PTH 1-34对鹿茸间充质干细胞成骨机制的转录组学分析

doi:10.7668/hbnxb.20194001

摘要/Abstract

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5 737个差异表达基因,其中1 877个上调,3 860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。

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

Abstract:

PTH 1-34 has an osteogenic effect on antler MSCs.The study aimed to explore the possible mechanism 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,induction group,and PTH 1-34 intervention group)with three replicates each.After the identification of markers and calcium nodules,total RNA was extracted using the TRIzol method and these libraries were double-end sequenced using Next-Generation Sequencing,an Illumina-based sequencing platform.Addition of 10 nmol/L PTH 1-34 for osteogenesis induction significantly increased alkaline phosphatase activity and calcium nodule production in antler mesenchymal 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 total of 5 737 differentially expressed genes were screened,of which 1 877 were up-regulated and 3 860 were down-regulated;GO enrichment analysis showed that the types of differential genes mainly included cell motility,proliferation and migration,bone development,angiogenesis and morphogenesis related under the action of biological processes;through screening,eight genes related to osteogenesis were found:RUNX2,BGN,ALPL,SPARC,OPN,CPNE1,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 sequencing results;transcription factors with high predicted correlation family was zf-C2H2,and the osteogenic transcription 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;possibly regulating angiogenesis and cellular autophagy by inhibiting the process of antler ossification.

Key words: Deer antler, Parathyroid hormone 1-34, Mesenchymal stem cell, Osteogenesis induction, Transcriptomics

邢宝瑞, 赵海平, 李光玉, 马泽芳, 孙红梅, 胡肖, 谭展清, 刘振. PTH 1-34对鹿茸间充质干细胞成骨机制的转录组学分析[J]. 华北农学报, 2024, 39(S1): 344-352. doi: 10.7668/hbnxb.20194001.

XING Baorui, ZHAO Haiping, LI Guangyu, MA Zefang, SUN Hongmei, HU Xiao, TAN Zhanqing, LIU Zhen. Transcriptomic Analysis of PTH 1-34 on the Osteogenesis Mechanism of Antler Mesenchymal Stem Cells[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(S1): 344-352. doi: 10.7668/hbnxb.20194001.

http://www.hbnxb.net/CN/Y2024/V39/IS1/344

图/表 9

图1 碱性磷酸酶和茜素红染色不同小写字母表示差异显著(P<0.05);不同大写字母表示差异极显著(P<0.01)。

Fig.1 Alkaline phosphatase and alizarin red staining Different lowercase letters indicate significant differences(P<0.05);Different capital letters indicate extremely significant differences(P<0.01).

表1 数据质量评估统计表

Tab.1 Statistical table of data quality assessment

样品 Sample	有效条目总数 Clean reads No.	有效数据/bp Clean data	有效条目占比/% Clean reads	有效数据占比/% Clean data
诱导组-1 Induction-1	44 172 308	6 670 018 508	93.45	93.45
诱导组-2 Induction-2	45 872 296	6 926 716 696	93.70	93.70
诱导组-3 Induction-3	45 166 788	6 820 184 988	93.97	93.97
PTH 1-34干预组-1 PTH 1-34 intervention-1	44 298 146	6 689 020 046	94.13	94.13
PTH 1-34干预组-2 PTH 1-34 intervention-2	46 686 304	7 049 63 1904	93.84	93.84
PTH 1-34干预组-3 PTH 1-34 intervention-3	45 340 436	6 846 405 836	93.72	93.72

图2 主成分分析横坐标为第一主成分,纵坐标为第二主成分。在图中的不同形状表示不同的样品,不同的颜色表示不同的分组。

Fig.2 Principal components analysis The horizontal coordinates are the first principal component and the vertical coordinates are the second principal component. Different shapes in the graph indicate different samples and different colors indicate different groupings.

表2 注释结果汇总

Tab.2 Summary of annotation results

数据库 Database	数目 Number	占比/% Percentage
NR	74 599	32.25
GO	64 944	28.08
KEGG	39 979	17.28
Pfam	18 891	8.17
eggNOG	72 335	31.27
Swissprot	43 865	18.96
所有数据库In all database	13 198	5.71

图3 差异表达基因聚类与火山图 A.红色表示高表达基因;绿色表示低表达基因。B.红点表示改组上调基因,蓝点表示改组下调基因,灰点表示非显著差异表达基因。

Fig.3 Cluster and volcano map of differentially expressed genes A.Red color indicates highly expressed genes,green color indicates lowly expressed genes;B.Red dots indicate reorganized up-regulated genes, blue dots indicate reorganized down-regulated genes,and gray dots indicate non-significant differentially expressed genes.

图4 GO和KEGG富集分析 A.差异基因GO功能富集分析;B.KEGG富集分析。根据富集结果,通过Rich factor、FDR值(P值校正值)和富集到此GO Term上的基因个数来衡量富集的程度。其中,横坐标Rich factor指该GO Term中富集到的差异基因个数与注释到的差异基因个数的比值。Rich factor越大,表示富集的程度越大。挑选FDR值最小的即富集最显著的前20个GO Term条目进行显示。

Fig.4 GO and KEGG enrichment analysis A.Differential gene GO enrichment analysis;B.KEGG pathway enrichment analysis.Based on the enrichment results,the degree of enrichment is measured by the Rich factor,FDR value(P-value correction)and the number of genes enriched on this GO Term.The Rich factor is the ratio of the number of enriched genes to the number of annotated genes in the GO Term,where the larger the Rich factor is,the greater the enrichment is.The top 20 GO Term entries with the smallest FDR values,i.e.the most significant enrichment,are selected for display.

图5 蛋白网络互作和差异转录因子功能分析 A、B.蛋白网络互作图,一条连线代表一组相互关系,节点大小表示该节点的度,红色代表上调,绿色代表下调。C.差异转录因子统计图,横坐标为不同转录因子家族,纵坐标为落到该转录因子家族的基因数目。

Fig.5 Functional analysis of protein network interactions and differential transcription factors A,B.Protein network interactions graph,a connecting line represents a set of interrelationships,the node size indicates the degree of that node,red represents up-regulation,green represents down-regulation.C.Statistical plot of differential transcription factors,the horizontal coordinates are different transcription factor families,the vertical coordinates are the number of genes falling into that transcription factor family.

表3 差异基因功能统计表

Tab.3 Functional statistics of differential genes

基因 Gene	名称 Name	生物功能 Biological function	文献 References
RUNX2	Runt相关转录因子2	成骨细胞分化和骨骼形态发生的转录因子,对成骨细胞成熟和软骨内成骨必不可少	[26⇓—28]
SP7/Osterix	转录因子Sp7	成骨细胞分化必需的转录激活因子	[29—30]
OPN/OCN	骨钙素	是骨矿物代谢的重要指标,与磷灰石和钙紧密结合	[31]
ALPL	碱性磷酸酶	代谢各种磷酸盐化合物,促进羟基磷灰石结晶的形成,在骨骼矿化中起关键作用	[32—33]
SPARC	骨连蛋白	成骨标记物,通过与细胞外基质和细胞因子的相互作用来调节细胞生长,与钙结合来促进成骨	[34]
BGN/SLRR1A	双糖链蛋白聚糖	成骨标记物,在维持骨稳态和骨重塑过程中发挥重要作用	[35]
CPNE1	钙离子依赖性的磷脂结合蛋白1	钙依赖性磷脂结合蛋白,在钙介导的细胞内过程中起作用	[36]
STRN4	钙调蛋白结合蛋白4	以钙依赖性的方式结合钙调素,可能起支架或信号蛋白的作用	[37]
VAPB	囊泡相关膜蛋白相关蛋白B/C	参与细胞钙稳态调节	[38]
WiPi2	WD重复结构域磷酸肌醇互作蛋白2	自噬机制的组成部分,控制细胞内主要降解过程,在此过程中,细胞质物质被包装成自噬体并递送到溶酶体进行降解	[39—40]
ATG14	自噬相关蛋白14	促进自噬体-溶酶体复合物的形成	[41—42]
UNC5B	网状蛋白受体UNC5B	在血管生成过程中作为网蛋白受体负调控血管分支	[43]
SHC1	SHC转化蛋白1	参与血管生成素受体TEK/TIE2下游信号传导,在内皮细胞迁移和芽生血管生成中发挥调节作用。促进破骨细胞的生成	[44—45]
FLNA	细丝蛋白A	在血管、心脏和大脑器官的发育过程中,在细胞和细胞的接触和黏附连接中起作用	[46]
TNFSF15	肿瘤坏死因子配体超家族成员15	介导NF-kappa-B的激活,抑制血管内皮生长和血管生成	[47]

表3 差异基因功能统计表

Tab.3 Functional statistics of differential genes

基因 Gene	名称 Name	生物功能 Biological function	文献 References
RUNX2	Runt相关转录因子2	成骨细胞分化和骨骼形态发生的转录因子,对成骨细胞成熟和软骨内成骨必不可少	[26⇓—28]
SP7/Osterix	转录因子Sp7	成骨细胞分化必需的转录激活因子	[29—30]
OPN/OCN	骨钙素	是骨矿物代谢的重要指标,与磷灰石和钙紧密结合	[31]
ALPL	碱性磷酸酶	代谢各种磷酸盐化合物,促进羟基磷灰石结晶的形成,在骨骼矿化中起关键作用	[32—33]
SPARC	骨连蛋白	成骨标记物,通过与细胞外基质和细胞因子的相互作用来调节细胞生长,与钙结合来促进成骨	[34]
BGN/SLRR1A	双糖链蛋白聚糖	成骨标记物,在维持骨稳态和骨重塑过程中发挥重要作用	[35]
CPNE1	钙离子依赖性的磷脂结合蛋白1	钙依赖性磷脂结合蛋白,在钙介导的细胞内过程中起作用	[36]
STRN4	钙调蛋白结合蛋白4	以钙依赖性的方式结合钙调素,可能起支架或信号蛋白的作用	[37]
VAPB	囊泡相关膜蛋白相关蛋白B/C	参与细胞钙稳态调节	[38]
WiPi2	WD重复结构域磷酸肌醇互作蛋白2	自噬机制的组成部分,控制细胞内主要降解过程,在此过程中,细胞质物质被包装成自噬体并递送到溶酶体进行降解	[39—40]
ATG14	自噬相关蛋白14	促进自噬体-溶酶体复合物的形成	[41—42]
UNC5B	网状蛋白受体UNC5B	在血管生成过程中作为网蛋白受体负调控血管分支	[43]
SHC1	SHC转化蛋白1	参与血管生成素受体TEK/TIE2下游信号传导,在内皮细胞迁移和芽生血管生成中发挥调节作用。促进破骨细胞的生成	[44—45]
FLNA	细丝蛋白A	在血管、心脏和大脑器官的发育过程中,在细胞和细胞的接触和黏附连接中起作用	[46]
TNFSF15	肿瘤坏死因子配体超家族成员15	介导NF-kappa-B的激活,抑制血管内皮生长和血管生成	[47]

图6 qRT-PCR验证RNA-seq数据

Fig.6 qRT-PCR validation of RNA-seq data

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