| 摘要: |
| 【目 的】探讨新吉细毛羊血浆外泌体（PLA-Exos）对小尾寒羊毛囊发育的影响，并通过转录组测序分析筛出皮肤组织中影响毛囊发育的关键miRNA，为miRNA对毛囊发育的调控机制研究提供理论依据。【方法】采用双相沉淀法提取新吉细毛羊 PLA-Exos，对其进行鉴定。构建小尾寒羊脱毛模型，在其左侧背部皮肤脱毛区域五点皮内注射新吉细毛羊PLA-Exos，每日一次，连续注射5 d，右侧对应脱毛区域皮内注射相同体积的PBS作为对照，注射结束后第21天采集皮肤组织，HE染色观察皮肤组织学变化。对皮肤样本进行转录组测序分析，使用DESeq2软件，以|log2 Fold Change|≥1 和错误发现率（FDR）≤0.05为标准筛选差异表达miRNA，利用miRanda和RNAhybrid预测其靶基因，并对靶基因进行GO功能注释和KEGG通路富集分析，进而构建差异miRNA-mRNA调控网络。利用实时荧光定量PCR（RT-qPCR）对转录组测序结果进行验证 。【结 果】成功分离出新吉细毛羊PLA-Exos，其粒径主要集中在30~150 nm，在106 nm处出现浓度峰值，表达表面标志蛋白TSG101和CD9。皮肤组织切片观察发现，PLA-Exos组毛囊数目较对照组明显增加。从皮肤组织中共鉴定出1 474个miRNA（已知miRNA 146 个，新预测1 328个），共筛选出18个差异表达miRNA，其中14个上调表达（novel-mir-335、novel-mir-957、novel-mir-855、novel-mir-561、novel-mir-797、novel-mir-1158、novel-mir-917、novel-mir-816、novel-mir-1119、novel-mir-616、novel-mir-1192、novel-mir-528、novel-mir-451和novel-mir-478），4个下调表达（novel-mir-1170、novel-mir-965、novel-mir-1352 和 novel-mir-156）。18个差异表达miRNA共预测到493个靶基因，GO功能注释分析显示，差异表达miRNA的靶基因主要涉及生物学过程中的细胞过程 、单一生物过程等条目及细胞组分和分子功能中的细胞 、细胞部分、结合与催化活性等条目；KEGG通路分析显示共富集在273个通路中，有6个miRNA及其靶基因富集于与毛囊调控密切相关的MAPK和Wnt信号通路。通过RT-qPCR对10个差异表达miRNA进行验证，结果与转录组测序数据一致 。【结 论】novel-mir-1192、novel-mir-1158、novel-mir-156、novel-mir-917、novel-mir-797和novel-mir-451及其靶基因是调控毛囊发育的候选基因，可能对绵羊毛发生长有重要影响。 |
| 关键词: 毛囊发育 miRNAs 血浆外泌体 新吉细毛羊 小尾寒羊 |
| DOI：10. 13207/j. jnwafu. 2026. 10. 003 |
| 分类号: |
| 基金项目:国家自然科学基金项目（32060781） |
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| Transcriptomic analysis and differential miRNA screening of the effect of plasma⁃derived exosomes in Xinji fine⁃wool sheep on promoting hair follicle development in Small⁃tailed Han sheep |
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LIU Wenqing1, ZHANG Caihong1, ZHANG Xinyu2, FU Jiaqi1, YUAN Min1, SUN Fuliang1,3
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1.College of Agriculture，Yanbian University，Yanji，Jilin 133002，China;2.nstitute of Special Animal and Plant Sciences，Chinese Academy of Agricultural Sciences，Changchun，Jilin 130112，China;3.Laboratory Animal Center，Yanbian University，Yanji，Jilin 133002，China
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| Abstract: |
| 【Objective】This study aims to investigate the effect of plasma-derived exosomes（PLA-Exos）from Xinji fine-wool sheep on hair follicle development in Small-tailed Han sheep. Through transcriptome sequencing analysis，key miRNAs influencing hair follicle development in skin tissues were screened，to provide a theoretical basis for research on the regulatory mechanisms of miRNAs in hair follicle development.【Method】PLA-Exos were extracted from Xinji fine-wool sheep using a two-step precipitation method，and were subse-quently characterized. A depilation model was established for Small-tailed Han sheep. On the depilated area of the left dorsal skin，PLA-Exos were intradermally injected at five points once daily for five consecutive days.On the corresponding depilated area of the right dorsal skin，the same volume of PBS was intradermally injected as the control. Skin tissue samples were collected on day 21 post-injection，and their histological changes were
observed via hematoxylin and eosin（HE）staining. Transcriptome sequencing analysis was performed on the skin samples. Differentially expressed miRNAs（DEmiRNAs）were screened using DESeq2 software with the criteria of |log 2 Fold Change|≥1 and false discovery rate（FDR）≤0.05. Their target genes were predicted by using miRanda and RNAhybrid. Gene ontology（GO）functional annotation and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analyses were conducted on the predicted target genes，and a regulatory network of DEmiRNA-mRNA was constructed subsequently. Real-time quantitative polymerase chain reaction（RT-qPCR）was used to validate the transcriptome sequencing results.【Result】PLA-Exos were successfully isolated from Xinji fine-wool sheep. Their particle size primarily ranged from 30 to 150 nm，with a concentration peak observed at 106 nm，and the surface marker proteins TSG101 and CD9 expressed. Histological observation of skin tissue sections revealed a significantly higher number of hair follicles in the PLA-Exos group compared to the control group（P<0.05）.A total of 1 474 miRNAs were identified in the skin tissues（146 known miRNAs and 1 328 novel predicted miRNAs）. Eighteen DEmiRNAs were identified，including 14 upregulated DEmiRNAs（novel-mir-335，novel-mir-957，novel-mir-855，novel-mir-561，novel-mir-797，novel-mir-1158，novel-mir-917，novel-mir-816，novel-mir-1119，novel-mir-616，novel-mir-1192，novel-mir-528，novel-mir-451，and novel-mir-478）and 4 downregulated DEmiRNAs（novel-mir-1170，novel-mir-965，novel-mir-1352，and novel-mir-156）. A total of 493 target genes were predicted for these 18 DEmiRNAs. GO func?tional annotation analysis revealed that the target genes of the DEmiRNAs were primarily associated with the biological process such as cellular process and single-organism process. The enriched cellular component and molecular function terms were predominantly associated with cells，cell parts，as well as binding and catalytic activities. KEGG pathway enrichment analysis revealed 273 pathways，among which 6 miRNAs and their target genes were notably enriched in the MAPK and Wnt signaling pathways，which were closely associated with hair follicle regulation. Validation of 10 DEmiRNAs by RT-qPCR yielded results consistent with the transcriptome
sequencing data.【Conclusion】Six DEmiRNAs（novel-mir-1192，novel-mir-1158，novel-mir-156，novel-mir-917，novel-mir-797，and novel-mir-451），along with their target genes，are identified as candidate genes regula-ting hair follicle development，which may significantly influence hair growth in sheep. |
| Key words: hair follicle develepment miRNAs plasma exosomes Xinji fine-wool sheep Small-tailed Han sheep |
