HH,Chen DQ,Wang YN,et al. New insights into TGF-β/Smad signaling in tissue fibrosis[J]. Chem Biol Interact,2018,292(25):76-83.
[19]张传山,杨舒婷,毕晓娟,等.泡型包虫病患者肝脏组织TGF-β1和Gadd45γ基因的表达及其在肝损伤中的作用研究[J].中国病原生物学杂志,2016,11(10):908-912.
[20]陈骏,钱云良. TGF-β/Smads通路与增生性瘢痕肌成纤维细胞分化[J].中国美容医学,2007,16(7):1000-1003.
[21] Yin S,Chen X,Zhang J,et al. The effect of Echinococcus granulosus on spleen cells and TGF-βexpression in the peripheral blood of BALB/c mice[J]. Parasite Immunol,2017,39(3):e12415.
[22] Pang NN,Zhang FB,Li SY,et al. TGF-β/Smad signaling p athway positively up-regulates the differentiation of Interleukin-9-producing CD4+T cells in human Echinococcus granulosus infection[J]. J Infect,2018,76(4):406-416.
[23] Zhang CS,Wang LM,Wang H,et al. Identification and characterization of functional Smad8 and Smad4 homologues from Echinococcus granulosus[J]. Parasitol Res,2014,113(10):3745-3757.
[24]单骄宇,热比亚·努力,李瑞,等.调节性T细胞转录因子Foxp3和IL-8在棘球蚴病患者肝组织病灶中的表达[J].中国寄生虫学与寄生虫病杂志,2018,36(3):218-223.
[25] Liu YM,Abudounnasier G,Zhang TC,et al. Increased expression of TGF-β1 in correlation with liver fibrosis during Echinococcus granulosus infection in mice[J]. Korean J Parasitol,2016,54(4):519-525.
[26]印双红,张俊波,陈小林,等.细粒棘球蚴感染中阻断TGF-β1受体对淋巴细胞的影响[J].中国免疫学杂志,2015,31(5):607-612.
[27]高富.细粒棘球绦虫重组蛋白P29诱导绵羊的免疫保护力及其免疫机制研究[D].银川:宁夏医科大学,2015.
[2] Cadavid Restrepo AM,Yang YR,McManus DP,et al. Environmental risk factors and changing spatial patterns of human seropositivity for Echinococcus spp. in Xiji County,Ningxia Hui Autonomous Region,China[J]. Parasit Vectors,2018,11(1):159.
[3] Kern P,Menezes da Silva A,Akhan O,et al. The echinococcoses:diagnosis,clinical management and burden of disease[J]. Adv Parasitol,2017,96:259-369.
[4]张静宵,马霄,刘玉芳,等.青海省棘球蚴病流行与分布情况调查[J].中国寄生虫学与寄生虫病杂志,2017,35(5):465-467.
[5]宋健,裴迎新,郭卫东,等. 2011-2017年内蒙古自治区棘球蚴病流行特征分析[J].中国寄生虫学与寄生虫病杂志,2018,36(6):560-564.
[6]阿达来提·托留汉,漫格库丽·哈提木拉提,阿合里江·卡依多拉,等.新疆塔城地区棘球蚴病流行现状调查[J].中国寄生虫学与寄生虫病杂志,2018,36(6):565-570.
[7]严信留,贡桑曲珍,伍卫平,等.西藏自治区村民和学生棘球蚴病知识态度和行为调查[J].中国寄生虫学与寄生虫病杂志,2018,36(1):38-42.
[8]王东,冯宇,李凡,等.甘肃省藏区人群棘球蚴病流行现状调查及分析[J].中国寄生虫学与寄生虫病杂志,2017,35(2):140-144.
[9]张梦媛,伍卫平,官亚宜,等.我国棘球蚴病疾病负担分析[J].中国寄生虫学与寄生虫病杂志,2018,36(1):15-19,25.
[10] Cadavid Restrepo AM,Yang YR,McManus DP,et al.Spatiotemporal patterns and environmental drivers of human echinococcoses over a twenty-year period in Ningxia Hui Autonomous Region,China[J]. Parasit Vectors,2018,11(1):108.
[11]何叶,尹家祥.棘球蚴病流行因素分析[J].中国热带医学,2017,17(4):418-420.
[12]张文宝,张壮志,郑雪婷,等.棘球蚴(包虫)病预防疫苗的研制与应用[J].中国人兽共患病学报,2018,34(9):834-838.
[13]师志云,李昭宇,卜阳,等.细粒棘球绦虫(中国大陆株)诊断抗原P-29基因的表达、纯化及免疫原性初步分析[J].中国人兽共患病学报,2009,25(11):1065-1067.
[14] Wang H,Li ZH,Gao F,et al. Immunoprotection of recombinant Eg.P29 against Echinococcus granulosus in sheep[J]. Vet Res Commun,2016,40(2):73-79.
[15] Zhao X,Zhang FB,Li ZW,et al. Bioinformatics analysis of EgA31 and EgG1Y162 proteins for designing a multi-epitope vaccine against Echinococcus granulosus[J]. Infect Genet Evol,2019:S1567-S1348(19)30057-7.
[16] Liu FX,Fan XX,Li L,et al. Development of recombinant goatpox virus expressing Echinococcus granulosus EG9 5 vaccine antigen[J]. J Virol Methods,2018,261:28-33.
[17]张静,曾静,李亮,等. Smad蛋白抑制剂SIS3对细粒棘球蚴原头节的作用研究[J].中国病原生物学杂志,2017,12(5):389-393.
[18] Hu HH,Chen DQ,Wang YN,et al. New insights into TGF-β/Smad signaling in tissue fibrosis[J]. Chem Biol Interact,2018,292(25):76-83.
[19]张传山,杨舒婷,毕晓娟,等.泡型包虫病患者肝脏组织TGF-β1和Gadd45γ基因的表达及其在肝损伤中的作用研究[J].中国病原生物学杂志,2016,11(10):908-912.
[20]陈骏,钱云良. TGF-β/Smads通路与增生性瘢痕肌成纤维细胞分化[J].中国美容医学,2007,16(7):1000-1003.
[21] Yin S,Chen X,Zhang J,et al. The effect of Echinococcus granulosus on spleen cells and TGF-βexpression in the peripheral blood of BALB/c mice[J]. Parasite Immunol,2017,39(3):e12415.
[22] Pang NN,Zhang FB,Li SY,et al. TGF-β/Smad signaling p athway positively up-regulates the differentiation of Interleukin-9-producing CD4+T cells in human Echinococcus granulosus infection[J]. J Infect,2018,76(4):406-416.
[23] Zhang CS,Wang LM,Wang H,et al. Identification and characterization of functional Smad8 and Smad4 homologues from Echinococcus granulosus[J]. Parasitol Res,2014,113(10):3745-3757.
[24]单骄宇,热比亚·努力,李瑞,等.调节性T细胞转录因子Foxp3和IL-8在棘球蚴病患者肝组织病灶中的表达[J].中国寄生虫学与寄生虫病杂志,2018,36(3):218-223.
[25] Liu YM,Abudounnasier G,Zhang TC,et al. Increased expression of TGF-β1 in correlation with liver fibrosis during Echinococcus granulosus infection in mice[J]. Korean J Parasitol,2016,54(4):519-525.
[26]印双红,张俊波,陈小林,等.细粒棘球蚴感染中阻断TGF-β1受体对淋巴细胞的影响[J].中国免疫学杂志,2015,31(5):607-612.
[27]高富.细粒棘球绦虫重组蛋白P29诱导绵羊的免疫保护力及其免疫机制研究[D].银川:宁夏医科大学,2015.