【作者】 陈伟；

【导师】 唐举玉；

【作者基本信息】 中南大学 ， 外科学（专业学位）， 2023， 硕士

【摘要】 目的:本研究旨在揭示酒精性股骨头坏死(alcohol-induced necrosis of the femoral head,AIONFH)患者血浆外泌体中miRNA的表达谱,并进一步探讨酒精刺激肝细胞分泌的外泌体对于血管内皮细胞(vascular endothelial cells,VECs)的潜在影响。方法:收集中国男性人群(10名AIONFH患者和10名健康对照组)的血液样本。使用试剂盒法分别提取正常人血浆外泌体(NC-Exos)和AIONFH患者血浆外泌体(AIONFH-Exos),并从电镜、粒径分析、Western Blot三个方面进行外泌体鉴定。提取外泌体总RNA,然后使用Illumina进行高通量测序,并绘制差异表达的miRNAs的聚类图、火山图。利用Target Scan预测所有差异表达的miRNAs的靶基因,通过GO功能富集分析、KEGG通路富集分析描绘出这些miRNAs的生物学功能和通路富集图谱,最后使用qRT-PCR对部分差异表达的miRNAs进行验证。使用试剂盒法提取正常肝细胞外泌体(NC-Exos)和酒精刺激肝细胞产生的外泌体(AML-Exos),并使用qRT-PCR检测AML-Exos中miR-155-5p的表达变化。分别通过划痕试验、CCK-8试验和流式细胞术检测AIONFH-Exos/AML-Exos对于VECs的迁移、增殖和凋亡的影响,并通过Western Blot和qRT-PCR检测AIONFH-Exos/AML-Exos干预VECs后miR-155-5p下游靶基因HIF-1α的表达变化。结果:外泌体鉴定结果显示,本研究成功提取了血浆外泌体和肝细胞外泌体。高通量测序发现,相较于NC-Exos,AIONFH-Exos中有82种miRNAs差异表达,其中31种miRNAs上调,而51种miRNAs下调。GO和KEGG分析结果提示,这些差异表达的miRNAs主要调节转录和细胞代谢,并调控如PI3K、MAPK、Wnt、Hippo、mTOR、TGF-β、HIF-1等经典调节骨稳态和血管生成的信号通路。qRT-PCR验证结果提示,miR-155-5p在AIONFH-Exos的上调最显著,且其靶向调控HIF-1α基因。更进一步的研究发现,AML-Exos中miR-155-5p特异性上调,且AIONFH-Exos和AML-Exos都抑制VECs的增殖和迁移,并促进其凋亡,同时下调其HIF-1α/VEGFA的表达。结论:AIONFH患者血浆外泌体中含有丰富的差异表达的miRNAs,其中miR-155-5p的表达差异最明显。在酒精刺激条件下,肝细胞分泌的外泌体可能通过运载miR-155-5p下调VECs中HIF-1α/VEGFA的表达,并抑制VECs的增殖和迁移,同时促进VECs的凋亡。图13幅,表8个,参考文献85篇更多还原

【Abstract】 Objective:The purpose of this study is to reveal the expression profile of miRNA in plasma exosomes of alcohol-induced osteonecrosis of the femoral head(AIONFH),and to further explore the potential effect of alcohol-stimulated hepatocyte exosomes on vascular endothelial cells(VECs).Methods:Blood samples from the Chinese male population(10AIONFH patients and 10 healthy controls)were collected.Plasma exosomes of normal people(NC-Exos)and patients with AIONFH(AIONFH-Exos)were extracted by kit and identified by transmission electron microscope(TEM),nanoparticle tracking analysis(NTA),and Western Blot.The total RNA of exosomes was extracted,and then Illumina was used for high-throughput sequencing.The cluster plot and volcano plot of differentially expressed miRNAs were drawn,and the target genes of all differentially expressed miRNAs were predicted by Target Scan.The biological functions and pathways of these miRNAs were described by GO functional enrichment analysis and KEGG pathway enrichment analysis.qRT-PCR was used to verify some differentially expressed miRNAs in AIONFH-Exos.Normal hepatocyte exosome(NC-Exos)and alcohol stimulated hepatocyte exosome(AML-Exos)were extracted by kit,and the expression of miR-155-5p in AML-Exos was detected by qRT-PCR.The effects of AIONFH-Exos/AML-Exos on the migration,proliferation and apoptosis of VECs were detected by scratch test,CCK-8 test and flow cytometry.Finally,the expression of HIF-1α,the downstream target gene of miR-155-5p,in VECs treated with AML-Exos was detected by Western Blot and qRT-PCR.Results:Exosomes identification showed that plasma exosomes and hepatocyte exosomes were extracted successfully.High-throughput sequencing showed that 82 miRNAs were differentially expressed in AIONFH-Exos compared with normal peripheral plasma exosomes(NC-Exos),of which 31 kinds of miRNAs were up-regulated and 51kinds of miRNAs were down-regulated.The results of GO and KEGG analysis suggest that these differentially expressed miRNAs mainly regulate transcription and cellular metabolism,and regulate classical signaling pathways influencing bone homeostasis and angiogenesis,such as PI3K,MAPK,Wnt,Hippo,mTOR,TGF-β,and HIF-1.The results of qRT-PCR showed that miR-155-5p,which targets to regulate HIF-1α,was up-regulated most significantly in AIONFH-Exos.Further studies showed that miR-155-5p was specifically up-regulated in alcohol-stimulated AML-Exos,and both AIONFH-Exos and AML-Exos inhibited the proliferation and migration of VECs,promoted their apoptosis,and down-regulated the expression of HIF-1α/VEGFA.Conclusion:There are abundant differentially expressed miRNAs in plasma exosomes of patients with alcohol-induced osteonecrosis of the femoral head,among which the miR-155-5p expression is the most significant.Under the condition of alcohol stimulation,the exosomes secreted by hepatocytes may down-regulate the expression of HIF-1α/VEGFA in VECs by carrying miR-155-5p,inhibit the proliferation and migration of VECs,and promote the apoptosis of VECs.更多还原