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中华妇幼临床医学杂志(电子版) ›› 2018, Vol. 14 ›› Issue (03) : 260 -269. doi: 10.3877/cma.j.issn.1673-5250.2018.03.003

所属专题: 文献

论著

乙酰肝素酶调控滋养细胞中细胞因子表达及其对滋养细胞作用的潜在分子机制预测
车光璐1, 王艳云2, 张林1,()   
  1. 1. 610041 成都,四川大学华西基础医学与法医学院免疫学教研室;610041 成都,四川大学华西第二医院分子与转化医学实验室
    2. 610041 成都,四川大学华西第二医院分子与转化医学实验室
  • 收稿日期:2017-12-22 修回日期:2018-02-12 出版日期:2018-06-01
  • 通信作者: 张林

Regulation of heparanase on the expression of cytokines and the prediction of its molecular mechanism in trophoblast cells

Guanglu Che1, Yanyun Wang2, Lin Zhang1,()   

  1. 1. Department of Immunology, West China School of Preclinical & Forensic Medicine, Sichuan University, Chengdu 610041, Sichuan Province, China; Laboratory of Molecular Translational Medicine, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
    2. Laboratory of Molecular Translational Medicine, West China Second University Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China
  • Received:2017-12-22 Revised:2018-02-12 Published:2018-06-01
  • Corresponding author: Lin Zhang
  • About author:
    Corresponding author: Zhang Lin, Email:
引用本文:

车光璐, 王艳云, 张林. 乙酰肝素酶调控滋养细胞中细胞因子表达及其对滋养细胞作用的潜在分子机制预测[J/OL]. 中华妇幼临床医学杂志(电子版), 2018, 14(03): 260-269.

Guanglu Che, Yanyun Wang, Lin Zhang. Regulation of heparanase on the expression of cytokines and the prediction of its molecular mechanism in trophoblast cells[J/OL]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2018, 14(03): 260-269.

目的

探究过表达滋养细胞中乙酰肝素酶(HPSE)对细胞因子表达的影响,并且初步预测HPSE对滋养细胞作用的潜在分子机制。

方法

选择早孕期人绒毛膜滋养细胞株HTR8/SVneo为研究对象,并构建HPSE过表达稳定转染HTR8/SVneo滋养细胞株,纳入检验组,以及HPSE正常表达稳定转染HTR8/SVneo滋养细胞株,纳入对照组。采用细胞因子抗体芯片半定量检测,对检验组和对照组滋养细胞株中343种细胞因子的变化情况进行检测。应用Image J软件分析和找出差异细胞因子蛋白。采用Western blotting检测2组滋养细胞株中表达水平差异最大的细胞因子AXL受体酪氨酸激酶表达水平,以验证细胞因子抗体芯片半定量检测结果。通过STRING蛋白数据库构建蛋白相互作用关系网络,并应用The Database for Annotation, Visualization and Integrated Discovery (DAVID)在线生物信息学分析软件,进行基因本体(GO)功能富集分析和京都基因与基因组百科全书(KEGG)信号通路分析。

结果

①细胞因子抗体芯片半定量检测结果显示,与对照组滋养细胞株相比,检验组滋养细胞株过表达HPSE后,其细胞因子表达下降,并且检验组与对照组滋养细胞株中细胞因子蛋白表达水平比值<0.666 7的细胞因子,即差异细胞因子共计15种,分别为AXL、细胞间黏附分子(ICAM)1、TEK受体酪氨酸激酶、尿激酶型纤溶酶原激活物受体(PLAUR)、金属蛋白酶组织抑制因子(TIMP)2、基质金属蛋白酶(MMP)9、淋巴细胞激活基因(LAG)3、肿瘤坏死因子受体超家族成员(TNFRSF)1B、TNFRSF1A、细胞毒性和调节性T细胞分子(CRTAM)、TIMP4、人血小板反应蛋白(THBS)1、白细胞介素17受体B(IL17RB)、连接黏附分子样蛋白(AMICA)及C-C类趋化因子16(CCL16)。②Western blotting检测结果显示,检验组滋养细胞株中AXL蛋白表达水平明显低于对照组滋养细胞株,并且差异有统计学意义(t=-6.931,P=0.020),这与细胞因子抗体芯片半定量检测结果一致。③通过STRING蛋白数据库分析结果发现,HPSE可通过血管内皮生长因子(VEGF)A,肿瘤蛋白p53(TP53)和CD44与MMP9产生关联,而MMP9又可直接或者间接与除LAG3、CCL16和IL17RB之外的其他12种差异细胞因子相关联,从而建立相互作用关系网络图。④细胞因子GO功能富集分析结果发现,差异细胞因子主要涉及细胞凋亡负调控和细胞外基质(ECM)分解等41个生物学过程。KEGG信号通路分析结果显示,差异细胞因子主要涉及癌症蛋白多糖信号通路和肿瘤坏死因子信号通路等6条信号通路。

结论

过表达HPSE可调控滋养细胞中细胞因子的表达,可能通过MMP9参与的癌症蛋白多糖信号通路影响滋养细胞的生物学功能。本研究结果可为后续研究HPSE对滋养细胞相关疾病影响,提供研究方向和研究基础。

Objective

To explore the influence of heparanase (HPSE) on the expression of cytokines and predict its underlying molecular mechanism in trophoblast cells.

Methods

The human first-trimester extravillous trophoblast cell line HTR8/SVneo cells were selected as research subjects of this study. And a HPSE over-expression stably transfected HTR8/SVneo cell line and a HPSE normal-expression stably transfected HTR8/SVneo cell line were constructed. They were enrolled into test group and control group, respectively. And then 343 cytokines were semi-quantitatively detected using human cytokine antibody array kit and analyzed by Image J software to discover differential cytokine proteins in trophoblast cell lines of two groups. Western blotting was performed to validate the semi-quantitative results of cytokine antibody array by detecting AXL receptor tyrosine kinase, which had the most significant difference between trophoblast cell lines of two groups. The protein-protein interaction network was constructed through STRING protein online database, and the gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed by The Database for Annotation, Visualization and Integrated Discovery (DAVID) online bioinformatics resources.

Results

①The semi-quantitative results of cytokine microarray showed that levels of 15 cytokines, including AXL, intercellular adhesion molecule (ICAM)1, TEK receptor tyrosine kinase, urokinase-type plasminogen activator receptor (PLAUR), tissue inhibitor of metalloproteinase (TIMP)2, matrix metalloprotein (MMP)9, lymphocyte-activation gene (LAG)3, tumor necrosis factor receptor superfamily (TNFRSF)1B, TNFRSF1A, cytotoxic and regulatory T cell molecule (CRTAM), tissue inhibitor of metalloproteinase (TIMP)4, human platelet reactive protein (THBS)1, interleukin 17 receptor B (IL17RB), junctional adhesion molecule-like protein (AMICA), and C-C chemotactic factor 16 (CCL16) were decreased in HPSE-overexpressed trophoblast cell line of test group, compared with the control trophoblast cell line of control group. And the ratio of levels of differential cytokines in trophoblast cell line between test group and control group was less than 0.666 7.②The result of Western blotting showed that the level of AXL in HPSE-overexpressed trophoblast cell line of test group was obviously lower than that in trophoblast cell line of control group, and the difference was statistically significant (t=-6.931, P=0.020), which was consistent with the semi-quantitative results of cytokines microarray. ③HPSE was associated with MMP9 by vascular endothelial growth factor (VEGF)A, tumor protein p53 (TP53) and CD44 by STRING protein online database. And MMP9 was directly or indirectly associated with other 12 types of differential cytokines except LAG3, CCL16 and IL17RB to establish an interaction network. ④GO enrichment analysis of cytokines showed that the differential cytokines were mainly involved in 41 biological processes, including negative regulation of apoptosis and extracellular matrix (ECM) disassembly. Results of KEGG signaling pathway analysis showed that the differential cytokines were mainly involved in 6 signaling pathways, including proteoglycan in cancer signaling pathway and tumor necrosis factor signaling pathway.

Conclusions

Overexpression of HPSE may regulate the expression of cytokines in trophoblast cells. And it may affect the biological functions of trophoblast cells through the proteoglycan in cancer signaling pathway, which MMP9 is involved in. This study can provide a research direction and basis for further study of the influences of HPSE in trophoblast cells associated diseases.

表1 细胞因子蛋白的细胞因子抗体芯片半定量检测结果分析
图1 细胞因子抗体芯片半定量检测结果图
图2 AXL蛋白蛋白质印迹法检测结果(图2A:AXL蛋白蛋白质印迹法检测图;图2B:2组AXL蛋白采用蛋白质印迹法检测的相对表达水平柱状图)
图3 乙酰肝素酶与15种差异细胞因子的相互作用网络图(图3A:HPSE与15种差异细胞因子的直接关系图;图3B:HPSE通过VEGFA、TP53和CD44与15种差异细胞因子的间接关系图)
图4 细胞因子GO功能富集和KEGG信号通路分析结果图(图4A:GO生物学功能分析;图4B:KEGG信号通路分析)
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