文章摘要
童朝刚,朱月琴,王亚男,潘学胜,芮晨辉,黄 艳.Psalmotoxin⁃1抑制活化的肝星状细胞过程中microRNA差异表达谱的变化分析[J].南京医科大学学报,2019,(4):513~519
Psalmotoxin⁃1抑制活化的肝星状细胞过程中microRNA差异表达谱的变化分析
Changes in differential expression profiles of microRNAs during the inhibition of activated hepatic stellate cells by psalmotoxin⁃1
投稿时间:2018-10-16  
DOI:10.7655/NYDXBNS20190409
中文关键词: 血小板衍生生长因子BB  微小RNA  肝星状细胞  高通量测序
英文关键词: platelet⁃derived growth factor⁃BB  microRNA  hepatic stellate cells  high⁃throughput sequencing
基金项目:安徽省自然科学基金杰青项目(1908085J30);安徽省高校自然科学研究项目(KJ2017A192);安徽省学术技术带头人后备人选科研活动经费资助项目(2015H040);高校优秀青年人才支持计划重点项目(gxyqZD2016049);安徽医科大学国家级大学生创新创业训练计划项目(201810366060);安徽医科大学校级青年拔尖人才支持计划(0601037104);青年英才双培工程(0601037206);临床医学(“5+3”一体化)专业“早期接触科研”训练计划立项制项目(2017?ZQKY?26)
作者单位
童朝刚 安徽医科大学附属巢湖医院普外科安徽 合肥 238000 
朱月琴 安徽医科大学药学院安徽 合肥 230032 
王亚男 安徽医科大学药学院安徽 合肥 230032 
潘学胜 安徽医科大学药学院安徽 合肥 230032 
芮晨辉 安徽医科大学药学院安徽 合肥 230032 
黄 艳 安徽医科大学药学院安徽 合肥 230032 
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中文摘要:
      目的:探讨酸敏感离子通道1a(acid?sensing ion channel 1a,ASIC1a)特异性阻断剂狼蛛毒素(psalmotoxin?1,PcTx?1)抑制血小板衍生生长因子BB(platelet derived growth factor?BB,PDGF?BB)活化的肝星状细胞(hepatic stellate cell,HSC)过程中微小RNA(microRNA,miRNA)差异表达谱及生物信息学分析。方法:取对数生长期的HSC细胞,分为对照组、模型组和PcTx组。对照组不做处理,模型组加入PDGF?BB(10 ng/mL)培养24 h,PcTx组给予PcTx?1刺激1 h后,再加入PDGF?BB(10 ng/mL)培养24 h。采用RT?PCR和Western blot检测α?平滑肌肌动蛋白(α?smooth muscle actin,α?SMA)、Ⅰ型胶原(collagen?Ⅰ)和ASIC1a的表达,MTT法检测细胞增殖情况,验证HSC细胞是否活化以及PcTx?1是否阻断ASIC1a的表达。提取的总RNA质检合格后进行高通量测序,筛选差异表达的miRNA。通过miRanda算法对差异miRNA进行靶基因预测,并对miRNA靶基因进行功能和代谢通路分析。结果:PcTx?1阻断ASIC1a后,ASIC1a的表达降低,HSC细胞活化的标志性基因α?SMA和collagen?Ⅰ的表达明显减少,提示PcTx?1可阻断PDGF诱导的HSC活化。与对照组比较,模型组miRNA表达谱中差异表达miRNA共有38个,其中上调6个,下调32个(P < 0.05)。与模型组比较,PcTx组miRNA表达谱中差异表达miRNA共有17个,其中上调1个,下调16个(P < 0.05)。结论:高通量筛选得到的PcTx?1抑制PDGF?BB活化HSC过程中miRNA差异表达谱,为肝纤维化的发病机制研究提供了新的靶点和思路。
英文摘要:
      Objective:To investigate the differential expression profile and bioinformatics analysis of microRNA(miRNA) in hepatic stellate cells(HSCs) activated by platelet?derived growth factor BB(PDGF?BB) inhibited by psalmotoxin?1(PcTx?1),blocker of acid?sensing ion channel 1a(ASIC1a). Methods:HSCs in logarithmic growth period were divided into the control group,the model group and the PcTx group. The control group was not treated,after the PcTx group was stimulated by PcTx?1 for 1 h,the model group and the PcTx group were cultured with PDGF?BB(10 ng/mL)medium for 24 h. RT?PCR and Western blot were used to detect the expression of α?smooth muscle actin(α?SMA),collagen?Ⅰ and ASIC1a,and MTT was used to detect the proliferation of cells. These results verified whether HSCs were activated and whether PcTx?1 reduced the expression of ASIC1a. High throughput sequencing of total RNA was carried out after quality control,and differential miRNAs were screened. The target genes of differential miRNAs were predicted by miRanda algorithm,then the functional significance of miRNA target genes and metabolic pathway involved in miRNA were analyzed. Results:After blocking ASIC1a by PcTx?1,the expression of ASIC1a decreased,and the expressions of α?SMA and collagen?Ⅰ in HSCs activation were obviously reduced. It suggested that PcTx?1 can block the activation of HSCs induced by PDGF?BB. In the miRNA expression profile of PDGF?stimulated HSCs,there were 38 differentially expressed miRNAs,of which 6 were up?regulated and 32 were down?regulated(P < 0.05). After ASIC1a was blocked by PcTx?1,there were 17 differentially expressed miRNAs in the miRNA expression profiles between the PcTx group and the model group,including 1 up?regulation and 16 down?regulation(P < 0.05). Conclusion:The differential expression profile of miRNA in HSCs activated by PDGF?BB inhibited by PcTx?1 in high?throughput screening,providing new targets and ideas for the study of pathogenesis of liver fibrosis.
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