细粒棘球蚴感染中TGF-β/Smad信号通路对IL-9调控作用的研究

摘要/Abstract

摘要：

目的检测细粒棘球蚴病患者肝组织中转化生长因子β（TGF-β）、Smad3、Smad7、转录因子PU.1和白细胞介素-9（IL-9）mRNA表达水平,初步探讨TGF-β/Smad信号通路在细粒棘球蚴感染病灶中对Th9细胞的调控作用。方法以新疆医科大学第一附属医院住院手术的30例棘球蚴病患者（CE患者组）和20例肝血管瘤患者（对照组）为研究对象,取手术中切除的肝脏组织,用Percoll法分离肝脏单个核细胞,实时荧光定量PCR分别检测CE患者组和对照组肝脏单个核细胞中TGF-β、Smad3、Smad7、PU.1和IL-9 mRNA的相对表达水平。采用Spearman相关性分析方法分析感染组肝脏单个核细胞中Smad3 mRNA的相对表达水平与PU.1 mRNA的相对表达水平的相关性。在CE患者组肝脏单个核细胞中加入Smad3抑制剂SIS3进行体外干预（SIS3阻断组）,另设PBS对照组,加入等量PBS,采用实时荧光定量PCR检测两组肝脏单个核细胞中PU.1和IL-9 mRNA的相对表达水平,ELISA检测细胞培养上清中IL-9水平。结果实时荧光定量PCR结果显示,CE患者组肝组织中单个核细胞的TGF-β、Smad3、PU.1和IL-9 mRNA相对表达水平分别为2.55 ± 1.32、2.26 ± 1.35、1.71 ± 0.66和1.67 ± 0.75,均高于对照组的1.54 ± 0.76、1.34 ± 0.51、1.27 ± 0.62和1.01 ± 0.50（P < 0.01或0.05）;Smad7的相对表达水平为0.49 ± 0.37,低于对照组的0.88 ± 0.50（P < 0.01）。Spearman相关性分析结果显示,CE患者组肝脏单个核细胞中Smad3 mRNA的相对表达水平与PU.1 mRNA的相对表达水平呈正相关（r = 0.397,P < 0.05）。用SIS3阻断TGF-β/Smad信号通路后,肝脏单个核细胞中PU.1和IL-9 mRNA的相对表达水平为1.00 ± 0.57和0.85 ± 0.50,低于PBS对照组的1.46 ± 0.64和1.32 ± 0.72（P < 0.01）。ELISA检测结果显示,细胞培养上清中IL-9水平为（128.45 ± 41.97）pg/ml,低于PBS对照组的（166.64 ± 57.90）pg/ml（P < 0.05）。结论细粒棘球蚴病患者肝脏组织中TGF-β、Smad2、Smad3、PU.1和IL-9表达上调,TGF-β/Smad通路可能参与调控细粒棘球蚴感染中IL-9的表达。

关键词: 细粒棘球蚴, 感染, TGF-β/Smad信号通路;, 转录因子PU.1, 白细胞介素-9

Abstract:

Objective To examine mRNA expression of TGF-β, Smad3, Smad7, PU.1 and Interleukin-9 (IL-9) in livers of patients with cystic echinococcosis, in order to explore the regulations of Th9 cells by the TGF-β/Smad signaling in Echinococcus granulosus infection. Methods The liver mononuclear cells were isolated from liver tissues obtained during surgery from 30 patients with cystic hydatid disease and 20 patients with hepatic hemangioma (control group) in the First Affiliated Hospital of Xinjiang Medical University using the Percoll method. Real-time fluorescent quantitative PCR was performed to detect the mRNA expression of TGF-β, Smad3, Smad7, PU.1 and IL-9 in the mononuclear cells. The relationship between Smad3 mRNA expression and PU.1 mRNA expression was assessed by Spearman correlation analysis. The Smad3 inhibitor SIS3 or PBS was added to the mononuclear cells of the infected group, then PU.1 and IL-9 mRNA expression were determined by real-time fluorescent quantitative PCR, and IL-9 level in the supernatant of cell culture was assessed by ELISA. Results The relative mRNA expression of TGF-β, Smad3, PU.1 and IL-9 in CE patients were 2.55 ± 1.32, 2.26 ± 1.35, 1.71 ± 0.66 and 1.67 ± 0.75, respectively, all significantly higher than those in the control group (1.54 ± 0.76, 1.34 ± 0.51, 1.27 ± 0.62 and 1.01 ± 0.50, P < 0.05 or 0.01). The relative mRNA expression of Smad7 in CE patients (0.49 ± 0.37) was significantly lower than that in the control group (0.88 ± 0.50; P < 0.01). The Smad3 mRNA expression and PU.1 mRNA expression were positively correlated (r = 0.397, P < 0.05). After blocking the TGF-β/Smad signaling pathway by SIS3, the expression of PU.1 and IL-9 mRNA in liver mononuclear cells (1.00 ± 0.57, 0.85 ± 0.50) both decreased compared with the PBS treatment (1.46 ± 0.64, 1.32 ± 0.72) (P < 0.01). ELISA results showed that the IL-9 level in the supernatant of cell culture [(128.45 ± 41.97) pg/ml] also reduced compared with the PBS treatment [(166.64 ± 57.90) pg/ml; P < 0.05]. Conclusion The expression of TGF-β, Smad2, Smad3, PU.1 and IL-9 up-regulated in patients with cystic hydatid disease and the TGF-β/Smad pathway may regulate the expression of IL-9 after Echinococcus granulosus infection.

Key words: Echinococcus granulosus, Infection, TGF-β/Smad signal pathway;, PU.1, IL-9

中图分类号:

R532.32

庞楠楠, 安梦婷, 张峰波, 赵骁, 闫芳, 丁剑冰. 细粒棘球蚴感染中TGF-β/Smad信号通路对IL-9调控作用的研究[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(5): 460-464.

Nan-nan PANG, Meng-ting AN, Feng-bo ZHANG, Xiao ZHAO, Fang YAN, Jian-bing DING. Regulations of IL-9 by the TGF-β/Smads signaling pathway in human Echinococcus granulosus infection[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2018, 36(5): 460-464.

图/表 2

参考文献 21

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基因 Gene	引物序列（5′→3′） Primer sequences (5′→3′)	退火温度/℃ Tm/℃	长度/bp Length/bp
TGF-β1	F:ACACCAACTATTGCTTCAG	55.7	159
	R:TGTCCAGGCTCCAAATG
Smad3	F: GTGCTCCATCTCCTACTAC	56.5	183
	R: CCTCTTCCGATGTGTCTC
Smad7	F:TTTGTGTATTTATTTCTTTCTCTC	54.5	194
	R:CACTCTCGTCTTCTCCTC
Smad2	F: TTTGCTGCTCTTCTGGCTCA	60	179
	R: AACCACTGTAGAGGTCCATTCA
PU.1	F: GGAGCCCGGCTGGATGTTAC	60.04	79
	R: CACCAGGTCTTCTGATGGCTGA
IL-9	F: CTCTAGCAGTCCACTTCACCAA	59.3	112
	R: ACAGCATGGGTCTGTCTTCT
β-Actin	F: TAGGCGGACTGTTACTGAGC	60	233
	R: TGCTCCAACCAACTGCTGTC