关键词: 诱导多能干细胞, 双峰驼, 丙戊酸, 细胞重编程

Abstract: 【Objective】 In order to improve the efficiency of the reprogramming process of bactrian camel fibroblasts and reduce the risk of tumorigenesis caused by the introduction of proto-oncogenes. In this experiment, valproic acid (VPA) was added to the fibroblast reprogramming process to explore the effect of small molecules on the reprogramming of Bactrian camel fibroblasts. 【Methods】 In view of this, we used March-aged Bactrian camel fetal fibroblasts, combined with classic induction combination OSKM (Oct4, Sox2, Klf4, and c-Myc) and EGFP five retrovirus reprogramming of bactrian camel fibroblasts (OSKM group), and collected the cells by adding VPA treatment for 7 days after the second viral infection (OSKM+VPA group). Endogenous and exogenous genes were examined using PCR to confirm the modification effect of retrovirus on Bactrian camel fibroblasts. Eight genes were randomly selected from those more significantly affected by VPA according to RNA-seq data to check whether their trends before and after VPA addition were consistent with the trends of RNA-seq data to verify the accuracy of RNA-seq data. The transcriptome sample genes were classified by GO analysis and significant enrichment pathways for target genes were clarified using KEGG pathway enrichment analysis and hypergeometric validation analysis. Total RNA was extracted from the collected cells and combined with RNA-seq and Real time-quantitative interpretation（RT-qPCR） techniques to detect the effect of VPA on the reprogramming of Bactrian camel fibroblasts. 【Results】 It was detected using PCR that the expression of endogenous and exogenous genes in different groups. The results showed that nSox2, Sox2, Oct4, Klf4 and c-Myc genes were expressed in both OSKM and OSKM+VPA groups, and the expression in OSKM+VPA group was higher than that in OSKM group, while they were not expressed in BCEFs group. Eight genes were randomly selected for testing, and the results showed that: three genes TP53, CCNB1 and CCD20, which are related to cell cycle signalling pathway, were down-regulated in expression after the addition of VPA; S100A4, CKS2, VIM and MMP9 genes, which are related to the phenotypic characteristics of cancer, were down-regulated in expression; VEGFC gene expression was up-regulated in PI3k-Akt signalling pathway. This expression trend is consistent with the trend of the histological data. The results showed that the expression of proliferation genes Mki67 and PCNA were down-regulated, while the expression of apoptosis gene CASP7 was up-regulated after the addition of VPA. KEGG and hypergeometric validation analyses of the transcriptome data were performed, and 959 differentially expressed genes were screened according to the analysis results, which were enriched in 276 signalling pathways, including eight signalling pathways with Q values less than 0.05: steroid biosynthesis, cell cycle, PPAR signalling pathway, progesterone-mediated oocyte maturation, fatty acid metabolism, ECM-receptor interactions, cell adhesion molecules and cholesterol metabolism. The 26 differentially expressed genes related to cell cycle, fatty acid metabolism, cell adhesion molecule and cholesterol metabolism were screened and four of them were randomly selected for testing, showing that VPA upregulated the expression of L1CAM, CNTN1 and NFASC genes in the Bactrian camel fibroblast adhesion molecule signalling pathway and enhanced intercellular interactions. It was also upregulated that the expression of CD36 gene in the fatty acid signaling pathway. 【Conclusion】 The results showed that the VPA to cell block before the split phase, to reduce risk differentiation during the process of reprogramming. Meanwhile, VPA affected several signaling pathways in the reprogramming process of Bactrian camel fibroblasts, and regulated the expression trend of related genes in the signaling pathways, which effectively improved the reprogramming efficiency of the cells and played an important role in the reprogramming of Bactrian camel fibroblasts.

Key words: induced pluripotent stem cells, bactrian camel, valproic acid, cell reprogramming