摘要
目的分析人富血小板血浆(PRP)调控人表皮干细胞(ESC)的靶基因。方法(1)收集陆军军医大学第一附属医院行泌尿外科手术的6例男性患者术后弃用的包皮组织,患者年龄为5~25岁,既往身体健康,无泌尿系统感染,采用快速贴壁法培养人ESC并进行形态学观察及鉴定。收集解放军南部战区总医院1名29岁女性健康志愿者静脉血40 mL,采用二次离心法提取PRP。(2)将培养成功的原代人ESC按照随机数字表法分为对照组和PRP处理组,每组3孔。对照组细胞不行特殊处理;PRP处理组待细胞贴壁12 h,在培养基中加入PRP,使其最终体积分数为2.5%。提取RNA应用RNA测序技术进行2组人ESC转录组测序和数据分析,以错误发现率<0.05、差异倍数≥4为标准应用Dr.Tom数据挖掘系统筛选差异表达基因,对获得的差异表达基因进行基因本体论(GO)富集分析找出显著富集的GO条目。再用京都基因和基因组(KEGG)信号通路注释分析进一步寻找差异表达基因可能参与的生物学过程或者代谢通路。最后,选取与再上皮化过程相关且差异表达明显的基因,利用实时荧光定量反转录PCR验证基因的差异表达情况。对数据行独立样本t检验。结果(1)培养细胞呈克隆样生长,形态为铺路石样,CD49f阳性率达95.132%、CD71阳性率为0.006%,证明ESC原代培养成功。(2)质控数据分析显示,选取样本质量较好,序列比对百分比较高,满足测序要求。(3)测序数据显示,2组间共有449个差异表达基因,其中上调基因354个、下调基因95个,进一步聚类分析确定2组间有18个显著上调基因和5个显著下调基因。GO富集分析以及KEGG信号通路注释分析表明,显著差异表达基因主要富集在表皮构建和角化过程,同时可能与白细胞介素17信号通路相关。(4)选取了与再上皮化过程相关且差异表达明显的角蛋白19、角蛋白10以及S100A7基因进行验证。实时荧光定量反转录PCR显示,与对照组比较,PRP处理组细胞角蛋白19 mRNA和S100A7 mRNA表达量明显升高(t=10.270、5.690,P<0.01),角蛋白10 mRNA表达量明显降低(t=7.306,P<0.01),与测序数据结果一致。结论PRP调节人ESC功能促进创面再上皮化涉及角蛋白19、角蛋白10以及S100A7等多个基因的转录调控,深入探讨PRP影响人ESC的可能调控网络将为其后续临床治疗提供依据。
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.
作者
许鹏程
贺伟峰
程飚
Xu Pengcheng;He Weifeng;Cheng Biao(Department of Burns and Plastic Surgery,General Hospital of Southern Theater Command of PLA,Guangzhou 510010,China;State Key Laboratory of Trauma,Burns and Combined Injury,Institute of Burn Research,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing Key Laboratory for Disease Proteomics,Chongqing 400038,China)
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2020年第10期915-922,共8页
Chinese Journal of Burns
基金
国家重点研发计划(2017YFC1103301)
军事医学创新工程专项(18CXZ029)
广东省科技计划(2015B020233012、2014B020212010)
国家自然科学基金面上项目(31872742)
军队医学科技青年培育计划(20QNPY024)。
关键词
富血小板血浆
高通量核苷酸测序
人表皮干细胞
转录组分析
Platelet-rich plasma
High-throughput nucleotide sequencing
Human epidermal stem cells
Transcriptome analysis
