基于转录组学的梅花鹿茸皮组织修复机制研究
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摘要
为研究不同生长时期梅花鹿东北亚种Cervus nippon hortulorum鹿茸茸皮差异基因表达变化,以期为鹿茸生长机制及组织修复的临床治疗提供理论依据,本研究采用Illumina测序技术和生物信息学方法对快速生长期和骨化期鹿茸生长中心茸皮进行转录组测序和差异基因表达分析。从快速生长期和骨化期茸皮中分别获得45 422 254条和44 530 430条clean reads,测序质量值分别为98.50和98.39;通过Trinity组装,分别获得44 352条和43 194条组装序列,平均长度分别为977 nt和1 135 nt。通过数据库比对和差异基因表达分析筛选出生长因子7种、转录因子25种和胶原分子6种。在快速生长期鉴定出多种参与组织修复的重要生长因子和转录因子,包括Pdgfa、Tgfβ3、Egfl7、Vegfb、Sox4、Sox12、Scx、E4f1、Tcf4和Elf4等。
We aimed to study the gene expression pattern of the velvet skin of sika deer(Cervus nippon hortulorum) antler at different growth stages, and to provide theoretical basis for the growth mechanism of the antler as well as the clinical treatments of tissue repair. Illumina RNA sequencing and bioinformatic tools were conducted to analyze the gene expression pattern of velvet skins at rapid growth and ossification stages. Finally, 45 422 254 and 44 530 430 clean reads with Q20 values of 98.50 and 98.39 were generated from the rapid growth and the ossification stages, respectively. A total of 44 352 and 43 194 Unigenes with average lengths of 977 nt and 1 135 nt were obtained by using Trinity software. We identified 7 growth factors, 25 transcription factors and 6 collagens by BLAST against the protein database. Among these genes, a series of genes(including Pdgfa, Tgfβ3, Egfl7, Vegfb, Sox4, Sox12, Scx, E4 f1, Tcf4 and Elf4) which play pivotal roles in regulating tissue repair were identified. Our current study provides a data basis for the mechanistic studies on antler growth and tissue repair.
We aimed to study the gene expression pattern of the velvet skin of sika deer(Cervus nippon hortulorum) antler at different growth stages, and to provide theoretical basis for the growth mechanism of the antler as well as the clinical treatments of tissue repair. Illumina RNA sequencing and bioinformatic tools were conducted to analyze the gene expression pattern of velvet skins at rapid growth and ossification stages. Finally, 45 422 254 and 44 530 430 clean reads with Q20 values of 98.50 and 98.39 were generated from the rapid growth and the ossification stages, respectively. A total of 44 352 and 43 194 Unigenes with average lengths of 977 nt and 1 135 nt were obtained by using Trinity software. We identified 7 growth factors, 25 transcription factors and 6 collagens by BLAST against the protein database. Among these genes, a series of genes(including Pdgfa, Tgfβ3, Egfl7, Vegfb, Sox4, Sox12, Scx, E4 f1, Tcf4 and Elf4) which play pivotal roles in regulating tissue repair were identified. Our current study provides a data basis for the mechanistic studies on antler growth and tissue repair.
引文
国家药典委员会.2015.中华人民共和国药典[M].北京:中国医药科技出版社.
张嵩,李峰.2013.不同规格鹿茸商品药材中氨基酸含量分析[J].中国中药杂志,38:1919-1923.
Bae IH,Park JW,Kim DY.2014.Enhanced regenerative healing efficacy of a highly skin-permeable growth factor nanocomplex in a full-thickness excisional mouse wound model[J].International Journal of Nanomedicine,9:4551-4567.
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Hu MS,Maan ZN,Wu JC,et al.2014.Tissue engineering and regenerative repair in wound healing[J].Annals of Biomedical Engineering,42:1494-1507.
Kavyanifar A,Turan S,Lie DC.2018.SoxC transcription factors:multifunctional regulators of neurodevelopment[J].Cell and Tissue Research,371:91-103.
Lefebvre V,Bhattaram P.2016.SOXC genes and the control of skeletogenesis[J].Current Osteoporosis Reports,14:32-38.
Li C,Clark DE,Lord EA,et al.2002.Sampling technique to discriminate the different tissue layers of growing antler tips for gene discovery[J].Anatomical Record,268:125-130.
Li C,Suttie JM.2000.Histological studies of pedicle skin formation and its transformation to antler velvet in red deer (Cervus elaphus)[J].Anatomical Record,260:62-71.
Li C,Zhao H,Liu Z,et al.2014.Deer antler-a novel model for studying organ regeneration in mammals[J].International Journal of Biochemistry & Cell Biology,56:111-122.
Li C.2010.Exploration of the mechanism underlying neogenesis and regeneration of postnatal mammalian skin-deer antler velvet[J].International Journal of Medical and Biological Frontiers,16:1-19.
Li C.2012.Deer antler regeneration:a stem cell-based epimorphic process[J].Birth Defects Research,Part C-Embryo Today:Reviews,96:51-62.
Lu R,Qu Y,Ge J,et al.2012.Transcription factor TCF4 maintains the properties of human corneal epithelial stem cells[J].Stem Cells,30:753-761.
Mu Z,Zhang S,He C,et al.2017.Expression of SoxC transcription factors during zebrafish retinal and optic nerve regeneration[J].Neuroscience Bulletin,33(1):53-61.
Sakabe T,Sakai K,Maeda T,et al.2018.Transcription factor scleraxis vitally contributes to progenitor lineage direction in wound healing of adult tendon in mice[J].Journal of Biological Chemistry,293:5766-5780.
Sivina M,Yamada T,Park CS,et al.2011.The transcription factor E74-like factor controls quiescence of endothelial cells and their resistance to myeloablative treatments in bone marrow[J].Arteriosclerosis Thrombosis and Vascular Biology,31:1185-1191.
Sudhagar A,Kumar G,El-Matbouli M.2018.Transcriptome analysis based on RNA-Seq in understanding pathogenic mechanisms of diseases and the immune system of fish:a comprehensive review[J].International Journal of Molecular Sciences,19:E245.DOI:10.3390/ijms19010245.
Suttie JM,Fennessy PF.1985.Regrowth of amputated velvet antlers with and without innervation[J].Journal of Experimental Zoology,234:359-366.
Trapnell C,Williams BA,Pertea G,et al.2010.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J].Nature Biotechnology,28:511-515.
Wang L,Feng Z,Wang X,et al.2010.DEGseq:an R package for identifying differentially expressed genes from RNA-seq data[J].Bioinformatics,26:136-138.
张嵩,李峰.2013.不同规格鹿茸商品药材中氨基酸含量分析[J].中国中药杂志,38:1919-1923.
Bae IH,Park JW,Kim DY.2014.Enhanced regenerative healing efficacy of a highly skin-permeable growth factor nanocomplex in a full-thickness excisional mouse wound model[J].International Journal of Nanomedicine,9:4551-4567.
Bourgard C,Albrecht L,Kayano ACAV,et al.2018.Plasmodium vivax biology:insights provided by genomics,transcriptomics and proteomics [J].Frontiers in Celluar and Infection Microbiology,8:34.DOI:10.3389/fcimb.2018.00034.
Chang KC,Hertz J.2017.SoxC transcription factors in retinal development and regeneration[J].Neural Regeneration Research,12:1048-1051.
Chu W,Zhao H,Li J,et al.2017.Custom-built tools for the study of deer antler biology[J].Frontiers in Bioscience (Landmark),22:1622-1633.
Costa-Silva J,Domingues D,Lopes FM.2017.RNA-Seq differential expression analysis:an extended review and a software tool[J].PLoS ONE,12:e0190152.DOI:10.1371/journal.pone.0190152.
Goguet-Rubio P,Seyran B,Gayte L,et al.2016.E4F1-mediated control of pyruvate dehydrogenase activity is essential for skin homeostasis[J].Proceedings of the National Academy of Sciences of the United States of America,113:11004-11009.
Grabherr MG,Haas BJ,Yassour M,et al.2011.Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nature Biotechnology,29:644-652.
Hrdlickova R,Toloue M,Tian B.2017.RNA-Seq methods for transcriptome analysis[J].Wiley Interdisciplinary Reviews RNA,8:e1364.DOI:10.1002/wrna.1364.
Hu MS,Maan ZN,Wu JC,et al.2014.Tissue engineering and regenerative repair in wound healing[J].Annals of Biomedical Engineering,42:1494-1507.
Kavyanifar A,Turan S,Lie DC.2018.SoxC transcription factors:multifunctional regulators of neurodevelopment[J].Cell and Tissue Research,371:91-103.
Lefebvre V,Bhattaram P.2016.SOXC genes and the control of skeletogenesis[J].Current Osteoporosis Reports,14:32-38.
Li C,Clark DE,Lord EA,et al.2002.Sampling technique to discriminate the different tissue layers of growing antler tips for gene discovery[J].Anatomical Record,268:125-130.
Li C,Suttie JM.2000.Histological studies of pedicle skin formation and its transformation to antler velvet in red deer (Cervus elaphus)[J].Anatomical Record,260:62-71.
Li C,Zhao H,Liu Z,et al.2014.Deer antler-a novel model for studying organ regeneration in mammals[J].International Journal of Biochemistry & Cell Biology,56:111-122.
Li C.2010.Exploration of the mechanism underlying neogenesis and regeneration of postnatal mammalian skin-deer antler velvet[J].International Journal of Medical and Biological Frontiers,16:1-19.
Li C.2012.Deer antler regeneration:a stem cell-based epimorphic process[J].Birth Defects Research,Part C-Embryo Today:Reviews,96:51-62.
Lu R,Qu Y,Ge J,et al.2012.Transcription factor TCF4 maintains the properties of human corneal epithelial stem cells[J].Stem Cells,30:753-761.
Mu Z,Zhang S,He C,et al.2017.Expression of SoxC transcription factors during zebrafish retinal and optic nerve regeneration[J].Neuroscience Bulletin,33(1):53-61.
Sakabe T,Sakai K,Maeda T,et al.2018.Transcription factor scleraxis vitally contributes to progenitor lineage direction in wound healing of adult tendon in mice[J].Journal of Biological Chemistry,293:5766-5780.
Sivina M,Yamada T,Park CS,et al.2011.The transcription factor E74-like factor controls quiescence of endothelial cells and their resistance to myeloablative treatments in bone marrow[J].Arteriosclerosis Thrombosis and Vascular Biology,31:1185-1191.
Sudhagar A,Kumar G,El-Matbouli M.2018.Transcriptome analysis based on RNA-Seq in understanding pathogenic mechanisms of diseases and the immune system of fish:a comprehensive review[J].International Journal of Molecular Sciences,19:E245.DOI:10.3390/ijms19010245.
Suttie JM,Fennessy PF.1985.Regrowth of amputated velvet antlers with and without innervation[J].Journal of Experimental Zoology,234:359-366.
Trapnell C,Williams BA,Pertea G,et al.2010.Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J].Nature Biotechnology,28:511-515.
Wang L,Feng Z,Wang X,et al.2010.DEGseq:an R package for identifying differentially expressed genes from RNA-seq data[J].Bioinformatics,26:136-138.