TLR7在小鼠毛囊增值中作用的初步探讨
摘要
研究目的:本课题基于前期研究发现TLR7受体在毛囊隆突部高表达并且给予TLR7激动剂咪喹莫特后小鼠毛囊数量增加及生长周期延长的发现拟通过RT-PCR的方法验证前期由基因芯片筛选出的可能的调控基因Fos,并通过使用JNK通路阻断剂SP600125在动物试验水平上进一步证实TLR7信号通路作用于毛囊干细胞的可能靶位基因,以期对TLR7信号通路对毛囊增殖调控的机制有初步的探讨。
方法:6周大C57雌性小鼠10只,随机分为使用咪喹莫特实验组与使用生理盐水对照组。4周后小鼠背部皮肤取材,将所取皮肤组织TRIZOL法提取RNA,RT-PCR方法分析两组样品Fos基因mRNA的表达差异。再将C57雌性小鼠20只,随机分为咪喹莫特处理组、DMSO对照组、SP00125组和咪喹莫特及SP600125混合组,处理4周后各组小鼠背部皮肤H&E染色观察组织学改变并行磷酸化组蛋白H3检测增殖情况。
结果:(1) Fos mRNA在咪喹莫特实验组的表达率显著高于对照组,差异有统计学意义(P<0.05)(2)阻断JNK信号通路后,再使用TLR7激动剂咪喹莫特,小鼠毛囊的增值水平较咪喹莫特处理组明显降低,但仍高于DMSO对照组。
结论:(1)咪喹莫特可能通过激活TLR7使JNK通路下游基因FOS的活化来达到促进毛囊增值的作用。
(2)TLR7激活后可能通过JNK、NF-κB和IRF等多条信号通路共同作用达到促进毛囊增殖分化的效果。
Research objectives: Our previous research found that TLR7 highly expressed in the hair follicle bulge. After using TLR7 agonist imiquimod, the number of hair follicles increased in mice, and extend the growth cycle. The basis of this subject in the preliminary work, further by RT-PCR method verified the possible regulation of gene Fos, and by using the SP600125 (JNK pathway inhibitor) in animal levels studies to further confirm and explore the role of TLR7 signaling pathway in hair follicle stem cells in the target gene view of the TLR7 signaling pathway on proliferation and regulation of hair follicle mechanism was discussed.
Methods: 6-week-old C57 female mice, 10 mice were randomly divided into the use of imiquimod in the experimental group and control group using saline. Back skin of mice after 4 weeks were sacrificed to the TRIZOL extraction from skin tissue RNA, RT-PCR to analyze two samples of mRNA expression of Fos gene differences.Then 20 C57 female mice were randomly divided into treatment group imiquimod, DMSO control group, SP00125 group and imiquimod &SP600125 mixed group.4 weeks after treatment the skin of mice in each group H & E staining of histological change the parallel detection of phosphorylated histone H3 proliferation.
Results: (1) Fos mRNA in the experimental group imiquimod significantly higher expression rate, the difference was statistically significant (P <0.05) (2) Block the JNK signaling pathway, then use the TLR7 agonist imiquimod, the proliferation level of mouse hair follicles than imiquimod treatment were significantly lower, but still higher than the DMSO control group.
Conclusions: (1) imiquimod may activates TLR7 to enhance the proliferation of the follicle progenitor via Fos activities. (2) TLR7 may activates JNK、NF-κB and IRF several other channels to promote joint action of the effect of proliferation and differentiation of hair follicles.
方法:6周大C57雌性小鼠10只,随机分为使用咪喹莫特实验组与使用生理盐水对照组。4周后小鼠背部皮肤取材,将所取皮肤组织TRIZOL法提取RNA,RT-PCR方法分析两组样品Fos基因mRNA的表达差异。再将C57雌性小鼠20只,随机分为咪喹莫特处理组、DMSO对照组、SP00125组和咪喹莫特及SP600125混合组,处理4周后各组小鼠背部皮肤H&E染色观察组织学改变并行磷酸化组蛋白H3检测增殖情况。
结果:(1) Fos mRNA在咪喹莫特实验组的表达率显著高于对照组,差异有统计学意义(P<0.05)(2)阻断JNK信号通路后,再使用TLR7激动剂咪喹莫特,小鼠毛囊的增值水平较咪喹莫特处理组明显降低,但仍高于DMSO对照组。
结论:(1)咪喹莫特可能通过激活TLR7使JNK通路下游基因FOS的活化来达到促进毛囊增值的作用。
(2)TLR7激活后可能通过JNK、NF-κB和IRF等多条信号通路共同作用达到促进毛囊增殖分化的效果。
Research objectives: Our previous research found that TLR7 highly expressed in the hair follicle bulge. After using TLR7 agonist imiquimod, the number of hair follicles increased in mice, and extend the growth cycle. The basis of this subject in the preliminary work, further by RT-PCR method verified the possible regulation of gene Fos, and by using the SP600125 (JNK pathway inhibitor) in animal levels studies to further confirm and explore the role of TLR7 signaling pathway in hair follicle stem cells in the target gene view of the TLR7 signaling pathway on proliferation and regulation of hair follicle mechanism was discussed.
Methods: 6-week-old C57 female mice, 10 mice were randomly divided into the use of imiquimod in the experimental group and control group using saline. Back skin of mice after 4 weeks were sacrificed to the TRIZOL extraction from skin tissue RNA, RT-PCR to analyze two samples of mRNA expression of Fos gene differences.Then 20 C57 female mice were randomly divided into treatment group imiquimod, DMSO control group, SP00125 group and imiquimod &SP600125 mixed group.4 weeks after treatment the skin of mice in each group H & E staining of histological change the parallel detection of phosphorylated histone H3 proliferation.
Results: (1) Fos mRNA in the experimental group imiquimod significantly higher expression rate, the difference was statistically significant (P <0.05) (2) Block the JNK signaling pathway, then use the TLR7 agonist imiquimod, the proliferation level of mouse hair follicles than imiquimod treatment were significantly lower, but still higher than the DMSO control group.
Conclusions: (1) imiquimod may activates TLR7 to enhance the proliferation of the follicle progenitor via Fos activities. (2) TLR7 may activates JNK、NF-κB and IRF several other channels to promote joint action of the effect of proliferation and differentiation of hair follicles.
引文
[1] Chuong CM.Molecular basis of epithelial appendage morphogenesis[M]. Molecular biology intelligence unit 1, Austin, Tex R.G. Landes.1998, p359-369.
[2]郑占才,王金燕,黄敬彦,等.毛根检查法观察斑秃毛发生长周期变化(附30例病例分析)[J] .中日友好医院学报.1991,03:012.
[3] Taylor G,Lehrer MS,Jensen PJ,et al.Involvement of follicular stem cells in forming not only the follicle but also the epidermis[J]. Cell.2000,102:451-461.
[4] Cotsarelis G.Epithelial stem cells:a folliculocentric view[J].Invest Dermatol.2006,126:1459-1468.
[5] Estrach S, Ambler CA, Lo Celso C,et al.Jagged 1 is aβ-catenin target gene required for ectopic hair follicle formation in adult epidermis[J]. Development.2006,133:4427–4438.
[6] Botchkarev V A,Kishimoto J.Molecular control of epithelial 2 mesenchymal interactions during hair follicle cycling[J].Investig Dermatol Sym PProc.2003,8:46-55.
[7] Gerard MJ,Beaudoin,Jeanne M,et al.Hairless triggers reactivation of hair growth by promoting Wnt signaling[J].Proc Natl Acad Sci USA.2005,102(10):14653 -14658.
[8] Lee J,Chuang TH,Redecke V,etal.Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7[J]. Proc Natl Acad Sci U S A 100.2003, 6646-6651.
[9]Takeda K,Akim S.TLR signaling pathway[J].Semin Immunol.2004, 16(1):3-9.
[10]Hemmi H,Kaisho T, Takeuchi O,et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway[J].Nat Immunol.2002,3(3):196-200.
[11]黄松明,张爱华,丁桂霞,等.血管紧张素Ⅱ通过JNK-c-JUN/AP-1信号通路调控系膜细胞转化生长因子β1表达[J].中华肾脏病杂志,2003,19(5):30l-304.
[12] Scapoli L,Ramos-Nino ME,Martinelli M,et a1.Src-dependent ERK5 andSrc/EGFR dependent ERKl/2 activation is required for cell proliferation by asbestos[J].Oncogene 2004,23(3):805-813.
[13]Kou ID,Shen R,Shishodia S,et a1.PTEN down regulates AP-1 and targets c-fos in human glioma cells via P13-kinase/Akt pathway[J].Mol Cell Biochem .2007,300(1-2):77-87
[14]Du X,Poltorak A,Wei Y,et al.Three novel mammalian Toll-like receptors: gene structure,expression,and evolution[J].Eur Cytokine Network.2000,11(3):362-371.
[15] Dunne A,O'Neill LA.The interleukin-1 receptor/Toll-like receptor superfamily:signal transduction during inflammation and host defense[J].Sci STKE.2003 Feb 25,2003(171):re3.
[16] Biochimica.The role of Jun,Fos and the AP-1 complex in cell proliferation and transformation. biophysica acta [J].1991,1072(2-3):129-57.
[17] N Rajaram,T K Kerppola.DNA bending by Fos-Jun and the orientation of heterodimer binding depend on the sequence of the AP-1 site[J]. EMBO J.1997 May 15,16(10):2917–2925.
[18] C Fisher,M R Byers,M J Iadarola,et al.Patterns of epithelial expression of Fos protein suggest important role in the transition from viable to cornifiedcell during keratinization[J]. Development.February 1991,111,253-2588
[19] Hendzel MJ,Wei Y,Mancini MA,et al.Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation[J]. Chromosoma. 1997 Nov,106(6):348-60.
[1]Cotsarelis G,Sun TT,Lavker RM. Label-retaining cells residue in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle and skin carcinogenesis[J].Cell,1990,61: 1329-1337.
[2]Kobayashi K,Rochat A,Barrandon Y.Segregation of keratinocyte colony-forming cells in the bulge of the rat vibrissa [J].Proc Natl Acad Sci U S A .1993,90:7391-5.
[3]Rochat A,Kobayashi K,Barrandon Y. Location of stem cells of human hair follicles by clonal analysis [J].Cell 1994,76:1063-73.
[4] Morris RJ,Potten CS.Highly persistent label-retaining cells in the hair follicle of mice and their fate following induction of anagen [J]. J Invest Dermatol.1999,112:470-5.
[5] Taylor G,Lehrer MS,Jensen PJ,et al. Involvement of follicular stem cells in forming not only the follicle but also the epidermis [J].Cell.2000,102:451-61.
[6] Michel M,Totok N,GodboutMJ,et al.Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin19 expressing cells are differentially localized in function of anatomic sites and their number varieswith donor age andculture stage[J].J Cell Sci.1996,109: 1017-1028.
[7]Lyle S,Christofidou-Solomidou M,Liu Y,et al.The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells[J].J Cell Sci.1998,111:3179-3188.
[8] Moll I. Proliferative potential of different keratinocytes of plucked human hair follicle[J].J Invest Der m atoll.1995,105 (1):142-21.
[9] Li A,Simmons PJ, Kaur P.Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype[J]. Proceedings of the National Academy of Sciences of the United States of America. 1998 Mar,3195(7):3902-7.
[10]Tani H, Morris RJ, Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype[J].Proc Natl Acad Sci USA.2000,97:10960–5.
[11]Trempus CS, Morris RJ,Bortner CD,et al.Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface markerCD34 [J].Invest Dermatol.2003,120(4):501-511.
[12] Pellegrini G,Dellambra E,Golisano O,et al.p63 identifies keratinocyte stem cells[J].Proc Natl Acad Sci USA.2001,18:3156-3161.
[13]Ohyama M, Terunuma A,Tock CL,Radonovich MF,et al. Characterization and isolation of stem cell-enriched human hair follicle bulge cells[J].J Clin Invest 2006.116:249–60.
[14]Taylor G,Lehrer MS,Jensen PJ,et al.Involvement of follicular stem cells in forming not only the follicle but also the epidermis[J].Cell. 2000,102:451-46.
[15] Oshima H,Rochat A,Kedzia C,et al.Morphogenesis and renewal of hair follicles from adult multipotent stem cells[J]. Cell.2001,104:233-245
[16] Liu Y,Lyle S,Yang X,et al.Keratin 15 promoter targets putative epithelial stem cells in the hair follicle bulge[J].J Invest Dermatol.2003 Nov,121(5):963-968.
[17] Tumbar T,Guasch G,Greco V,et al.Defining the epithelial stem cell niche in skin[J].Science.2004 Jan 16;303(5656):359-363.
[18] Amoh Y,Li L,Yang M,et al. Nascent blood vessels in the skin arise fromnestin-expressing hair-follicle cells[J].Proc Natl Acad Sci USA. 2004,101:13291-13295.
[19] Amoh Y,Li L,Katsuoka K,et al.Multipotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons[J]. Proc Natl Acad Sci USA.2005,102:5530-5534.
[20] Amoh Y,Li L,Campillo R,et al.Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves[J].Proc Natl Acad Sci USA.2005,102:17734.
[21] Richardson GD,Arnott EC,Whitehouse CJ,et al.Cultured cells from the adulthuman hair follicle dermis can be directed toward adipogenic and osteogenic differentiation [J].J Invest Dermatol.2005,124:1090-1091.
[22] Salic A,Lee E,Mayer L,et al.Control ofβ-catenin stability: reconstitution of the cytoplasmic steps of thewnt pathway in Xenopus egg extracts[J].Mol Cell.2000,5: 523-532.
[23] Zhou P,Jacobs J,Byrne C,et al. Hair morphogenesis and gene expression are regulated by lymphoid enhancer factor 1(LEF-1) [J].Genes Dev.1995,9: 700-713.
[24]van Genderen C,Okamura RM,Fari?as I,et al.Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice [J].Genes Dev.1994,8(22):2691-2703.
[25]Wilson C,Cotsarelis G,Wei ZG,et al.Cells within the bulge region of mouse hair follicle transiently proliferate during early anagen: heterogeneity and functional difference of various hair cycles[J]. Differentiation.1994,55:127-136.
[2]郑占才,王金燕,黄敬彦,等.毛根检查法观察斑秃毛发生长周期变化(附30例病例分析)[J] .中日友好医院学报.1991,03:012.
[3] Taylor G,Lehrer MS,Jensen PJ,et al.Involvement of follicular stem cells in forming not only the follicle but also the epidermis[J]. Cell.2000,102:451-461.
[4] Cotsarelis G.Epithelial stem cells:a folliculocentric view[J].Invest Dermatol.2006,126:1459-1468.
[5] Estrach S, Ambler CA, Lo Celso C,et al.Jagged 1 is aβ-catenin target gene required for ectopic hair follicle formation in adult epidermis[J]. Development.2006,133:4427–4438.
[6] Botchkarev V A,Kishimoto J.Molecular control of epithelial 2 mesenchymal interactions during hair follicle cycling[J].Investig Dermatol Sym PProc.2003,8:46-55.
[7] Gerard MJ,Beaudoin,Jeanne M,et al.Hairless triggers reactivation of hair growth by promoting Wnt signaling[J].Proc Natl Acad Sci USA.2005,102(10):14653 -14658.
[8] Lee J,Chuang TH,Redecke V,etal.Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7[J]. Proc Natl Acad Sci U S A 100.2003, 6646-6651.
[9]Takeda K,Akim S.TLR signaling pathway[J].Semin Immunol.2004, 16(1):3-9.
[10]Hemmi H,Kaisho T, Takeuchi O,et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway[J].Nat Immunol.2002,3(3):196-200.
[11]黄松明,张爱华,丁桂霞,等.血管紧张素Ⅱ通过JNK-c-JUN/AP-1信号通路调控系膜细胞转化生长因子β1表达[J].中华肾脏病杂志,2003,19(5):30l-304.
[12] Scapoli L,Ramos-Nino ME,Martinelli M,et a1.Src-dependent ERK5 andSrc/EGFR dependent ERKl/2 activation is required for cell proliferation by asbestos[J].Oncogene 2004,23(3):805-813.
[13]Kou ID,Shen R,Shishodia S,et a1.PTEN down regulates AP-1 and targets c-fos in human glioma cells via P13-kinase/Akt pathway[J].Mol Cell Biochem .2007,300(1-2):77-87
[14]Du X,Poltorak A,Wei Y,et al.Three novel mammalian Toll-like receptors: gene structure,expression,and evolution[J].Eur Cytokine Network.2000,11(3):362-371.
[15] Dunne A,O'Neill LA.The interleukin-1 receptor/Toll-like receptor superfamily:signal transduction during inflammation and host defense[J].Sci STKE.2003 Feb 25,2003(171):re3.
[16] Biochimica.The role of Jun,Fos and the AP-1 complex in cell proliferation and transformation. biophysica acta [J].1991,1072(2-3):129-57.
[17] N Rajaram,T K Kerppola.DNA bending by Fos-Jun and the orientation of heterodimer binding depend on the sequence of the AP-1 site[J]. EMBO J.1997 May 15,16(10):2917–2925.
[18] C Fisher,M R Byers,M J Iadarola,et al.Patterns of epithelial expression of Fos protein suggest important role in the transition from viable to cornifiedcell during keratinization[J]. Development.February 1991,111,253-2588
[19] Hendzel MJ,Wei Y,Mancini MA,et al.Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation[J]. Chromosoma. 1997 Nov,106(6):348-60.
[1]Cotsarelis G,Sun TT,Lavker RM. Label-retaining cells residue in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle and skin carcinogenesis[J].Cell,1990,61: 1329-1337.
[2]Kobayashi K,Rochat A,Barrandon Y.Segregation of keratinocyte colony-forming cells in the bulge of the rat vibrissa [J].Proc Natl Acad Sci U S A .1993,90:7391-5.
[3]Rochat A,Kobayashi K,Barrandon Y. Location of stem cells of human hair follicles by clonal analysis [J].Cell 1994,76:1063-73.
[4] Morris RJ,Potten CS.Highly persistent label-retaining cells in the hair follicle of mice and their fate following induction of anagen [J]. J Invest Dermatol.1999,112:470-5.
[5] Taylor G,Lehrer MS,Jensen PJ,et al. Involvement of follicular stem cells in forming not only the follicle but also the epidermis [J].Cell.2000,102:451-61.
[6] Michel M,Totok N,GodboutMJ,et al.Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin19 expressing cells are differentially localized in function of anatomic sites and their number varieswith donor age andculture stage[J].J Cell Sci.1996,109: 1017-1028.
[7]Lyle S,Christofidou-Solomidou M,Liu Y,et al.The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells[J].J Cell Sci.1998,111:3179-3188.
[8] Moll I. Proliferative potential of different keratinocytes of plucked human hair follicle[J].J Invest Der m atoll.1995,105 (1):142-21.
[9] Li A,Simmons PJ, Kaur P.Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype[J]. Proceedings of the National Academy of Sciences of the United States of America. 1998 Mar,3195(7):3902-7.
[10]Tani H, Morris RJ, Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype[J].Proc Natl Acad Sci USA.2000,97:10960–5.
[11]Trempus CS, Morris RJ,Bortner CD,et al.Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface markerCD34 [J].Invest Dermatol.2003,120(4):501-511.
[12] Pellegrini G,Dellambra E,Golisano O,et al.p63 identifies keratinocyte stem cells[J].Proc Natl Acad Sci USA.2001,18:3156-3161.
[13]Ohyama M, Terunuma A,Tock CL,Radonovich MF,et al. Characterization and isolation of stem cell-enriched human hair follicle bulge cells[J].J Clin Invest 2006.116:249–60.
[14]Taylor G,Lehrer MS,Jensen PJ,et al.Involvement of follicular stem cells in forming not only the follicle but also the epidermis[J].Cell. 2000,102:451-46.
[15] Oshima H,Rochat A,Kedzia C,et al.Morphogenesis and renewal of hair follicles from adult multipotent stem cells[J]. Cell.2001,104:233-245
[16] Liu Y,Lyle S,Yang X,et al.Keratin 15 promoter targets putative epithelial stem cells in the hair follicle bulge[J].J Invest Dermatol.2003 Nov,121(5):963-968.
[17] Tumbar T,Guasch G,Greco V,et al.Defining the epithelial stem cell niche in skin[J].Science.2004 Jan 16;303(5656):359-363.
[18] Amoh Y,Li L,Yang M,et al. Nascent blood vessels in the skin arise fromnestin-expressing hair-follicle cells[J].Proc Natl Acad Sci USA. 2004,101:13291-13295.
[19] Amoh Y,Li L,Katsuoka K,et al.Multipotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons[J]. Proc Natl Acad Sci USA.2005,102:5530-5534.
[20] Amoh Y,Li L,Campillo R,et al.Implanted hair follicle stem cells form Schwann cells that support repair of severed peripheral nerves[J].Proc Natl Acad Sci USA.2005,102:17734.
[21] Richardson GD,Arnott EC,Whitehouse CJ,et al.Cultured cells from the adulthuman hair follicle dermis can be directed toward adipogenic and osteogenic differentiation [J].J Invest Dermatol.2005,124:1090-1091.
[22] Salic A,Lee E,Mayer L,et al.Control ofβ-catenin stability: reconstitution of the cytoplasmic steps of thewnt pathway in Xenopus egg extracts[J].Mol Cell.2000,5: 523-532.
[23] Zhou P,Jacobs J,Byrne C,et al. Hair morphogenesis and gene expression are regulated by lymphoid enhancer factor 1(LEF-1) [J].Genes Dev.1995,9: 700-713.
[24]van Genderen C,Okamura RM,Fari?as I,et al.Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice [J].Genes Dev.1994,8(22):2691-2703.
[25]Wilson C,Cotsarelis G,Wei ZG,et al.Cells within the bulge region of mouse hair follicle transiently proliferate during early anagen: heterogeneity and functional difference of various hair cycles[J]. Differentiation.1994,55:127-136.
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