肝细胞生长因子促进人毛囊生长的作用研究

Study on the effect of hepatocyte growth factor on promoting the growth of human hair follicles

目的观察肝细胞生长因子(HGF)对人毛囊生长的影响,并探讨其促进毛发生长的作用机制。方法毛囊来源于中国医学科学院整形外科医院4例除皱术后患者废弃的正常头皮组织,分离单根毛囊进行体外器官培养。以常规培养为对照组,培养液中添加浓度为10 ng/ml的HGF作为实验组,显微镜下测量不同培养天数毛囊的生长长度;通过观察培养毛囊毛球部毛基质与毛乳头的形态,评价HGF对毛囊生长周期的影响。分离培养人毛囊上皮细胞,应用流式细胞术对其表面标志进行鉴定;以常规培养的毛囊上皮细胞为对照组,以培养基添加10 ng/ml的HGF处理48 h后的毛囊上皮细胞为实验组,收集细胞提取RNA,转录组测序分析HGF对毛囊上皮细胞基因表达的影响,应用real-time PCR对测序分析结果进行验证。各项定量数据以均值±标准差表示,2组间比较应用独立样本t检验,P<0.05为差异具有统计学意义。结果随着体外培养时间的延长,毛囊生长趋于停止;毛囊体外培养7、10、14 d时,对照组毛囊生长长度(单位0.1 mm)分别为12.210±4.191、13.710±3.518、14.000±4.057,实验组HGF促进毛发生长,生长长度分别为16.700±5.143、18.800±4.917、23.000±7.196,差异具有统计学意义(t=2.353,P<0.05;t=2.962,P<0.01;t=3.910,P<0.01);分离培养的毛囊上皮细胞呈典型的铺路石样形态,表达上皮细胞表面标志分子CD49f;转录组测序结果显示,毛囊上皮细胞高表达上皮细胞标志基因KRT14、KRT5、KRT6A、CDH1、SOX9、CD49f,FPKM值分别为6012±2141、4072±1369、3896±1991、95.06±21.48、101.30±38.52、162.00±47.83;不表达或极低表达间质细胞标志基因THY1、DPP4、CDH2(N-cadherin)、ACTA2、PDGFRA、COL1A1、COL3A1,FPKM值分别为0.740±0.825、0.632±0.765、0.000±0.034、1.674±1.235、0.000±0.014、2.526±3.531、0.000±0.015;与对照组相比,实验组经HGF处理后毛囊上皮细胞中改变的基因显著富集于细胞周期及细胞分裂相关生物学过程,相关基因的表达下调;Real-Time PCR验证显示CENPA、CDC20、UBE2C、CDK1、AURKB、NDC80分别降为对照组的0.689±0.053(t=10.170,P<0.001)、0.676±0.121(t=4.652,P<0.01)、0.761±0.148(t=2.785,P<0.05)、0.599±0.153(t=4.530,P<0.05)、0.706±0.113(t=4.507,P<0.05)、0.579±0.092(t=7.931,P<0.01)倍,差异具有统计学意义。结论HGF促进毛囊体外器官培养的生长并延长其生长时间;但在细胞水平上抑制毛囊上皮细胞增殖相关基因表达。

Objective This study aims to observe the effect of hepatocyte growth factor(HGF)on the growth of human hair follicles,and explore its mechanism of promoting hair growth.Methods Hair follicles were isolated from the normal scalp tissue discarded by 4 rhytidectomy patients in the Plastic Surgery Hospital of the Chinese Academy of Medical Sciences,and the isolated single hair follicles were cultured in vitro.Normal culture condition was used as the control group,and HGF with a concentration of 10 ng/ml was added to the culture medium as the experimental group.The growth length of hair follicles in different culture days was measured under a microscope.The effect of HGF on the growth cycle of hair follicles was evaluated by observing the morphology of hair matrix and dermal papilla of hair follicle.Hair follicle epithelial cells(HFECs)were identified by flow cytometry.The normal cultured HFECs were used as the control groups,while the HFECs treated with 10 ng/ml HGF for 48h as the experimental groups.HFECs were collected to extract RNA and transcriptome sequencing was applied to detect the effects of HGF on gene expression of HFECs.Real-time PCR was used to verify the sequencing results.All quantitative data were displayed as Mean±SD deviation,the independent sample t test was applied for the comparison between the two groups and the difference was statistically significant when P<0.05.Results With the extension of culture time in vitro,hair follicle growth tends to stop.HGF promoted the growth of cultured hair follicles,and the growth length(unit 0.1 mm)of hair follicles were statistically different between the experimental and control group at 7 d(16.700±5.143 vs.12.210±4.191,t=2.353,P<0.05),10 d(18.800±4.917 vs.13.710±3.518,t=2.962,P<0.01)and 14 d(23.000±7.196 vs.14.000±4.057,t=3.910,P<0.01).HFECs we cultured displayed cuboidal morphology and highly expressed epithelial cell surface marker CD49f.RNA-seq showed HFECs highly expressed epithelial cell marker genes including KRT14,KRT5,KRT6A,CDH1,SOX9 and CD49f,the FPKM of which were 6012±2141,4072±1369,3896±1991,95.06±21.48,101.30±38.52,162.00±47.83 respectively.HFECs did not or hardly express mesenchymal markers as THY1,DPP4,CDH2(N-cadherin),ACTA2,PDGFRA,COL1A1and COL3A1,the FPKM of which were 0.740±0.825,0.632±0.765,0.000±0.034,1.674±1.235,0.000±0.014,2.526±3.531,0.000±0.015,respectively.GO analysis showed the differential expressed genes between HGF treated and normal cultured HFECs were enriched in cell cycle-related biological processes such as nuclear division and chromosome separation.Real-Time PCR further verified that the expression of CENPA、CDC20、UBE2C、CDK1、AURKB、NDC80 in HGF treated HFECs were significantly downregulated to 0.689±0.053(t=10.17,P<0.001)、0.676±0.121(t=4.652,P<0.01)、0.761±0.148(t=2.785,P<0.05)、0.599±0.153(t=4.530,P<0.05)、0.706±0.113(t=4.507,P<0.05)、0.579±0.092(t=7.931,P<0.01)of the control group,respectively.Conclusions HGF can effectively promote the growth of cultured human hair follicles in vitro,but downregulate the cell cycle-related genes of hair follicle epithelial cells.