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

doi:10.12140/j.issn.1000-7423.2019.03.013

摘要/Abstract

摘要：

目的观察重组蛋白P29（rP29）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rP29免疫组（rP29组）、佐剂组、PBS组,每组12只。rP29组小鼠皮下注射rP29蛋白（10 μg/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rP29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,rP29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,rP29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,rP29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）。rP29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 rP29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。

关键词: 细粒棘球蚴, 转化生长因子β1;, TGF-β/Smad信号通路;

Abstract:

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 rP29 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 rP29 (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 mRNA 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 rP29 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 rP29 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 rP29 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, p-Smad 2,3 and Smad 4) were also reduced except for Smad 7 which was up-regulated.

Key words: Echinococcus granulosus, Transforming growth factor-β1;, TGF-β/Smad signaling pathway;

中图分类号:

R383.33

马锐, 刘成, 徐士梅, 朱明星, 赵嘉庆, 万巧凤, 朱佳佳, 赵巍. 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[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(3): 316-321.

Rui MA, Cheng LIU, Shi-mei XU, Ming-xing ZHU, Jia-qing ZHAO, Qiao-feng WAN, Jia-jia ZHU, Wei ZHAO. Effect of recombinant protein P29 on the TGF-β/Smad signaling pathway in the liver of mice infected with Echinococcus granulosus[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2019, 37(3): 316-321.

图/表 2

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