目的:本工作的目的是找到一种在体外安全可靠地扩增人牙髓细胞(hDPC)的有效方法。这里,我们检查了层粘连蛋白-511(iMatrix-511)的新型重组E8片段在hDPC中关于活力和细胞扩散的作用。Further,我们使用RNA测序(RNA-seq)研究了其在hDPC中的作用的潜在机制。
方法:从无龋的上颌第三磨牙获得hDPCs(n=3)。进行CCK-8测定以测量在iMatrix-511和两种其他ECM蛋白上培养的细胞的活力。用相差显微镜观察细胞形态。在IlluminaNovaseqTM6000平台上进行在iMatrix-511或非包被对照上培养的hDPC的RNA-seq。
结果:iMatrix-511(0.5μg/cm2)在一定程度上增强了hDPC的活力,优于COL-1和明胶。在iMatrix-511上短期培养hDPC产生233个差异表达基因(DEGs)。前12个上调最多的基因是XIAP,AL354740,MRFAP1,AC012321,KCND3,TMEM120B,AC009812,GET1-SH3BGR,CNTN3,AC090409,GEN1和PIK3IP1,而前12个最下调的基因是SFN,KRT17,RAB4B-EGLN2,CSTA,KCTD11,ATP6V1G2-DDX39B,AC010323,SBSN,LYPD3,FOSB,AC022400和CHI3L1。qPCR验证证实了GEN1、KCND3、PIK3IP1和MRFAP1的显著上调。进行了基因本体论(GO)和京都基因和基因组百科全书(KEGG)分析,富含各种细胞外基质相互作用的基因,雌激素和脂肪代谢相关的功能和途径。
结论:iMatrix-511促进了hDPC的扩散和增殖。它增强抗凋亡基因的表达,同时抑制表皮发育相关基因的表达。
OBJECTIVE: The aim of the present work was to find an efficient method for safe and reliable expansion of human dental pulp cells (hDPCs) in vitro. Here, we examined the effect of a novel recombinant E8 fragment of Laminin-511 (iMatrix-511) in hDPCs regarding viability and cell spreading. Further, we investigated the underlying mechanisms governing its effects in hDPCs using RNA sequencing (RNA-seq).
METHODS: hDPCs were obtained from caries-free maxilla third molars (n = 3). CCK-8 assay was conducted to measure the viability of cells cultured on iMatrix-511 and two other ECM proteins. Cell morphology was observed by phase contrast microscope. RNA-seq of hDPCs cultured on iMatrix-511 or noncoated control was performed on Illumina NovaseqTM 6000 platform.
RESULTS: iMatrix-511 (0.5 μg/cm2) enhanced the viability of hDPCs to an extent better than COL-1 and gelatin. Short term culture of hDPCs on iMatrix-511 resulted in 233 differentially expressed genes (DEGs). The top 12 most upregulated genes were XIAP, AL354740, MRFAP1, AC012321, KCND3, TMEM120B, AC009812, GET1-SH3BGR, CNTN3, AC090409, GEN1 and PIK3IP1, whereas the top 12 most downregulated genes were SFN, KRT17, RAB4B-EGLN2, CSTA, KCTD11, ATP6V1G2-DDX39B, AC010323, SBSN, LYPD3, FOSB, AC022400 and CHI3L1. qPCR validation confirmed the significant upregulation of GEN1, KCND3, PIK3IP1 and MRFAP1. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed, with genes enriched in various extracellular matrix interaction, estrogen and fat metabolism-related functions and pathways.
CONCLUSIONS: iMatrix-511 facilitated spreading and proliferation of hDPCs. It enhances expression of anti-apoptotic genes, while inhibits expression of epidermis development-related genes.