目的 基于轉(zhuǎn)錄組學(xué)探討含胎牛血清（FBS）培養(yǎng)基、無(wú)血清培養(yǎng)基（SFM）培養(yǎng)給間充質(zhì)干細(xì)胞（MSCs）生物學(xué)活性帶來(lái)的差異。方法 從3例產(chǎn)婦臍帶制備人臍帶MSCs并進(jìn)行表面標(biāo)志物及三系分化鑒定；用含10% FBS的DMEM/F-12完全培養(yǎng)基及SFM連續(xù)培養(yǎng)MSCs，取P4代用轉(zhuǎn)錄組測(cè)序，開(kāi)展全局轉(zhuǎn)錄組譜分析，進(jìn)行細(xì)胞的基因表達(dá)差異分析，使用DAVID對(duì)差異表達(dá)的基因進(jìn)行基因本體（GO）功能富集分析；使用R包c(diǎn)lusterProfiler的GSEA函數(shù)進(jìn)行基因集富集分析以探索通路的功能變化。提取3個(gè)批次的FBS組、SFM組的P4代MSCs mRNA，¹²⁵IPCR（qRT-PCR）法對(duì)轉(zhuǎn)錄組學(xué)的差異基因進(jìn)行驗(yàn)證。結(jié)果 制備的MSCs經(jīng)鑒定均符合要求；轉(zhuǎn)錄組測(cè)序分析結(jié)果顯示，不同培養(yǎng)條件顯著改變MSCs轉(zhuǎn)錄組情況，F(xiàn)BS組高表達(dá)的代表性基因有趨化因子介導(dǎo)蛋白1（CXCL1）、白細(xì)胞介素1β（IL1B）、G蛋白耦聯(lián)受體家族C5組成員A（GPRC5A）、CUB結(jié)構(gòu)域含量蛋白1（CDCP1）、緩激肽B1受體（BDKRB1），SFM組高表達(dá)的代表性基因有24-脫氫膽固醇還原酶（DHCR²4）、N-乙酰谷氨酸合成酶（NAGS）、早幼粒細(xì)胞白血病鋅指蛋白（ZBTB16）、跨膜蛋白119（TMEM119）。SFM培養(yǎng)的MSCs高表達(dá)的基因顯著富集在DNA復(fù)制、細(xì)胞增殖和代謝相關(guān)的生物過(guò)程，而傳統(tǒng)血清培養(yǎng)的MSCs高表達(dá)的基因顯著富集在細(xì)胞外基質(zhì)矩陣、對(duì)細(xì)胞因子刺激的反應(yīng)、對(duì)炎癥反應(yīng)及信號(hào)傳導(dǎo)等具有生物功能性的生物過(guò)程。差異基因的qRT-PCR檢測(cè)結(jié)果與轉(zhuǎn)錄組分析結(jié)果一致。結(jié)論 MSCs的基因表達(dá)會(huì)因培養(yǎng)基是否含血清而變化，盡管SFM培養(yǎng)的MSCs展現(xiàn)了更強(qiáng)的增殖能力，但FBS培養(yǎng)基培養(yǎng)的MSCs更具有生物功能性。

Objective To explore the differences in the biological activity of mesenchymal stem cells (MSCs) cultured in fetal bovine serum (FBS)-containing medium and serum-free medium (SFM) based on transcriptomics. Methods MSCs were isolated from the umbilical cords of three patients and identified by surface markers and tri-lineage differentiation. MSCs were continuously cultured in DMEM/F-12 complete medium containing 10% FBS and SFM. The fourth passage (P4) cells were subjected to transcriptome sequencing for global transcriptome profiling and differential gene expression analysis. DAVID was used for Gene Ontology (GO) functional enrichment analysis of differentially expressed genes. Gene Set Enrichment Analysis (GSEA) was performed using the clusterProfiler R package to explore functional changes in pathways. The mRNA of P4 MSCs from three batches of FBS and SFM groups was extracted, and real-time fluorescence quantitative PCR (qRT-PCR) was used to verify the differentially expressed genes identified by transcriptomics. Results The isolated MSCs met the requirements after identification. Transcriptome sequencing analysis showed that different culture conditions significantly altered the transcriptome of MSCs. Representative genes highly expressed in the FBS group included chemokine ligand 1 (CXCL1), interleukin 1β (IL1B), G protein-coupled receptor family C group 5 member A (GPRC5A), CUB domain-containing protein 1 (CDCP1), and bradykinin B1 receptor (BDKRB1). Representative genes highly expressed in the SFM group included 24-dehydrocholesterol reductase (DHCR24), N-acetylglutamate synthase (NAGS), promyelocytic leukemia zinc finger protein (ZBTB16), and transmembrane protein 119 (TMEM119). Genes highly expressed in SFM-cultured MSCs were significantly enriched in biological processes related to DNA replication, cell proliferation, and metabolism, while genes highly expressed in FBS-cultured MSCs were significantly enriched in biological processes with biological functions such as extracellular matrix organization, response to cytokine stimulation, and inflammatory response and signal transduction. The qRT-PCR results of the differentially expressed genes were consistent with the transcriptome analysis results. Conclusion Analysis of transcriptome sequencing data showed that the gene expression of MSCs varied according to the medium. Although MSCs cultured in SFM showed stronger proliferation ability, MSCs cultured in FBS medium were more biologically functional.

R965

天津市科技計(jì)劃項(xiàng)目細(xì)胞制品的成藥性及轉(zhuǎn)化研究項(xiàng)目（23ZGCXQY00050）；天津市科技計(jì)劃項(xiàng)目細(xì)胞和基因治療產(chǎn)品概念驗(yàn)證平臺(tái)建設(shè)項(xiàng)目（24ZYCGCG00600）