摘要
目的研究小分子药物Tideglusib对表皮干细胞增殖能力和细胞周期的影响及其机制。方法将人表皮干细胞采用随机数字表法随机分为对照组,小分子药物Tideglusib 50 nmol/L、100 nmol/L、200 nmol/L组,培养48 h后,通过细胞计数试剂盒8(CCK-8)实验检测细胞增殖能力变化(n=6)。通过RNA测序检测转录组水平变化,以P.adj<0.05为差异表达基因筛选标准,分析差异表达基因,并通过基因本体论(GO)富集分析差异表达基因对表皮干细胞生物过程、细胞组成、分子功能的影响(n=3)。通过实时定量聚合酶链式反应(qRT-PCR)验证差异表达基因驱动蛋白家族成员14(KIF14)的转录水平变化,通过蛋白质印迹法和免疫荧光染色验证KIF14蛋白表达水平的变化(n=3)。通过蛋白质印迹法探究蛋白激酶B(PKB)、磷酸化蛋白激酶B(p-PKB)表达水平的变化(n=3)。数据比较采用独立样本t检验,单因素方差分析和Tukey-post-hoc检验。结果培养48 h后,CCK-8实验结果表明,同对照组相比,50 nmol/L组的小分子药物Tideglusib对表皮干细胞增殖能力的影响,差异无统计学意义(P>0.05),100 nmol/L组和200 nmol/L组的小分子药物Tideglusib可明显促进表皮干细胞的增殖(P<0.05)。测序数据显示,同对照组相比,小分子药物Tideglusib 200 nmol/L组共有107个差异表达基因,其中106个表达上调,1个表达下调。GO富集分析表明,差异表达基因主要同有丝分裂与细胞周期相关。通过流式细胞仪检测发现,小分子药物Tideglusib可呈浓度依赖性地提升表皮干细胞进入有丝分裂期的比例,50 nmol/L组的小分子药物Tideglusib对表皮干细胞的细胞周期的影响无统计学差异(P>0.05),100 nmol/L和200 nmol/L的小分子药物Tideglusib可明显提高表皮干细胞处于有丝分裂期的比例,差异有统计学意义(P<0.05)。通过qRT-PCR验证细胞周期相关差异表达基因KIF14发现,小分子药物Tideglusib对KIF14转录的促进作用呈浓度依赖性,50 nmol/L组的Tideglusib即可促进KIF14的转录(P<0.05)。蛋白质印迹法检测证明,小分子药物Tideglusib对KIF14蛋白表达水平的影响呈浓度依赖性,50 nmol/L组的小分子药物Tideglusib即可提高KIF14的蛋白表达水平(P<0.05)。免疫荧光染色表明,小分子药物Tideglusib对KIF14相对荧光强度的影响呈浓度依赖性,50 nmol/L组的Tideglusib对KIF14相对荧光强度的影响无统计学差异(P>0.05),100 nmol/L组和200 nmol/L组的小分子药物Tideglusib可明显提高表皮干细胞KIF14的相对荧光强度(P<0.05)。通过蛋白质印迹法检测KIF14上游蛋白PKB与p-PKB表达水平发现,不同浓度的小分子药物Tideglusib对表皮干细胞PKB的总蛋白水平不产生影响,差异无统计学意义(P>0.05),50 nmol/L组的小分子药物Tideglusib对表皮干细胞p-PKB表达的影响差异无统计学意义(P>0.05),100 nmol/L组和200 nmol/L组的小分子药物Tideglusib可明显提高表皮干细胞p-PKB的表达。结论小分子药物Tideglusib可提升有丝分裂期细胞比例,促进表皮干细胞的增殖,其机制可能与通过上调PI3K/PKB/KIF14信号通路调控表皮干细胞的细胞周期有关。
Objective To study the effect of the small molecule drug Tideglusib on the proliferation ability and cell cycle of epidermal stem cells and its mechanism.Methods The human epidermal stem cells were randomly divided into the control group,and the small molecule drug Tideglusib 50 nmol/L,100 nmol/L,and 200 nmol/L groups with the random number table method.After 48 hours of culture,changes in cell proliferation ability were assessed through the cell counting kit-8(CCK-8)experiment(n=6).Transcriptome level changes were detected by RNA sequencing,with P.adj<0.05 as the screening criteria for differentially expressed genes.Gene ontology enrichment was used to analyze the effect of differentially expressed genes on the biology process,cell composition,molecular function of epidermal stem cells(n=3).The transcription level of the differentially expressed gene KIF14 was verified by qRT-PCR,and the protein expression level of KIF14 was verified by Western blotting and immunofluorescence staining(n=3).The expression levels of PKB and p-PKB were explored by Western blotting(n=3).Results After 48 hours of culture,the CCK-8 experiment showed that the difference of the effect on the proliferation of epidermal stem cells between 50 nmol/L group of small molecule drug Tideglusib and the control group was not statistically significant(P>0.05),while 100 nmol/L group and 200 nmol/L group Tideglusib could significantly promote the proliferation of epidermal stem cells(P<0.05).RNA sequencing data showed that compared with the control group,the small molecule drug Tideglusib 200 nmol/L group had a total of 107 differentially expressed genes,106 of which were up-regulated and 1 down-regulated.GO enrichment analysis showed that differentially expressed genes were mainly related to mitosis and cell cycle.Through flow cytometry,small molecule drug Tideglusib could increase the proportion of epidermal stem cells entering mitosis in a concentration-dependent manner.There was no statistical difference in the effect of 50 nmol/L group of small molecule drug Tideglusib on the cell cycle of epidermal stem cells(P>0.05),100 nmol/L and 200 nmol/L Tideglusib could significantly increase the proportion of epidermal stem cells in mitosis(P<0.05).Validation of the cell cycle-related differentially expressed gene KIF14 by qRT-PCR found that small molecule drug Tideglusib could promote KIF14 transcription in a concentration-dependent manner,and 50 nmol/L group of small molecule drug Tideglusib could promote KIF14 transcription(P<0.05).Western blotting proved that small molecule drug Tideglusib had a concentration-dependent effect on the expression level of KIF14 protein,and 50 nmol/L group of small molecule drug Tideglusib could increase the protein expression level of KIF14(P<0.05).Immunofluorescence staining showed that Tideglusib had a concentration-dependent effect on the relative fluorescence intensity of KIF14.There was no statistical difference in the effect of 50 nmol/L group of small molecule drug Tideglusib on the relative fluorescence intensity of KIF14(P>0.05).Small molecule drug Tideglusib at 100 nmol/L group and 200 nmol/L group could significantly increase the relative fluorescence intensity of KIF14(P<0.05).The expression levels of PKB and p-PKB,the upstream protein of KIF14 were detected by Western blotting.It was found that small molecule drug Tideglusib at different concentrations did not affect the total protein level of PKB(P>0.05).The 50 nmol/L group of small molecule drug Tideglusib had little effect on the expression of p-PKB in epidermal stem cells(P>0.05).Small molecule drug Tideglusib at 100 nmol/L group and 200 nmol/L group could significantly increase the expression of p-PKB in epidermal stem cells(P<0.05).Conclusions The small molecule drug Tideglusib can increase the proportion of mitotic cells and promote the proliferation of epidermal stem cells.The mechanism may be related to the regulation of the cell cycle of epidermal stem cells by up-regulating the PI3K/PKB/KIF14 signaling pathway.
作者
孙佳辰
马景龙
申传安
张文
刘馨竹
Sun Jiachen;Ma Jinglong;Shen Chuanan;Zhang Wen;Liu Xinzhu(Department of Burns and Plastic Surgery,Fourth Medical Center of PLA General Hospital,Beijing 100089,China)
出处
《中华损伤与修复杂志(电子版)》
CAS
2021年第5期389-397,共9页
Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)
基金
北京市自然科学基金重点项目(7171009)
首都临床特色应用研究与成果推广重点课题(Z17110001017146)
国家重点研发计划(2017YFC1103503)
“十三五”军队后勤科研项目重大项目(ALB18J001)
“十三五”军队重点学科专业建设项目(A350109)。