铁皮石斛多糖对人脐静脉内皮细胞基因表达谱的影响研究
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摘要
目的:探讨铁皮石斛多糖对人脐静脉内皮细胞(HUVEC)基因表达谱的影响。方法:以HUVEC为对象,采用MTS法检测不同剂量铁皮石斛多糖(50、100、200、400、800μg/mL)对HUVEC细胞增殖活性的影响,并计算30%细胞生长抑制浓度(IC30)以筛选后续试验剂量;采用基因表达谱芯片技术检测铁皮石斛多糖作用24 h后HUVEC细胞基因表达谱的变化情况,筛选差异表达基因;利用DAVID生物信息学资源数据库对差异表达前5位的基因进行GO富集分析和KEGG通路富集分析;以免疫相关差异表达基因为对象,采用实时荧光定量聚合酶链反应法(qRT-PCR)对芯片检测结果进行验证。结果:经100、200、400、800μg/mL铁皮石斛多糖作用后,HUVEC细胞的存活率均显著降低(P<0.05或P<0.01);IC30值为408μg/mL。经400μg/mL(据IC30值确定)铁皮石斛多糖作用后,HUVEC细胞中共有差异表达基因91个,其中表达上调的有84个、表达下调的有7个。上、下调表达差异前5位的基因分别为SELE、CCL2、CXCL6、IL8、ICAM1以及VWCE、CPT1A、CLU、CCL14、CINS4,主要与免疫调节、炎症反应等有关。HUVEC细胞中的差异表达基因主要分布于胞外区域,富集于细胞因子的产生及反应、刺激反应等生物学过程,发挥趋化因子及其受体活性等分子功能;其中上调表达基因SELE、ICAM1、CXCL2等主要富集于肿瘤坏死因子(TNF)信号通路、甲型H1N1流感、单纯疱疹病毒感染等通路,下调表达基因CCL14主要富集于趋化因子信号通路。qRT-PCR验证结果显示,ICAM1基因的相对表达量显著升高,CCL14基因的相对表达量显著降低(P<0.05),与芯片检测结果一致。结论:经铁皮石斛多糖处理后,HUVEC细胞共有91个基因表达差异明显,且以表达上调为主。表达差异基因可能通过TNF信号通路、甲型H1N1流感、单纯疱疹病毒感染等通路参与机体免疫调节。
OBJECTIVE:To investigate the effects of Dendrobium officinale polysaccharides on gene expression profile of HUVEC. METHODS:HUVEC was selected as objects. MTS method was used to detect the effects of different doses of D.officinale polysaccharides(50,100,200,400,800 μg/mL) on the proliferation activity of HUVEC. The growth inhibitory concentration of 30% cells(IC30)was calculated to screen the dose of follow-up tests. cDNA microarray assay was used to detect the changes of gene expression profile for HUVEC after treated with D. officinale polysaccharides for 24 h,so as to screen differentially expressed genes. GO enrichment analysis and KEGG pathway enrichment analysis were performed for top 5 differentially expressed genes by using DAVID bioinformatics resource database. Real-time fluorescence quantitative polymerase chain reaction(q RT-PCR) was used to validate the results of microarray detection with immunity-related differentially expressed genes as objects. RESULTS:After treated with 100,200,400,800 μg/mL D. officinale polysaccharides,survival rate of HUVEC were decreased significantly(P<0.05 or P<0.01). IC30 value was 408 μg/mL. After treated with 400 μg/mL(by IC30)D. officinale polysaccharides,there were 91 differentially expressed genes in HUVEC cells,of which 84 were up-regulated and 7 were down-regulated. Top 5 genes of up-regulated and down-regulated expression were SELE,CCL2,CXCL6,IL8,ICAM1 as well as VWCE,CPT1 A,CLU,CCL14,CINS4,which may be mainly associated with immune conditions and inflammatory responses. The differentially expressed genes mainly distributed in extracellular domain, and were enriched in biological processes such as production and response of cytokines and stimulus response,and played molecular functions such as chemokine and its receptor activity. The up-regulated genes as SELE,ICAM1 and CXCL2 were mainly enriched in TNF signaling pathway,influenza A(H1 N1),herpes simplex virus infection and other pathways. The down-regulated gene CCL14 was mainly enriched in chemokine signaling pathway. Results of qRT-PCR validation tests showed that relative expression of ICAM1 was increased significantly,while that of CCL14 was decreased significantly(P<0.05),which was in agreement with microarray detection results. CONCLUSIONS:After treated with D. officinale polysaccharides,the expression of 91 genes in HUVEC cells are different significantly,mainly being up-regulated. The differentially expressed genes may participate in immune regulation through TNF signaling pathway,influenza A(H1 N1)and herpes simplex virus infection.
OBJECTIVE:To investigate the effects of Dendrobium officinale polysaccharides on gene expression profile of HUVEC. METHODS:HUVEC was selected as objects. MTS method was used to detect the effects of different doses of D.officinale polysaccharides(50,100,200,400,800 μg/mL) on the proliferation activity of HUVEC. The growth inhibitory concentration of 30% cells(IC30)was calculated to screen the dose of follow-up tests. cDNA microarray assay was used to detect the changes of gene expression profile for HUVEC after treated with D. officinale polysaccharides for 24 h,so as to screen differentially expressed genes. GO enrichment analysis and KEGG pathway enrichment analysis were performed for top 5 differentially expressed genes by using DAVID bioinformatics resource database. Real-time fluorescence quantitative polymerase chain reaction(q RT-PCR) was used to validate the results of microarray detection with immunity-related differentially expressed genes as objects. RESULTS:After treated with 100,200,400,800 μg/mL D. officinale polysaccharides,survival rate of HUVEC were decreased significantly(P<0.05 or P<0.01). IC30 value was 408 μg/mL. After treated with 400 μg/mL(by IC30)D. officinale polysaccharides,there were 91 differentially expressed genes in HUVEC cells,of which 84 were up-regulated and 7 were down-regulated. Top 5 genes of up-regulated and down-regulated expression were SELE,CCL2,CXCL6,IL8,ICAM1 as well as VWCE,CPT1 A,CLU,CCL14,CINS4,which may be mainly associated with immune conditions and inflammatory responses. The differentially expressed genes mainly distributed in extracellular domain, and were enriched in biological processes such as production and response of cytokines and stimulus response,and played molecular functions such as chemokine and its receptor activity. The up-regulated genes as SELE,ICAM1 and CXCL2 were mainly enriched in TNF signaling pathway,influenza A(H1 N1),herpes simplex virus infection and other pathways. The down-regulated gene CCL14 was mainly enriched in chemokine signaling pathway. Results of qRT-PCR validation tests showed that relative expression of ICAM1 was increased significantly,while that of CCL14 was decreased significantly(P<0.05),which was in agreement with microarray detection results. CONCLUSIONS:After treated with D. officinale polysaccharides,the expression of 91 genes in HUVEC cells are different significantly,mainly being up-regulated. The differentially expressed genes may participate in immune regulation through TNF signaling pathway,influenza A(H1 N1)and herpes simplex virus infection.
引文
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[12]HUANG X,NIE S,CAI H,et al.Study on Dendrobium officinale O-acetyl-glucomannan(Dendronan?):PartⅥ:protective effects against oxidative stress in immunosuppressed mice[J].Food Res Int,2015.DOI:10.1016/j.foodres,2015.01.035.
[13]石梅兰.铁皮石斛对高糖环境下HUVECs氧化应激损伤的保护作用[D].衡阳:南华大学,2012.
[14]张红玉,戴关海,马翠,等.铁皮石斛多糖对S180肉瘤小鼠免疫功能的影响[J].浙江中医杂志,2009,44(5):380-381.
[15]SUN XL,LAW BY,DE SEABRA RODRIGUES DIASIR,et al.Pathogenesis of thromboangiitis obliterans:gene polymorphism and immunoregulation of human vascular endothelial cells[J].Atherosclerosis,2017.DOI:10.1016/j.atherosclerosis.2017.08.009.
[16]Parameswaran N,Suresh R,Bal V,et al.Lack of ICAM-1on APCs during T cell priming leads to poor generation of central memory cells[J].J Immunol,2005,175(4):2201-2211.
[2]HUANG K,LI Y,TAO S,et al.Purification,characterization and biological activity of polysaccharides from Dendrobium officinale[J].Molecules,2016.DOI:10.3390/molecules20160701.
[3]LIN X,SHAW PC,SZE SC,et al.Dendrobium officinale polysaccharides ameliorate the abnormality of aquaporin5,pro-inflammatory cytokines and inhibit apoptosis in the experimental Sj?gren’s syndrome mice[J].Int Immunopharmacol,2011,11(12):2025-2032.
[4]XIANG L,STEPHEN SZE CW,NG TB,et al.Polysaccharides of dendrobium officinale inhibit TNF-α-induced apoptosis in A-253 cell line[J].Inflamm Res,2012,26(3):313-324.
[5]何小群,黄锁义,肖刚,等.铁皮石斛多糖对癫痫大鼠海马NF-κB和IL-10表达的影响[J].中华中医药学刊,2018,36(7):123-125.
[6]程京,邢婉丽.生物芯片技术[M].北京:清华大学出版社,2004:291-303.
[7]丁艳.铁皮石斛对高糖环境下人脐静脉内皮细胞产生NO、TNF-Α和ICAM-1的影响[D].衡阳:南华大学,2013.
[8]BRAZMA A,VILO J.Gene expression data analysis[J].Microbes Infect,2001,3(10):823-829.
[9]YANAIHARA N,HIRATA Y,YAMAGUCHI N,et al.Antitumor effects of interleukin-6(IL-6)/interleukin-6 receptor(IL-6R)signaling pathway inhibition in clear cell carcinoma of the ovary[J].Mol Carcinog,2016,55(5):832-841.
[10]HUBBARD AK,ROTHLEIN R.Intercellular adhesion molecule-1(ICAM-1)expression and cell signaling cascades[J].Free Radic Biol Med,2000,28(9):1379-1386.
[11]YI L,ZHENG Y,LI T,et al.Chemokines CCL2,3,14stimulate macrophage bone marrow homing,proliferation and polarization in multiple myeloma[J].Oncotarget,2015,6(27):24218-24229.
[12]HUANG X,NIE S,CAI H,et al.Study on Dendrobium officinale O-acetyl-glucomannan(Dendronan?):PartⅥ:protective effects against oxidative stress in immunosuppressed mice[J].Food Res Int,2015.DOI:10.1016/j.foodres,2015.01.035.
[13]石梅兰.铁皮石斛对高糖环境下HUVECs氧化应激损伤的保护作用[D].衡阳:南华大学,2012.
[14]张红玉,戴关海,马翠,等.铁皮石斛多糖对S180肉瘤小鼠免疫功能的影响[J].浙江中医杂志,2009,44(5):380-381.
[15]SUN XL,LAW BY,DE SEABRA RODRIGUES DIASIR,et al.Pathogenesis of thromboangiitis obliterans:gene polymorphism and immunoregulation of human vascular endothelial cells[J].Atherosclerosis,2017.DOI:10.1016/j.atherosclerosis.2017.08.009.
[16]Parameswaran N,Suresh R,Bal V,et al.Lack of ICAM-1on APCs during T cell priming leads to poor generation of central memory cells[J].J Immunol,2005,175(4):2201-2211.