Exploration of the mechanism of human platelet-rich plasma in regulating and controlling human epidermal stem cells for promoting wound re-epithelialization at transcriptome level

Abstract

Objective: To analyze target genes of human platelet-rich plasma (PRP) in regulating and controlling human epidermal stem cells (ESCs). Methods: (1) The discarded foreskin tissues were collected from 6 male patients of the First Affiliated Hospital of Army Medical University after urological surgery. The patients aged 5 to 25 years with good health and without urinary system infection. Human ESCs were cultivated using quick attachment method, and were subjected to morphological observation and identification. Venous blood sample in the volume of 40 mL was collected from a female healthy volunteer (aged 29 years) of General Hospital of Southern Theater Command of PLA, and PRP was extracted by second centrifugation method. (2) The successfully cultured primary human ESCs were divided into control group and PRP group according to the random number table, with 3 wells in each group. The cells in control group were not specially treated. In PRP group, PRP was added to the ESC medium to achieve final volume fraction of 2.5% after the cells were adhered for 12 hours. RNA was extracted, and transcriptome sequencing and data analysis of human ESCs of two groups were performed using RNA sequencing technology. Using the false discovery rate less than 0.05 and the fold change more than or equal to 4 as the standard, the differentially expressed genes were screened by Dr. Tom data mining system. Gene ontology (GO) enrichment analysis was performed on the obtained differentially expressed genes to find out the GO entries with significant enrichment. Then Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation analysis was used to further analyze the biological processes or metabolic pathways in which differentially expressed genes might be involved. Finally, the genes related to re-epithelialization and significantly differentially expressed were selected, and the differential expression of genes was verified by real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR). Data were statistically analyzed with independent-samples t test. Results: (1) The cultured cells were cloned with a paving stone-like shape and positive rate of CD49f of 95.132% and CD71 of 0.006%, which proved that the primary culture of ESCs was successful. (2) The quality control data analysis showed that the selected samples had better quality and higher sequence alignment rate, which met the requirements of sequencing. (3) Sequencing data showed that there were a total of 449 differentially expressed genes between the two groups, including 354 up-regulated genes and 95 down-regulated genes. Further cluster analysis determined that there were 18 significantly up-regulated genes and 5 significantly down-regulated genes between the two groups. GO enrichment analysis and KEGG pathway annotation analysis showed that the significantly differentially expressed genes were mainly enriched in the epidermis construction and keratinization process, which also might be related to interleukin 17 signaling pathway. (4) Keratin 19, keratin 10, and S100A7 genes which were related to the process of re-epithelialization and significantly differentially expressed were selected for verification. Real-time fluorescent quantitative RT-PCR showed that compared with those of control group, the mRNA expressions of keratin 19 and S100A7 of cells in PRP group were significantly increased (t=10.270, 5.690, P<0.01), while the mRNA expression of keratin 10 was significantly decreased (t=7.306, P<0.01), which was consistent with the result of sequencing data. Conclusions: PRP regulates function of human ESCs and promotes wound re-epithelialization involving transcriptional regulation of multiple genes, including keratin 19, keratin 10, and S100A7. In-depth exploration of the possible regulatory network of PRP affecting human ESCs will provide the basis for its subsequent clinical application.