多房棘球蚴病患者肝病灶组织差异表达基因的研究

doi:10.12140/j.issn.1000-7423.2021.01.007

摘要/Abstract

摘要：

目的探讨多房棘球蚴感染小鼠肝差异基因在多房棘球蚴病患者肝病灶组织中的表达情况及意义。方法选取高通量基因表达数据库中编号为GSE24376的小鼠感染多房棘球蚴基因芯片数据集,分析感染小鼠和未感染小鼠之间的差异表达基因,对差异表达基因进行基因本体（GO）分析。取6例多房棘球蚴病患者肝病灶组织和距离病灶组织2 cm的周围肝组织,采用荧光定量PCR检测2个高表达差异基因mRNA相对转录水平。取45份多房棘球蚴病患者石蜡包埋肝组织样品（炎性反应和肝纤维化改变严重的病灶29份,较少炎性细胞浸润和肝纤维化病变的肝组织16份）,采用免疫组化法分析2个高表达差异基因的表达情况。用SPSS 21.0统计学软件对数据进行统计分析。结果生物信息学分析结果显示,共筛选出21个差异表达基因,其中9个基因表达上调,12个基因表达下调。GO分析结果显示,差异基因主要位于细胞内囊泡及细胞外基质等细胞成分中,参与了中性粒细胞的脱颗粒及活化等生物过程,并且具有CC趋化因子受体结合活性及趋化因子活性。高表达差异基因CHI3L3（在人体中为CHI3L1）和CCL8荧光定量PCR结果显示,6例多房棘球蚴病患者肝病灶组织中CHI3L1和CCL8 mRNA相对转录水平均高于病灶周围肝组织（P < 0.01）,最高倍数分别达7倍和9倍。免疫组化分析结果显示：29份多房棘球蚴病患者肝组织炎性反应和肝纤维化改变严重的病灶样品中,有20份（占69%）呈CHI3L1蛋白高表达,其主要着色部位位于细胞浆;有22份（占76%）呈CCL8蛋白高表达。16份多房棘球蚴病患者肝组织轻微炎症样品中,2份（占13%）呈CHI3L1蛋白阳性;4份（占33%）呈CCL8蛋白阳性。炎性反应和肝纤维化改变严重的病灶组织中,CHI3L1和CCL8蛋白的阳性表达率分别达96%和97%,均高于轻微炎症肝组织的3%和8%（P < 0.01）。结论 CHI3L1和CCL8蛋白在多房棘球蚴病患者肝组织中表达增高,提示二者在机体对虫体的免疫应答过程中发挥了一定作用。

关键词: 多房棘球蚴, 感染, 差异表达基因, CHI3L3, CCL8

Abstract:

Objective To investigate the expression profile of differentially expressed genes in liver lesions of alveolar echinococcosis patients, based on the findings that these genes were indentified in mice infected with alveolar metacestode. Methods The gene chip dataset (No. GSE24376) in the high-throughput gene expression database was selected from the gene expression omnibus (GEO), to compare the genes with differential expression between infected and uninfected mice. The differentially expressed genes underwent gene ontology (GO) analysis. Lesion tissues and adjacent tissues 2 cm from the lesion were collected from 6 patients with E. multilocularis cyst infection. Fluorescence quantitative PCR was performed to detect the relative mRNA levels of two highly expressed differential genes. Forty-five paraffin-embedded liver tissue samples from patients with multilocular hydatid infection (29 samples with severe inflammatory response and liver fibrosis, and 16 with relatively normal liver tissue with less inflammatory cell infiltration and liver fibrosis) underwent immunohistochemistry to analyze the expression of the two highly expressed differential genes. Statistical analysis was performed on the data with SPSS 21.0 statistical software. Results As compared to the normal mice, 21 differentially expressed genes were found, including 9 overexpressed genes and 12 downregulated genes in liver tissues of infected mice. The results of GO analysis showed that the differentially expressed genes were expressed in vesicle lumen, cytoplasmic vesicle lumen and extracellular matrix, participated in biological processes of neutrophil degranulation and activation, and possessed CC chemokine receptor binding activity and chemokine activity. The fluorescence quantitative PCR results showed that the mRNA expression levels of CHI3L3 (CHI3L1 in humans) and CCL8, which had higher differential expression, were higher in the liver lesion tissues than in the surrounding tissues of the 6 patients with multilocular hydatid infection (P < 0.01), reaching an utmost 7 and 9 folds, respectively. The results of immunohistochemistry showed that among the 29 lesion samples with severe inflammatory reaction and liver fibrosis, 20 samples (69%) showed high expression of CHI3L1 protein, with staining mainly located in the cytoplasm, and 22 samples showed high levels of CCL8 protein (76%). Of the 16 relatively normal liver tissue samples, only 2 (13%) were positive for CHI3L1 protein, and only 4 were positive for CCL8 protein (33%). In the lesions with severe inflammatory response and liver fibrosis, the positive expression rate of CHI3L1 and CCL8 proteins reached 96% and 97%, respectively, which were higher than 3% and 8% in normal liver tissues (P < 0.01). Conclusion The expression of CHI3L1 and CCL8 was upregulated in the liver tissues of patients with E. multilocularis, suggesting that they play a role in the immune response to parasites.

Key words: Echinococcus multilocularis, Infection, Differentially expressed genes, CHI3L3, CCL8

中图分类号:

R383.33

朱吉海, 曹得萍, 赵珺, 刘珺, 史虎祥, 刘燕. 多房棘球蚴病患者肝病灶组织差异表达基因的研究[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(1): 48-54.

ZHU Ji-hai, CAO De-ping, ZHAO Jun, LIU Jun, SHI Hu-xiang, LIU Yan. Investigation of differentially expressed genes in liver tissues of patients with alveolar echinococcosis[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2021, 39(1): 48-54.

图/表 5

表1

表2

GSE24376基因芯片多房棘球蚴感染小鼠与健康小鼠的差异表达基因

差异表达基因 Differentially expressed genes	倍数变化 Fold change	P值 P value	注释 Annotation
CHI3L3	6.53	5.57E-07	几丁质酶3-3 Chitinase 3-like 3
CCL8	4.84	4.42E-06	C-C基序趋化配体8 C-C motif chemokine ligand 8
RETNLA	4.82	2.85E-05	抵抗素-α Resistin like alpha
SLPI	3.31	2.58E-04	分泌白细胞肽酶抑制剂 Secretory leukocyte peptidase inhibitor
CCL12	2.77	3.80E-04	趋化因子(C-C基序)配体12 Chemokine (C-C motif) ligand 12
CLEC4D	2.04	6.35E-04	c型凝集素域家族4成员D C-type lectin domain family 4 member D
EAR11	2.02	3.45E-04	嗜酸粒细胞相关,核糖核酸酶A家族,成员11 Eosinophil-associated, ribonuclease A family, member 11
UBD	1.93	6.82E-04	泛素D Ubiquitin D
CCL17	1.57	7.62E-04	C-C基序趋化配体17 C-C motif chemokine ligand 17
HSD3B4	-4.66	1.46E-05	3-和甾体德尔塔-异构酶4 3 beta-and steroid delta-isomerase 4
SULT5A1	-3.55	2.34E-04	磺基转移酶家族5A,成员1 Sulfotransferase family 5A, member 1
EGFR	-3.06	1.66E-04	表皮生长因子受体 Epidermal growth factor receptor
A1BG	-2.97	3.78E-04	alpha-1-B糖蛋白 Alpha-1-B glycoprotein
CYP17A1	-2.64	7.22E-05	细胞色素P450家族17亚家族A成员1 Cytochrome P450 family 17 subfamily A member 1
AHSG	-2.27	1.14E-04	α2-HS糖蛋白 Alpha 2-HS glycoprotein
SERPINA1D	-2.25	2.64E-04	丝氨酸（或半胱氨酸）肽酶抑制剂,分支A,成员1D Serine (or cysteine) peptidase inhibitor, clade A, member 1D
APOE	-2.23	1.88E-04	载脂蛋白e Apolipoprotein E
ALB1	-1.80	6.16E-04	阿宾娜1 ALBINA 1
NSG1	-1.79	7.12E-04	神经元小泡转运相关1 Neuronal vesicle trafficking associated 1
2310005P05RIK	-1.62	6.78E-04	内切酶/外切酶/磷酸酶家族含域蛋白1 Endonuclease/exonuclease/phosphatase family domain containing 1
FBP1	-1.54	8.26E-04	果糖二磷酸酶1 Fructose-bisphosphatase 1

表2

图1

图2

图3

参考文献 16

[1]	Cadavid Restrepo AM, Yang YR McManus DP, et al. The landscape epidemiology of echinococcoses[J]. Infect Dis Poverty, 2016,5:13.
[2]	Nunnari G, Pinzone MR, Gruttadauria S, et al. Hepatic echinococcosis: clinical and therapeutic aspects[J]. World J Gastroenterol, 2012,18(13):1448-1458.
[3]	Xu XL, Wang ZX, Wang Z, et al. Treatment of complicated hepatic alveolar echinococcosis: our experience of 98 cases[J]. Chin J Parasitol Parasit Dis, 2018,36(6):552-559. (in Chinese)
[3]	( 许晓磊, 王志鑫, 王展, 等. 98例复杂性肝多房棘球蚴病外科治疗方案的比较分析[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(6):552-559.)
[4]	Wen H, Vuitton L, Tuxun T, et al. Echinococcosis: advances in the 21st century[J]. Clin Microbiol Rev, 2019,32(2):e00075-18.
[5]	Xie WJ, Lv GD, Li Y, et al. Gene expression profile analysis of the liver in BALB/C mice infected with Echinococcus multilocularis by cDNA microarray[J]. Chin J Exp Surg, 2010,27(2):189-191. (in Chinese)
[5]	( 谢文娟, 吕国栋, 李瑶, 等. 泡球蚴感染小鼠所致肝损伤基因表达谱的变化[J]. 中华实验外科杂志, 2010,27(2):189-191.)
[6]	Wang L. Study on form mechanism of fibrosis capsule wall around Hepatichydaid[D]. Shihezi: Shihezi University, 2010. ( in Chinese)
[6]	( 王蕾. 肝细粒棘球蚴周围纤维囊壁形成机制的初步探索[D]. 石河子: 石河子大学, 2010.)
[7]	Pan W, Zhang YM, Sun FF, et al. Changes of phenotype and phagocytosis of peritoneal macrophages in mice infected with the larval-stage of Echinococcus granulosus[J]. Chin J Parasitol Parasit Dis, 2016,34(4):315-318. (in Chinese)
[7]	( 潘伟, 张玉梅, 孙芬芬, 等. 细粒棘球蚴感染小鼠腹腔巨噬细胞表型及吞噬功能的变化[J]. 中国寄生虫学与寄生虫病杂志, 2016,34(4):315-318.)
[8]	Liu HD, Wang HB, Fan HN, et al. Alveolar echinococcosis and immune evasion[J]. Chin J Parasitol Parasit Dis, 2018,36(6):655-660. (in Chinese)
[8]	( 刘寒冬, 王宏宾, 樊海宁, 等. 多房棘球蚴病的免疫逃避机制[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(6):655-660.)
[9]	Schuster S, Hurrell B, Tacchini-Cottier F. Crosstalk between neutrophils and dendritic cells: a context-dependent process[J]. J Leukoc Biol, 2013,94(4):671-675.
[10]	Cotterell SE, Engwerda CR, Kaye PM. Enhanced hematopoietic activity accompanies parasite expansion in the spleen and bone marrow of mice infected with Leishmania donovani[J]. Infect Immun, 2000,68(4):1840-1848.
[11]	Liu J, Cen JJ, Lv ZY, et al. The chemokine CCL8 and its progress in disease research[J]. J Trop Med, 2015,15(9):1287-1291. (in Chinese)
[11]	( 刘记, 岑俊杰, 吕志跃, 等. 趋化因子CCL8及其在疾病研究中的进展[J]. 热带医学杂志, 2015,15(9):1287-1291.)
[12]	Liu K, Jin M, Ye S, et al. CHI3L1 promotes proliferation and improves sensitivity to cetuximab in colon cancer cells by down-regulating p53[J]. J Clin Lab Anal, 2020,34(1):e23026.
[13]	Libreros S, Iragavarapu-Charyulu V. YKL-40/CHI3L1 drives inflammation on the road of tumor progression[J]. J Leukoc Biol, 2015,98(6):931-936.
[14]	Fantino E, Gangell CL, Hartl D, et al. Airway, but not serum or urinary, levels of YKL-40 reflect inflammation in early cystic fibrosis lung disease[J]. BMC Pulm Med, 2014,14:28.
[15]	Burman J, Raininko R, Blennow K, et al. YKL-40 is a CSF biomarker of intrathecal inflammation in secondary progressive multiple sclerosis[J]. J Neuroimmunol, 2016,292:52-57.
[16]	Tanaka Y, Matsumoto I, Inoue A, et al. Antigen-specific over-expression of human cartilage glycoprotein 39 on CD4⁺CD25⁺ forkhead box protein 3⁺ regulatory T cells in the generation of glucose-6-phosphate isomerase-induced arthritis [J]. Clin Exp Immunol, 2014,177(2):419-427.

基因名称Gene name	引物序列（5′ →3′） Primer sequence (5′ →3′)
CCL8	F：AACATGAAGGTCTCCGCTGG
	R：GCAGCAGGTGATTGGGGTAG
CHI3L1	F：GTTTTCCCAGTCACGAC
	R：CAGGAAACAGCTATGAC
β-actin	F：TTAGTTGCGTTACACCCTTTC
	R：ACCTTCACCGTTCCAGTTT