转录组水平探讨人富血小板血浆调控人表皮干细胞促创面再上皮化的机制  

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

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)

机构地区：[1]解放军南部战区总医院烧伤整形科,广州510010 [2]陆军军医大学(第三军医大学)第一附属医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆市疾病蛋白质组学重点实验室,400038

出　　处：《中华烧伤杂志》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)。

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,�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-s

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 

分 类 号：R644[医药卫生—外科学] R619+.6[医药卫生—临床医学] R329.2+8