| 陈江,张兵,冯燕,等.白及提取物对流感病毒感染MDCK细胞基因表达的干预研究[J].浙江中医药大学学报,2019,43(5):481-492. |
| 白及提取物对流感病毒感染MDCK细胞基因表达的干预研究 |
| Intervention Study of the Extracts from the Bletilla Striata on Gene Expression of MDCK Cells Infected with Influenza Virus |
| DOI：10.16466/j.issn1005-5509.2019.05.022 |
| 中文关键词: 白及 流感病毒H3N2 MDCK细胞 基因表达 |
| 英文关键词: Bletilla striata H3N2 MDCK cells gene expression |
| 基金项目:浙江省科技厅公益类项目(2015C33113) |
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| 中文摘要: |
| [目的]研究白及提取物对流感病毒感染的MDCK细胞基因表达的影响，探讨以宿主细胞为靶点的抗流感病毒作用机制。[方法] 建立甲型流感病毒H3N2感染的MDCK细胞模型，以白及提取物进行干预。Trizol法提取细胞总RNA，经琼脂糖甲醛变性凝胶电泳检测其完整性后，利用基因表达谱芯片进行表达谱分析，与未加药物的病毒对照组进行表达谱散点图比对，采用blindDescription统计学模型，以变化倍数(fold change，FC)≥2为上调基因，≤0.5为下调基因。对差异表达的基因按照细胞组分、分子功能、生物学过程进行GO功能聚类分析，并采用KEGG数据库分析差异基因在甲型流感病毒通路、磷脂酰肌醇-3-激酶-蛋白激酶B(phosphoinositide-3-kinase-protein kinase B，PI3K-AKT)信号通路、视黄酸诱导基因-1样受体(retinoic acid inducible gene-1-like receptor，RIG-1-like)信号通路中的定位。对表达具有显著差异的基因，以荧光定量PCR对基因表达情况进行验证。[结果] 经白及提取物干预后，流感病毒感染的MDCK细胞中856个基因表达上调，占基因总数的2.62%；1 158个基因下调，占基因总数的3.54%。GO聚类分析结果显示差异基因均涉及细胞的生理功能。KEGG分析显示，甲型流感病毒通路中热休克蛋白70(heat-shock proteins 70，HSP70)基因等11个差异基因上调，黑色素瘤分化相关抗原5(melanoma differentiation-associated antigen 5，MDA5)基因等15个基因下调；PI3K-AKT信号通路中受体酪氨酸激酶(receptor tyrosine kinase，RTK)、磷脂酰肌醇-3-激酶(phosphoinositide-3-kinase，PI3K)等15个基因上调；RIG-1-like信号通路中MDA5、RIG-1等9个基因下调。荧光定量PCR显示，与病毒对照组比较，白及提取物作用后MHC Ⅱ类转录激活因子(MHC class Ⅱ transactivator,CⅡTA)、HSP70、PI3K、核因子κB抑制因子(inhibitor of nuclear factor κB, IκB)等基因表达上调，表达量分别提高 4.32倍、12.13倍、17.88倍、18.25倍；干扰素调节因子7(interferon regulatory factor 7,IRF7)、Toll样受体3(Toll-like receptor 3,TLR3)、RIG、MDA5、双链RNA依赖的蛋白激酶(double-stranded RNA-dependent protein kinase，PKR)等基因下调，分别下降2.50倍、1.83倍、20.53倍、2.37倍、6.23倍。荧光定量PCR和基因表达谱分析结果相符。[结论] 流感病毒感染MDCK细胞后，白及提取物可以通过调控宿主细胞信号通路的相关基因表达水平，从而发挥抗流感病毒效果。 |
| 英文摘要: |
| [Objective] To study the intervention of gene expression of MDCK cells infected with influenza virus A/Sydney/5/97(H3N2) by extracts from the Bletilla striata, and to furtherly discuss the mechanism of anti-influenza virus targeting host cells. [Methods] The MDCK cell model of influenza virus A/Sydney/5/97(H3N2) infection was established, and the extracts from the Bletilla striata were used to intervene. The total RNA was extracted by Trizol method, and its integrity was detected by formaldehyde denayured gel electrophoresis, and the chips were used to analyze gene expression profile, and the expression spectrum scatter plot was compared with the untreated virus control group. The blindDescription statistical model was used, and the fold change ≥2 was the up-regulated gene, and ≤0.5 is the down-regulated gene. The differentially expressed genes were clustered by GO function clustering analysis according to cell components, molecular functions and biological processes. And the KEGG database was used to analyze the positioning of differential genes in the influenza virus A pathway, phosphoinositide-3-kinase-protein kinase B(PI3K-AKT) signaling pathway and retinoic acid inducible gene-1-like receptor (RIG-1-like) signaling pathway. For the genes with significant differences expression, primers were designed to verify the expression of the gene by Real-time PCR. [Results] After influenza virus-infected MDCK cells were intervened with the extracts from the Bletilla striata. 856 genes were up-regulated, accounting for 2.62% of the total genes; 1 158 genes were down-regulated, accounting for 3.54% of the total genes; GO cluster analysis showed that differential genes were all involved the physiological function of the cell. The KEGG pathway map analysis showed that in the influenza virus A pathway, 11 differentially expressed genes such as heat-shock proteins 70(HSP70) were up-regulated, 15 genes such as melanoma differentiation-associated antigen 5(MDA5) were down-regulated. Fifteen genes including receptor tyrosine kinase (RTK) and PI3K were up-regulated in the PI3K-AKT signaling pathway. And 9 genes such as MDA5 and RIG-1 were down-regulated in the RIG-1-like signaling pathway. The results of Real-time PCR showed that compared with the virus control group, the expressions of MHC class Ⅱ transactivator(CⅡTA), HSP70, PI3K, inhibitor of nuclear factor κB(IκB) and other genes were up-regulated after the action of the extracts from the Bletilla striata, and the expression levels were increased by 4.32 times, 12.13 times, 17.88 times and 18.25 times, respectively. The genes such as interferon regulatory factor 7(IRF7), Toll-like receptor 3(TLR3), RIG, MDA5 and double-stranded RNA-dependent protein kinase(PKR) were down-regulated, and the expression levels were decreased by 2.50 times, 1.83 times, 20.53 times, 2.37 times and 6.23 times, respectively. The results of Real-time PCR and gene expression profiling were consistent.[Conclusion] After influenza virus infection in MDCK cells, the extracts from the Bletilla striata can exert anti-influenza virus effects by regulating the expression levels of related genes in the host cells’ signaling pathway. |
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