基于转录组学分析Vero悬浮细胞并用表皮生长因子改善细胞自噬水平

Transcriptomic analysis of suspended Vero cells and reduction of cellular autophagy by epidermal growth factor

摘要:

目前悬浮驯化的Vero细胞存在细胞活率低和倍增时间长等问题。为了探究Vero悬浮细胞生长缓慢且活率低的主要原因，本研究对Vero悬浮细胞(命名为Vero-XF)和Vero贴壁细胞(命名为Vero-AD)进行转录组学分析，筛选悬浮培养时影响细胞生长的差异基因，并通过外源添加表皮生长因子(epidermal growth factor,EGF)改善Vero-XF生长。结果表明，与Vero-AD组相比，Vero-XF组有7 376个显著变化的差异基因。京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析发现细胞自噬和线粒体自噬最为显著。选择11个差异表达基因，进行实时荧光定量PCR (quantitative real-time polymerase chain reaction,qRT-PCR)验证，在Vero-XF中ATG9B、WIPI2、LAMP2、OPTN、Rab7a和DEPTOR显著上调，ATG4D显著下调，与转录组分析结果一致；另外ATG101、ATG2A和STX17显著上调，NBR1则差异不显著。Western blotting检测Vero-XF与Vero-AD中自噬标志蛋白LC3和P62的表达，其中Vero-XF的LC3Ⅱ/Ⅰ表达量显著上调，P62表达量显著下调，自噬水平较高。最后通过外源添加EGF改善Vero-XF自噬。结果表明，10、20、30 μg/L的EGF使得Vero-XF自噬水平分别降低了22.35%、48.15%、71.29%；比生长速率增加了15.48%、33.33%、57.14%；细胞凋亡率下降了2.84%、15.46%、16.23%。本研究结果初步揭示了Vero-XF自噬机制的激活是导致其生长缓慢的原因之一，为后续Vero细胞的悬浮培养提供了参考。

Abstract:

The culture of suspended Vero cells is facing difficulties such as low cell viability and long doubling time. To investigate the main reasons for the slow growth and low viability of suspended Vero cells, this study conducted transcriptomic analysis of suspended Vero cells (Vero-XF) and adherent Vero cells (Vero-AD) to screen the differentially expressed genes (DEGs) affecting the growth of suspended cells. In addition, epidermal growth factor (EGF) was supplemented to the culture system to improve the growth of Vero-XF. The results showed that compared with the Vero-AD group, the Vero-XF group had 7 376 significant DEGs. Kyoto encyclopedia of genes and genomes enrichment analysis revealed that the DEGs were mainly enriched in the autophagy and mitophagy pathways. Eleven DEGs were selected and verified by quantitative real-time PCR, which showed up-regulated expression of ATG9B, WIPI2, LAMP2, OPTN, Rab7a, and DEPTOR and down-regulated expression of ATG4D, being consistent with the results of transcriptomic analysis. In addition, the Vero-XF group showed significantly up-regulated expression of ATG101, ATG2A, and STX17 and insignificant change in the expression of NBR1, compared with the Vero-AD group. The protein levels of LC3 and P62 in Vero-XF and Vero-AD were determined by Western blotting, which showed up-regulated expression of LC3II/Ⅰ and down-regulated expression of P62 in Vero-XF, indicating a higher level of autophagy. Finally, the exogenous supplementation of EGF at 10, 20, and 30 μg/L in the culture system reduced the autophagy level of Vero-XF by 22.35%, 48.15%, and 71.29%, increased the specific growth rate by 15.48%, 33.33%, and 57.14%, and decreased the apoptosis rate by 2.84%, 15.46%, and 16.23%, respectively. The results of this study preliminarily reveal that the activation of autophagy is one of the reasons for the slow growth of Vero-XF, which provides reference for the subsequent culture of suspended Vero cells.