成纤维细胞生长因子21及其受体1、2在小鼠毛囊形成期的定位和表达
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摘要
本试验旨在通过研究成纤维细胞生长因子21(fibroblast growth factor 21,FGF21)及其受体FGFR1、FGFR2在胚胎小鼠毛囊形成阶段中的定位与表达,从而探究其在小鼠毛囊形成中的作用。试验采用免疫组织化学、实时荧光定量PCR及Western blotting方法研究FGF21、FGFR1、FGFR2蛋白和mRNA在胚胎期13～18d(E13～E18)小鼠皮肤中的表达。结果显示:(1)FGF21蛋白在E13主要表达于表层细胞、基底层细胞及结缔组织中,E14表达于表层细胞和基板,E15在毛芽中有微量表达,E16、E17表达于毛钉中,E18主要表达于未成熟毛囊细胞;FGFR1和FGFR2蛋白在E13～E18于表层细胞、基底层、基板、毛芽、毛钉、未成熟毛囊和结缔组织中均有表达。(2)实时荧光定量PCR及Western blotting结果显示:FGF21mRNA和蛋白在E14相对表达量最高;FGFR1mRNA及蛋白在E16～E17相对表达量较高;FGFR2mRNA及蛋白的相对表达量在E13至E18均呈上升趋势,在E18相对表达量最高。结果提示:在小鼠胚胎期毛囊形成和发育过程中,FGF21可能在诱导毛囊启动过程中发挥一定的作用;FGFR1可能促进毛钉形成;FGFR2可能在毛囊形成和成熟中发挥重要作用。
This study had investigated the localization and expression of fibroblast growth factor21(FGF21)with its receptors FGFR1 and FGFR2in the formation phase of embryonic mice hair follicles.E13-E18 embryonic mice back skin were used to determine the mRNA and protein relative expression levels of FGF21,FGFR1 and FGFR2by immunohistochemistry,Real-time PCR and Western blotting analysis.The results showed that:(1)FGF21expressed in epidermis,basal layer and connective tissue at E13;Expressed in epidermis and placode at E14;Slightly expressed in hair bud at E15;Expressed in hair peg at E16 and E17;Mainly expressed in immature hair follicle at E18.FGFR1 and FGFR2expressed in epidermis,basal layer,placode,hair bud,hair peg,immature hair follicle and connective tissue at E13-E18.(2)The results of Real-time PCR and Western blotting showed that the relative expression levels of FGF21 mRNA and protein were higher at E14 than that of any other stages;The relative expression levels of FGFR1 mRNA and protein were higher at E16-E17;And the relative expression of FGFR2 mRNA and protein showed as-cending trend from E13 to E18and was highest at E18.In summary,during the embryonic hair follicle formation and development,FGF21 might play a role in inducing hair follicle activation;FGFR1might promote hair peg formation;FGFR2might be necessary to induce hair follicle formation and hair follicle maturation.
This study had investigated the localization and expression of fibroblast growth factor21(FGF21)with its receptors FGFR1 and FGFR2in the formation phase of embryonic mice hair follicles.E13-E18 embryonic mice back skin were used to determine the mRNA and protein relative expression levels of FGF21,FGFR1 and FGFR2by immunohistochemistry,Real-time PCR and Western blotting analysis.The results showed that:(1)FGF21expressed in epidermis,basal layer and connective tissue at E13;Expressed in epidermis and placode at E14;Slightly expressed in hair bud at E15;Expressed in hair peg at E16 and E17;Mainly expressed in immature hair follicle at E18.FGFR1 and FGFR2expressed in epidermis,basal layer,placode,hair bud,hair peg,immature hair follicle and connective tissue at E13-E18.(2)The results of Real-time PCR and Western blotting showed that the relative expression levels of FGF21 mRNA and protein were higher at E14 than that of any other stages;The relative expression levels of FGFR1 mRNA and protein were higher at E16-E17;And the relative expression of FGFR2 mRNA and protein showed as-cending trend from E13 to E18and was highest at E18.In summary,during the embryonic hair follicle formation and development,FGF21 might play a role in inducing hair follicle activation;FGFR1might promote hair peg formation;FGFR2might be necessary to induce hair follicle formation and hair follicle maturation.
引文
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[13]MIZUTANI T,IKEGAMI M,NAGAHATA R,et al.Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epide[J].Nature Medicine,2005,11(12):1351-1354.
[14]PARAKKAL P F.Role of macrophages in collagen resorption during hair growth cycle[J].Journal of Ultrastructural Research,1969,29(3-4):210-217.
[15]MLLERRVER S,HANDJISKI B,VEEN C V D,et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages[J].Journal of Investigative Dermatology,2001,117(1):3-15.
[16]ZHANG H,ZHANG S,ZHAO H,et al.Ovine hair follicle stem cells derived from single vibrissae reconstitute haired skin[J].International Journal of Molecular Sciences,2015,16(8):17779-17797.
[17]DE B J.Enzymes of carbohydrate metabolism in human epidermal tumors 2enzymes of the glucose oxidation pathway,from triose-phosphate to pyruvate[J].Journal of Cutaneous Pathology,2010,1(4):176-179.
[18]许英蕾,孙建义.成纤维细胞生长因子与其受体的研究进展[J].药物生物技术,2004,11(3):194-198.XU Y L,SUN J Y.Research progress of fibroblast growth factors(FGFs)and their receptors(FGFRs)[J].Pharmaceutical Biotechnology,2004,11(3):194-198.(in Chinese)
[19]TERRANOVA C,NARLA S T,LEE Y W,et al.Global developmental gene programing involves a nuclear form of fibroblast growth factor receptor-1(FG-FR1)[J].PLoS One,2015,10(4):e0123380.
[20]伊海英,孙晓艳,付小兵,等.胎儿皮肤发育中成纤维细胞生长因子受体-2表达的免疫组织化学研究[J].第三军医大学学报,2008,30(21):2008-2010.YI H Y,SUN X Y,FU X B,et al.Distribution and expression of FGFR2in human fetal skin by immunohistochemical method[J].Acta Academiae Medicinae Militaris Tertiae,2008,30(21):2008-2010.(in Chinese)
[21]REVEST J M,SPENCERDENE B,KERR K,et al.Fibroblast growth factor receptor 2-Ⅲb acts upstream of Shh and Fgf4and is required for limb bud maintenance but not for the induction of Fgf8,Fgf10,Msx1,or Bmp4[J].Developmental Biology,2001,231(1):47-62.
[22]ZHANG X,IBRAHIMI O A,OLSEN S K,et al.Receptor specificity of the fibroblast growth factor family:The complete mammalian FGF family*[J].Journal of Biological Chemistry,1996,271(25):15292-15297.
[23]程笳琪.FGF7亚家族对小鼠毛囊形成和发育作用的研究[D].太谷:山西农业大学,2017.CHENG J Q.Study on function of fibroblast growth factor 7subfamily on hair follicle formation and development in mouse[D].Taigu:Shanxi Agricultural University,2017.(in Chinese)
[2]ITOH N,ORNITZ D M.Evolution of the Fgf and Fgfr gene families[J].Trends in Genetics,2004,20(11):563-569.
[3]PRESTA M,DELL’ERA P,MITOLA S.Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis[J].Cytokine and Growth Factor Reviews,2005,16(2):431-444.
[4]张耀方,肖业臣,王会岩.FGF21生物学特性的研究进展[J].中国生物制品学杂志,2009,22(6):618-621.ZHANG Y F,XIAO Y C,WANG H Y.Biological characteristics of fibroblast growth factor 21[J].Chinese Journal of Biologicals,2009,22(6):618-621.(in Chinese)
[5]刘旭,张平,张晓枫,等.利用CRISPR/Cas9系统构建FGF21基因敲除小鼠模型[J].遗传,2018,40(1):66-74.LIU X,ZHANG P,ZHANG X F,et al.Construction of FGF21knockout mouse models by the CRISPR/Cas9system[J].Hereditas,2018,40(1):66-74.(in Chinese)
[6]TSUBOI R,YAMAZAKI M,MATSUDA Y,et al.Antisense oligonucleotide targeting fibroblast growth factor receptor(FGFR)-1stimulates cellular activity of hair follicles in an in vitro,organ culture system[J].International Journal of Dermatology,2007,46(3):259-263.
[7]PETIOT A,CONTI F J,GROSE R,et al.A crucial role for Fgfr2-Ⅲb signaling in epidermal development and hair follicle patterning[J].Development,2003,130(22):5493-5501.
[8]刘颖,常露程,白云,等.内皮素3对小鼠黑色素沉着相关基因表达的影响[J].中国畜牧兽医,2018,45(3):581-589.LIU Y,CHANG L C,BAI Y,et al.Effects of EDN3on the expression of melanin-related genes in mouse melanocytes[J].China Animal Husbandry&Veterinary Medicine,2018,45(3):581-589.(in Chinese)
[9]BARTOSOVA L.Biology of hair growth[J].Seminars in Reproductive Medicine,1984,4(2):41-58.
[10]HARDY M H,VIELKIND U.Changing patterns of cell adhesion molecules during mouse pelage hair follicle development.Follicle morphogenesis in wild-type mice[J].Cells Tissues Organs,1996,157(3):169-182.
[11]HANDJISKI B K,EICUMLLER S,HOFMANN U,et al.Alkaline phosphatase activity and localization during the murine hair cycle[J].British Journal of Dermatology,1994,131(3):303-310.
[12]ROBINS E J,BREATHNACH A S.Fine structure of the human foetal hair follicle at hair-peg and early bulbous-peg stages of development[J].Journal of Anatomy,1969,104(3):553-569.
[13]MIZUTANI T,IKEGAMI M,NAGAHATA R,et al.Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epide[J].Nature Medicine,2005,11(12):1351-1354.
[14]PARAKKAL P F.Role of macrophages in collagen resorption during hair growth cycle[J].Journal of Ultrastructural Research,1969,29(3-4):210-217.
[15]MLLERRVER S,HANDJISKI B,VEEN C V D,et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages[J].Journal of Investigative Dermatology,2001,117(1):3-15.
[16]ZHANG H,ZHANG S,ZHAO H,et al.Ovine hair follicle stem cells derived from single vibrissae reconstitute haired skin[J].International Journal of Molecular Sciences,2015,16(8):17779-17797.
[17]DE B J.Enzymes of carbohydrate metabolism in human epidermal tumors 2enzymes of the glucose oxidation pathway,from triose-phosphate to pyruvate[J].Journal of Cutaneous Pathology,2010,1(4):176-179.
[18]许英蕾,孙建义.成纤维细胞生长因子与其受体的研究进展[J].药物生物技术,2004,11(3):194-198.XU Y L,SUN J Y.Research progress of fibroblast growth factors(FGFs)and their receptors(FGFRs)[J].Pharmaceutical Biotechnology,2004,11(3):194-198.(in Chinese)
[19]TERRANOVA C,NARLA S T,LEE Y W,et al.Global developmental gene programing involves a nuclear form of fibroblast growth factor receptor-1(FG-FR1)[J].PLoS One,2015,10(4):e0123380.
[20]伊海英,孙晓艳,付小兵,等.胎儿皮肤发育中成纤维细胞生长因子受体-2表达的免疫组织化学研究[J].第三军医大学学报,2008,30(21):2008-2010.YI H Y,SUN X Y,FU X B,et al.Distribution and expression of FGFR2in human fetal skin by immunohistochemical method[J].Acta Academiae Medicinae Militaris Tertiae,2008,30(21):2008-2010.(in Chinese)
[21]REVEST J M,SPENCERDENE B,KERR K,et al.Fibroblast growth factor receptor 2-Ⅲb acts upstream of Shh and Fgf4and is required for limb bud maintenance but not for the induction of Fgf8,Fgf10,Msx1,or Bmp4[J].Developmental Biology,2001,231(1):47-62.
[22]ZHANG X,IBRAHIMI O A,OLSEN S K,et al.Receptor specificity of the fibroblast growth factor family:The complete mammalian FGF family*[J].Journal of Biological Chemistry,1996,271(25):15292-15297.
[23]程笳琪.FGF7亚家族对小鼠毛囊形成和发育作用的研究[D].太谷:山西农业大学,2017.CHENG J Q.Study on function of fibroblast growth factor 7subfamily on hair follicle formation and development in mouse[D].Taigu:Shanxi Agricultural University,2017.(in Chinese)
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