FGF5和FGF21对绒山羊毛乳头细胞增殖的影响

doi:10.3864/j.issn.0578-1752.2025.04.015

摘要/Abstract

摘要：

【背景】绒山羊毛囊周期分为生长期、退行期和休止期。成纤维生长因子5（fibroblast growth factor5, FGF5）和成纤维生长因子21（fibroblast growth factor21, FGF21）是毛囊周期转换的重要调控因子。然而，FGF5和FGF21在皮肤组织中的定位以及调控毛囊周期转换的作用机制仍不清楚，而且FGF21在毛囊发育过程中的作用尚存在争议。毛乳头细胞（dermal papilla cells, DPCs）位于毛囊的毛乳头区域，当其功能缺失时，毛囊不能发育，使毛囊周期停滞在休止期，导致脱发。【目的】通过检测FGF5和FGF21在绒山羊皮肤组织中的表达定位，探究FGF5和FGF21对DPCs增殖的影响，并对其调控机制进行解析，丰富成纤维生长因子家族基因调控毛囊周期转换的机制，为进一步阐明绒山羊毛囊周期转换的分子机制提供理论依据。【方法】利用实时荧光定量PCR（real time quantitative PCR, RT-qPCR）和酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测FGF5、FGF21及其受体1（fibroblast growth factor receptor1, FGFR1）在绒山羊生长期和休止期皮肤组织中的表达情况，结合单细胞转录组测序数据分析其在绒山羊皮肤组织中的表达定位，并利用组织免疫荧光技术进行验证；通过构建过表达腺病毒载体在绒山羊DPCs中过表达FGF5和FGF21，利用MTT、EdU、流式细胞周期等方法检测其对绒山羊DPCs增殖的影响，并利用RT-qPCR、细胞免疫荧光和Western blotting检测FGFR1及增殖相关基因的表达情况；进一步利用RT-qPCR、细胞免疫荧光和Western blotting等方法探究FGF5和FGF21影响绒山羊DPCs增殖的作用机制。【结果】FGF5、FGF21及其受体FGFR1在绒山羊生长期皮肤组织中的表达量显著高于休止期（P<0.05）；FGF5、FGF21及其受体FGFR1均定位于绒山羊DPCs，提示FGF5和FGF21可能参与调控绒山羊DPCs的生物学功能；FGF5和FGF21过表达腺病毒在绒山羊DPCs中的过表达效果良好，其处理时间在84 h最佳（P<0.001）；FGF5和FGF21能够显著降低绒山羊DPCs的细胞活力，减少EdU⁺细胞比率。过表达FGF5减少了绒山羊DPCs中S期和G2期的细胞比率，而FGF21仅影响绒山羊DPCs中G2期的细胞比率（P<0.05）；过表达FGF5和FGF21促进其受体FGFR1的表达，并显著抑制增殖相关基因KI67和PCNA的表达（P<0.05）以及Wnt/β-catenin信号通路关键因子β-catenin的表达，其下游转录因子TCF3和JUN的表达也显著下降，而且Wnt/β-catenin信号通路中增殖相关基因MYC和CYCLIND1的表达也受到抑制（P<0.05）。【结论】FGF5、FGF21及其受体FGFR1均定位于绒山羊DPCs中，是调控绒山羊DPCs增殖的重要因子。研究证实FGF21和FGF5在绒山羊皮肤组织中的表达模式相近，通过调控Wnt/β-catenin信号通路抑制绒山羊DPCs的增殖。

关键词: 绒山羊, FGF5, FGF21, DPCs, 增殖, Wnt/β-catenin信号通路

Abstract:

【Background】The hair follicle cycle in goat is divided into three phases: the growth phase (anagen), the regression phase (catagen), and the resting phase (telogen). Fibroblast growth factor 5 (FGF5) and fibroblast growth factor 21 (FGF21) are important regulators in goat hair cycle. However, the precise localization of FGF5 and FGF21 in goat skin tissue and their regulatory mechanisms remain unclear during hair follicle cycle transition. Dermal papilla cells (DPCs) located in the dermal papilla region, are essential for hair follicle development. When DPCs lose their function, hair follicle remains in the telogen, leading to hair loss.【Objective】Therefore, this study aimed to identify the expression and localization of FGF5 and FGF21 in Cashmere goat skin tissue, understand their roles in DPCs, and to analyze their regulatory mechanisms. This would enrich the knowledge of fibroblast growth factor family proteins in hair follicle cycle regulation and provide a theoretical basis for further elucidating the molecular mechanisms of Cashmere goat hair follicle cycle transition. 【Method】Real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to measure the expression levels of FGF5, FGF21 and their receptor 1 (FGFR1) in the skin tissue of Cashmere goats at anagen and telogen. Single-cell transcriptome sequencing data were analyzed to determine the expression and localization of FGF5, FGF21 and FGFR1, which were validated through immunofluorescence methods. Adenovirus was explored to overexpress FGF5 and FGF21 in DPCs. The effects on DPCs proliferation were measured using MTT, EdU and flow cytometry. The expressions of FGFR1 and proliferation-related genes were detected using RT-qPCR, immunofluorescence and western blotting methods. RT-qPCR, immunofluorescence and western blotting were used to investigate the effect of FGF5 and FGF21 on the proliferation of DPCs. 【Result】FGF5, FGF21 and FGFR1 exhibited higher expression levels in anagen than that in telogen, all of which were located within DPCs. Both FGF5 and FGF21 restrained DPCs viability, which significantly reduced the number of EdU-positive cells. FGF5 decreased the percentage of DPCs in G2 and S phases, while FGF21 specifically affected the G2 phase. The expression of FGFR1 was significantly increased following the overexpression of FGF5 and FGF21. Moreover, the expressions of cell proliferation markers KI67 and PCNA were also significantly inhibited with the overexpression of FGF5 and FGF21. Additionally, the expression of β-catenin, a key component of Wnt/β-catenin signaling pathway, was reduced. Thereby the activity of transcription factors TCF3 and JUN was inhibited, which were the downstream effectors of Wnt/β-catenin signaling pathway. Thus the inhibition further led to the decreased expression of proliferation-related genes MYC and CYCLIND1. 【Conclusion】FGF5 and FGF21 played a critical role as regulatory factors in controlling the biological function of DPCs. This study provided the first evidences that FGF21, similar to FGF5, restrained the proliferation of DPCs by suppressing the Wnt/β-catenin signaling pathway. These findings contributes to our understanding of how fibroblast growth factors influence hair follicle cycle transition, particularly in the context of Cashmere goat hair growth and cashmere production.

Key words: cashmere goat, FGF5, FGF21, DPCs, proliferation, Wnt/β-catenin signaling pathway

王妞, 史昕冉, 张卫东, 王昕. FGF5和FGF21对绒山羊毛乳头细胞增殖的影响[J]. 中国农业科学, 2025, 58(4): 819-830.

WANG Niu, SHI XinRan, ZHANG WeiDong, WANG Xin. Effect of FGF5 and FGF21 on Proliferation of Dermal Papilla Cells in Cashmere Goat[J]. Scientia Agricultura Sinica, 2025, 58(4): 819-830.

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基因名称 Gene symbol	GenBank登录号 Accession No.	引物序列 Primer sequence (5′-3′)	产物长度 Production size (bp)	退火温度 Tm (℃)
β-actin	NM_001314342.1	5′-GCAGACAGGATGCAGAAAGA-3′	96	56
β-actin	NM_001314342.1	5′-GCAAGTACTCCGTGTGGATT-3′	96	56
FGF5	NM_001291973.1	5′-CCGCGATACACAGAACTGAA-3′	129	58
FGF5	NM_001291973.1	5′-ATCTTGGCAGAAAGTGGGTAG-3′	129	58
FGF21	XM_005692688.3	5′-GCGGGTCTGTCTGGATATAAA-3′	118	62
FGF21	XM_005692688.3	5′-CTCAGATGGCTGCTTCAGTA -3′	118	62
FGFR1	XM_018041774.1	5′-GAGGCTACAAGGTCCGTTATG-3′	125	62
FGFR1	XM_018041774.1	5′-GAGCTGGTAGGTATGGTTGATG-3′	125	62
FGFR3	XM_018045593.1	5′-GTCCGAGATGGAGATGATGAAG-3′	103	58
FGFR3	XM_018045593.1	5′-GTACTCCACCAGCACATACAG-3′	103	58
CTNNB1	XM_018066894.1	5′-CTGGACTCTGGAATCCATTCTG-3′	103	59
CTNNB1	XM_018066894.1	5′-GATACCACCCAAGTCCTGTATG-3′	103	59
C-JUN	XM_018044742.1	5′-CCAAGAACTGCATGGACCTAA-3′	111	56
C-JUN	XM_018044742.1	5′-AGATCGCTTCTGTAGTACTCCT-3′	111	56
C-MYC	XM_018058563.1	5′-GAAGAGGCGAGAACAGTTGAA-3′	86	58
C-MYC	XM_018058563.1	5′-CTATTGGAGGGAGGAACTGAAC-3′	86	58
CCND1	XM_018043271.1	5′-GCAGTCTTAGGCATCCTGT-3′	131	58
CCND1	XM_018043271.1	5′-CCTAGCCGAGAGGTTACAT-3′	131	58
PCNA	XM_005688167.3	5′-GCAGACGCGGCAGTATTA-3′	107	62
PCNA	XM_005688167.3	5′-CTCATCCTCTGAAGCCTAAGC-3′	107	62
MKI67	XM_005698601.3	5′-CTCAGGAAGCCTACACATACAC-3′	108	58
MKI67	XM_005698601.3	5′-AGCATCTTGGGAAACTCCAG-3′	108	58
TCF3	XM_018050411.1	5′-CCTGACTCCTATGGTGATCTTG-3′	131	58
TCF3	XM_018050411.1	5′-AAGAAGGACCTCAAAGCTCC-3′	131	58

抗体名称 Name of antibody	生产厂商 Manufacturer	货号 Cat No.
FGF5 Polyclonal Antibody	Proteintech	18171-1-AP
FGF21 Polyclonal Antibody	Proteintech	26272-1-AP
FGFR1 Monoclonal Antibody	Proteintech	60325-1-Ig
GAPDH Polyclonal Antibody	Proteintech	10494-1-AP
Beta Catenin Polyclonal Antibody	Proteintech	51067-2-AP
C-MYC Polyclonal Antibody	Proteintech	10828-1-AP
Cyclin D1 Polyclonal Antibody	Proteintech	26939-1-AP
PCNA Polyclonal Antibody	Proteintech	24036-1-AP
Goat Anti-Rabbit IgG H&L (Alexa Fluor® 555)	Abcam	ab150078
Goat Anti-Mouse IgG H&L (Alexa Fluor® 647)	Abcam	ab150115