[1]龙禹哲,郭鸿浩,杨双亚,等.骨髓间充质干细胞源外泌体对大鼠颈动脉球囊损伤后lncRNAs表达的影响[J].第三军医大学学报,2020,42(23):2320-2329.
 LONG Yuzhe,GUO Honghao,YANG Shuangya,et al.Effect of BMSCs-derived exosomes on expression of lncRNAs in rat carotid artery after balloon injury[J].J Third Mil Med Univ,2020,42(23):2320-2329.
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骨髓间充质干细胞源外泌体对大鼠颈动脉球囊损伤后lncRNAs表达的影响(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
42卷
期数:
2020年第23期
页码:
2320-2329
栏目:
基础医学
出版日期:
2020-12-15

文章信息/Info

Title:
Effect of BMSCs-derived exosomes on expression of lncRNAs in rat carotid artery after balloon injury
作者:
龙禹哲郭鸿浩杨双亚陈文明邓文文石蓓
遵义医科大学附属医院心内科
Author(s):
LONG Yuzhe GUO Honghao YANG Shuangya CHEN Wenming DENG Wenwen SHI Bei
Department of Cardiology, Affiliated Hospital of Zunyi Medical University, Zunyi, Guizhou Province, 563000, China
 
关键词:
骨髓间充质干细胞外泌体颈动脉损伤长链非编码RNA
Keywords:
bone marrow mesenchymal stem cells exosomes carotid artery injury long non-coding RNA
分类号:
R361.1; R394.3; R543.402
文献标志码:
A
摘要:

目的探讨骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)源外泌体(exosomes)对大鼠颈动脉球囊损伤后内膜增生的调控作用及其长链非编码RNA(long non-coding RNAs,lncRNAs)表达的影响。方法采用差速超速离心法提取大鼠BMSCs源外泌体,并通过Western blot和透射电镜对其进行鉴定。SD大鼠分为3组:①假手术组(只分离左颈总动脉血管);②损伤组(左颈总动脉采用球囊损伤);③外泌体组(建立大鼠颈动脉球囊损伤模型后原位注射0.25 μg/μL外泌体400 μL),14 d后采用HE染色观测颈动脉内膜/中膜面积比(intima/media,I/M)、免疫组化检测PCNA表达、Western blot检测血管内膜增殖及表型转化相关指标的变化。采用lncRNA-mRNA共表达谱芯片表达变化及其生物信息学分析筛查假手术组和损伤组大鼠颈动脉组织中差异表达的lncRNAs和mRNAs,对其潜在分子机制进行探讨。采用qRT-PCR检测各组颈动脉组织中候选lncRNAs的表达。结果电镜结果显示BMSCs源外泌体呈现“茶杯垫样”改变,Western blot结果显示所提取的外泌体中CD63、Alix、HSP70蛋白表达阳性。与损伤组相比,经BMSCs源外泌体处理后大鼠颈动脉I/M显著减少(P<0.05),增殖相关蛋白PCNA、Cyclin D1表达下调(P<0.05)。lncRNA-mRNA共表达谱芯片检测结果,与假手术组相比,损伤组有1 083个lncRNAs表达上调,629个lncRNAs表达下调,2 004个mRNAs表达上调,2 487个mRNAs表达下调(Fold Change>2.0,P<0.05)。GO和KEGG通路分析结果显示,多条lncRNAs与mRNAs相互关联,通过调控相应的靶mRNAs,参与血管平滑肌收缩、肌动蛋白细胞骨架调节和细胞周期等多种生物学过程,与球囊损伤后血管再狭窄的机制有关。结合生物信息学分析,采用qRT-PCR检测3组大鼠颈动脉组织中上述差异性表达最为显著的7个lncRNAs,结果表明,与损伤组相比,外泌体组XR_593268上调(P<0.05),XR_602080、XR_602012、XR_591017均下调(P<0.05)。结论BMSCs源外泌体抑制大鼠颈动脉球囊损伤后内膜增生,其潜在机制可能与XR_602080、XR_591017通过Gsk3b、Cdkn2b、Ccna2等分子调控平滑肌细胞周期变化有关。

Abstract:

ObjectiveTo investigate the regulatory effect of bone marrow mesenchymal stem cells (BMSCs) derived exosomes on intimal hyperplasia and expression of long non-coding RNAs (lncRNAs) in rat model of carotid artery balloon injury and its potential mechanism. MethodsRat BMSCs-derived exosomes were extracted from male SD rats by differential centrifugation, and then identified by Western blotting and transmission electron microscopy. Then a total of 36 male SD rats were randomly divided into sham-operation group, model group (carotid artery balloon injury), and model+exosome treatment group (in situ injection of 400 μL  0.25 μg/μL exosomes), with 12 rats in each group. In 14 d after the treatment, all rats were sacrificed to harvest the carotid artery. HE staining was used to observe the intima/media (I/M) area ratio of carotid artery. Immunohistochemical assay was used to detect the expression of PCNA, and Western blotting was applied for the expression of the proteins involved in the intimal proliferation and phenotypic transformation. LncRNA-mRNA co-expression microarray and bioinformatics analysis were employed to analyze the differentially expressed lncRNAs and mRNAs in carotid artery tissues of the rats from the sham operation group and the model group for the potential molecular mechanism. qRT-PCR was performed to detect the expression of candidate lncRNAs in the carotid artery tissues. ResultsTransmission electron microscopy displayed that the BMSCs-derived exosomes presented “coaster-like” changes. Western blotting results confirmed that CD63, Alix and HSP70 were expressed in the extracted exosomes. The treatment of BMSCs-derived exosomes resulted in significant reduce in the I/M area ratio of carotid artery (P<0.05) and decreased expression of proliferation-related proteins PCNA and Cyclin D1 (P<0.05). The results of lncRNA-mRNA co-expression microarray showed that there were 1 083 up- and 629 down-regulated lncRNAs, and 2 004 up- and 2 487 down-regulated mRNA (Fold change>2.0, P<0.05) in the model group when compared with the sham operation group. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that these multiple lncRNAs were correlated with mRNAs, and the corresponding target mRNAs are involved in many biological processes, such as vascular smooth muscle contraction, actin cytoskeleton regulation and cell cycle, which are related to the mechanism of vascular restenosis after balloon injury. In combination with bioinformatics analysis, qRT-PCR detected 7 lncRNAs with significant differential expression, and the results showed that XR_593268 was up-regulated (P<0.05), and XR_602080, XR_602012 and XR_591017 were down-regulated (P<0.05) in the exosome treatment group compared with the model group. ConclusionThe BMSCs-derived exosomes inhibit intimal hyperplasia in rats after balloon injury of carotid artery. The potential mechanism may be related to the regulation of smooth muscle cell cycle changes by lncRNAs XR_602080 and XR_591017 through Gsk3b, Cdkn2b and Ccna2.

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更新日期/Last Update: 2020-12-04