| Objective: Taking thymosin β10 from sika deer as the research object,to explore the effect of thymosin β10 from sika deer on the growth of osteocytes,to clarify the mechanism of thymosin β10 from sika deer to promote osteoblast apoptosis.To provide theoretical support for elucidating the biological activity and mechanism of thymosin β10 in sika deer.Methods: The sika deer antlers of sika deer at three different growth stages of primary stage(PS),rapid growth stage(RG)and ossification stage(OS)were used as samples,and the total RNA and total protein of sika deer antler were extracted by modified Trizol method and lysis method respectively;Illumina Hi Seq(?) 2000 data platform and i TRAQ technology for transcriptome sequencing and proteome sequencing of sika deer antler samples from three periods;SPSS 22.0 software was used for joint analysis of transcriptome data and proteome data;bioinformatics analysis method was used to predict the structure of thymosinβ10 protein;genetic engineering methods were used to construct Tβ10 gene overexpression vector derived from sika deer,and RNAi Technology was used to synthesize si RNA sequences targeting Tβ10 gene in vitro;The effects of overexpression and knockout of Tβ10gene on the activity of mouse osteoblasts and chondrocytes were detected by CCK8 method,flow cytometry analysis,and mitochondrial membrane potential assay(JC-1).Apoptosis-related genes and their signaling pathways were screened by transcriptome sequencing;the expression levels of apoptosis-related genes and proteins were detected by fluorescence quantitative PCR and Western blot analysis,respectively.Results: 1.Established the transcriptome database and proteome database of sika deer antler in the primary stages,rapid growth and ossification stages,and obtained a total of210,437 Unigenes and 2,261 proteins.Taking the primary stages as a control,the results of differential gene and differential protein analysis showed that 11,294 and 4,924 significantly differential genes and 236 and 211 differential proteins were obtained in PSvs RG group and PSvs OS group,respectively.2.Combined analysis of transcriptome and proteome data found that 54 and 51 DEGs encoded their corresponding DAPs in PSvs RG and PSvs OS groups,respectively.Pearson correlation analysis was performed on these genes/proteins,and the results showed that the transcriptome was weakly correlated with the proteome.After further screening of the obtained genes/proteins,eight candidate genes/proteins related to the growth and development of sika deer antler were screened out.Among them,thymosin β10 gene can be used as a candidate gene for further study.3.The structural analysis of sika deer thymosin β10 protein showed that it encodes 41 amino acids,the molecular weight is 4823.47 Da,the theoretical isoelectric point is 6.18,and there is an actin binding motif at residues 7-41;The amino acid sequence of Tβ10 of sika deer has the closest amino acid relationship with Tβ10 of Sus scrofa、Bos taurus and Cervus canadensis.4.According to the protein sequence of Tβ10 in the database,without changing its sequence,changing its codon preference and optimizing the sequence,the eukaryotic overexpression vector pc DNA3.1(-)-Tβ10 was successfully constructed;targeting osteoblasts and chondrocytes were synthesized and screened to obtain the si RNA-Tβ10fragment with the highest silencing efficiency;the optimal conditions for overexpression and silencing of Tβ10 gene were determined.5.In osteoblast MC3T3,overexpression of Tβ10 gene inhibited cell proliferation,and the cell survival rate was the lowest when the transfection amount of overexpression plasmid reached 0.2 μg;Silencing the Tβ10 gene promoted cell proliferation.When the transfection amount reached 3 pmol,the cell survival rate was the highest.6.In chondrocytes,overexpression of Tβ10 gene promoted cell proliferation,and the cell survival rate was the highest when the transfection amount of the overexpression plasmid reached 0.08 μg;Silencing the Tβ10 gene in chondrocytes inhibited cell proliferation.When the transfection amount reached 1 pmol,the cell survival rate was the lowest.7.Transcriptome sequencing was performed on the osteoblasts overexpressing and knocking out Tβ10 gene and the control group,and the difference analysis results showed that 26,223 and 6,345 differential genes were obtained in the control group vs the overexpression group and the control group vs the silence group,respectively.The enrichment analysis of the differential genes found that the differential genes in the control group vs the overexpression group were mainly involved in the regulation of biological processes such as cell cycle and apoptosis;The differential genes screened in the control group vs the silence group were mainly involved in the regulation of amino acid biosynthesis,apoptosis.After further screening,genes encoding apoptosis-related proteins were obtained,including: Fas/Fas L,Bcl-2,Bax and Caspase-3.8.The expression levels of apoptosis-related genes at m RNA and protein levels were detected by fluorescence quantitative PCR and western blot analysis.The results showed that in osteoblasts overexpressing Tβ10 gene,the expression of pro-apoptotic genes at m RNA and protein levels increased significantly with the increase of transfection amount,and the expression of Bcl-2 gene,which inhibited apoptosis,was significantly reduce;In osteoblasts with silenced Tβ10 gene,the expression of pro-apoptotic genes at m RNA and protein levels decreased with the increase of transfection amount,while the expression of apoptosis-inhibiting Bcl-2 gene increased significantly.Conclusion:1.It is a feasible method to establish sika deer antler transcriptome database and proteome database in different periods and screen candidate genes/proteins related to sika deer antler growth and development by multi-omics combined analysis method.2.Overexpression of Tβ10 decreased cell survival in mouse osteoblasts,whereas knockout of Tβ10 increased cell survival,It indicates that Tβ10 gene is a pro-apoptotic factor for osteoblasts.3.Overexpression of the Tβ10 gene in mouse chondrocytes increased cell survival,whereas knockout of the Tβ10 gene decreased chondrocyte survival,It shows that Tβ10 gene is an anti-apoptotic factor for chondrocytes.4.The overexpressed Tβ10 gene in osteoblasts may lead to apoptosis of osteoblasts by amplifying Fas signal.5.Sika deer antler thymosin β10 has an important regulatory effect on bone growth and development. |
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