PTH 1-34对鹿茸间充质干细胞成骨机制的转录组学分析  

Transcriptomic Analysis of PTH 1-34 on the Osteogenesis Mechanism of Antler Mesenchymal Stem Cells

作　　者：邢宝瑞 赵海平 李光玉 马泽芳[1] 孙红梅[2] 胡肖 谭展清 刘振 XING Baorui;ZHAO Haiping;LI Guangyu;MA Zefang;SUN Hongmei;HU Xiao;TAN Zhanqing;LIU Zhen(College of Animal Science and Technology,Qingdao Agricultural University,Qingdao 266109,China;Institute of Specialty Products,Chinese Academy of Agricultural Sciences,Changchun 130022,China;Jinan Stomatological Hospital,Jinan 250001,China)

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109 [2]中国农业科学院特产研究所,吉林长春130022 [3]济南市口腔医院,山东济南250001

出　　处：《华北农学报》2024年第S1期344-352,共9页Acta Agriculturae Boreali-Sinica

基　　金：青岛农业大学高层次人才引进项目(1121009);山东省现代农业产业技术体系特种经济动物产业技术体系(SDAIT-21-01)。

摘　　要：PTH 1-34对鹿茸间充质干细胞具有促成骨作用,旨在利用高通量无参转录组测序方法探索PTH 1-34促进鹿茸间充质干细胞成骨的可能机制。选用第3代鹿茸间充质干细胞,按试验要求随机分为3组(对照组、诱导组、PTH 1-34干预组),每组3个重复,诱导组添加成骨诱导液诱导鹿茸间充质干细胞成骨分化,PTH 1-34干预组在诱导组基础上添加10 nmol/L PTH 1-34。培养21 d,采用碱性磷酸酶和茜素红染色的方法对标志物和钙结节进行鉴定,鉴定完毕后,使用TRIzol法进行总RNA提取并采用第二代测序技术(Next-Generation Sequencing),基于Illumina测序平台,对这些文库进行双末端测序。添加浓度为10 mmol/L PTH 1-34进行成骨诱导,可明显增加鹿茸间充质干细胞碱性磷酸酶活性和钙结节的产生量。因与NCBI所发布的鹿科动物基因序列比对效率低于50%,选择了无参转录组测序。通过对差异基因富集分析,共筛选出5737个差异表达基因,其中1877个上调,3860个下调;GO富集分析显示,在生物过程作用下,差异基因的种类主要包括细胞运动、细胞增殖、细胞迁移、骨发育、血管生成和形态发生相关;通过筛选,找到了8个与成骨相关基因:RUNX2、BGN、ALPL、SPARC、OPN、CPNE1、STRN4、VAPB;2个与细胞自噬相关基因:WiPi2、ATG14;4个与血管生成相关基因:UNC5B、SHC1、FLNA、TNFSF15,使用qRT-PCR方法进行验证,结果与测序结果一致;转录因子预测相关性较高的家族为zf-C2H2,并筛选到了成骨转录因子SP7。PTH 1-34可通过上调RUNX2、SP7、BGN、ALPL、SPARC、OPN、CPNE1、STRN4、VAPB的mRNA表达来促进鹿茸间充质干细胞的成骨分化;可能通过抑制血管生成和细胞自噬来调节鹿茸的骨化过程。PTH 1-34 has an osteogenic effect on antler MSCs.The study aimed to explore the possible mecha-nism of PTH 1-34 promoting osteogenesis in antler MSCs by using the high-throughput non-reference transcriptome sequencing method.The 3rd generation antler MSCs were randomly divided into three groups(control group,induc-tion group,and PTH 1-34 intervention group)with three replicates each.After the identification of markers and cal-cium nodules,total RNA was extracted using the TRIzol method and these libraries were double-end sequenced u-sing Next-Generation Sequencing,an Illumina-based sequencing platform.Addition of 10 nmol/L PTH 1-34 for oste-ogenesis induction significantly increased alkaline phosphatase activity and calcium nodule production in antler mes-enchymal stem cells.The non-reference transcriptome sequencing was selected due to less than 50%efficiency of gene sequence comparison with deer family published by NCBI.Through differential gene enrichment analysis,a to-tal of 5737 differentially expressed genes were screened,of which 1877 were up-regulated and 3860 were down-regulated;GO enrichment analysis showed that the types of differential genes mainly included cell motility,prolifera-tion and migration,bone development,angiogenesis and morphogenesis related under the action of biological proces-ses;through screening,eight genes related to osteogenesis were found:RUNX2,BGN,ALPL,SPARC,OPN,CPNEI,STRN4,VAPB;two genes related to cellular autophagy:WiPi2,ATG14;four genes related to angiogenesis:UNC5B,SHC1,FLNA,TNFSF15,which were validated using qRT-PCR method,and the results were consistent with the se-quencing results;transcription factors with high predicted correlation family was zf-C2H2,and the osteogenic tran-scription factor SP7 was screened.PTH 1-34 could promote osteogenic differentiation of antler mesenchymal stem cells by upregulating the mRNA expression of RUNX2,SP7,BGN,ALPL,SPARC,OPN,CPNE1,STRN4,VAPB;pos-sibly regulating angiogenesis and cellular autophagy by inhibiting the process of antler ossifi

关 键 词：鹿茸 甲状旁腺素1-34 间充质干细胞 成骨诱导 转录组学 

分 类 号：S825[农业科学—畜牧学]