目的了解基质细胞衍生因子-1(SDF-1)在体外对表皮干细胞(ESCs)生长增殖的促进作用，为创面促愈药物研发提供理论依据。方法体外分离、培养ESCs，通过ESCs的免疫组化、免疫荧光染色对ESCs进行鉴定;RT-PCR检测ESCs中基质细胞衍生因子-1受体(CXCR4)基因的表达;取培养至第三代的ESCs分组，通过台盼蓝拒染实验、Edu标记法观察在不同剂量SDF-1的刺激作用和CXCR4拮抗剂存在下，A～E组及A’～E’组间ESCs生长增殖的差异。结果应用特制的ESCs培养基能培养出活力强、纯度高的ESCs;培养的ESCs表达K19、CD29、integrinα6，不表达CD71;CXCR4基因在ESCs中阳性表达;体外细胞实验证实SDF-1对ESCs有促进增殖作用，SDF-1浓度为100μg/L时作用最强，差异有统计学意义(P＜0.01);CXCR4拮抗剂AMD3100存在时，SDF-1对ESCs的促增殖作用失效。结论SDF-1对体外培养扩增的ESCs有促进增殖作用;SDF-1合理应用，有可能在皮肤治疗中发挥促愈作用。
Objective To explore the effect of stromal cell-derived factor-1(SDF-1) on proliferation of the epidermal stem cells(ESCs) in vitro, to provide an important theortical basis for development of new drugs which excels in promoting healing. Methods ESCs were isolated and cultured from healthy human foreskins which were identified by immunohistochemistry and immunofluorescence staining. The CXCR4 gene expression in ESCs was determined by RT-PCR. The passage cells were divided into several groups. The functions of the SDF-1under different dose affected on ESCs in different groups was investigated by Trypan Blue staining and Edu incorporation experiment. Furthermore, the performances of ESCs in different groups were observed when they suffered from the CXCR4 antagonist. Results The ESCs were successfully cultured with special condition culture medium, and the cells expressed K19, CD29 and integrinα6, and negative in CD71. CXCR4 gene was also expressed in ESCs. The SDF-1could promote the proliferation of the ESCs in vitro with dose dependent in a certain range, and the effect was most remarkable in 100μg/L groups(P＜0.01). After adding the CXCR4 antagonist AMD3100 into the medium, SDF-1no longer served any useful functions. Conclusion The SDF-1may promote the proliferation of ESCs in vitro and it could be used in clinical treatment as a new drug for skin wound healing.
[1] Janes S M, Lowell S,Hutter C. Epidermal stem cells [J].Pathol,2002,197(4):479-491.
[2] Yu L,Cecil J,Peng S B,et al.Identification and expression of novel isoforms of human stromal cell derived factor1 [J].Gene,2006,374(2):174-179.
[3] Siddiqui A R, Bernstein J M. Chronic wound infection: facts and controversies [J]. Clin Dermatol, 2010, 28(5):519-526.
[4] Veronique M, Francos A A, Dominique G,et al. Role of wound healing myofibroblasts on re-epithelialization of human skin [J]. Burns, 2000,26(1):3-12.
[5] Medina R J, Kataoka K, Takaishi M, et al. Isolation of epithelial stem cells from dermis by a three-dimensional culture system [J]. Cell Biochem, 2006, 98(1):174-184.
[6] 唐红英,伍素华,陈建,等.整合素β1对人ESC增殖与分化的影响[J].中华实验外科杂志,2007,24(1):22-24.
[7] 程大胜,夏照帆,唐洪泰,等.一种ESC检测方法的建立[J].解放军医学杂志,2003,28 (6):566-567.
[8] Balkwill F. The significance of cancer cell expression of the chemokine receptor CXCR4 [J]. Seminars Cancer Biology,2004,14(3):171-179.
[9] Sauve K, Lepage J, Sanchez M, et al. Positive feedback activation of estrogen receptors by the CXCL12-CXCR4 pathway [J].Cancer Research,2009, 69(14):5793-5800.
[10] Liu H, Pan Z, Li A,et al. Roles of chemokine receptor 4(CXCR4) and chemokine ligand 12 (CXCL12) in metastasis of hepatocellular carcinoma cells [J]. Cell Mol Immunol,2008,5(5):373-378.
[11] Yu Y, Arora A, Min W, et al.EdU incorporation is an alternative non-radioactive assay to [(3)H]thymidine uptake for in vitro measurement of mice T-cell proliferations [J]. Immunol Methods, 2009,350(1-2):9-35.
[12] Kaiser C L, Kamien A J,Shah P A, et al.5-Ethynyl-2-deoxyuridine labeling detects proliferating cells in the regenerating avian cochlea [J]. Laryngoscope,2009, 119(9):1770-1775.
[13] Fuchs E. Finding ones niche in the skin[J].Cell Stem Cell, 2009,4(6):499-502.
[14] Fuchs E, Chen T. A matter of life and death: self-renewal in stem cells [J]. EMBO Rep, 2013,14(1):39-48.
[15] 李建福,付小兵, 盛志勇,等.创面愈合过程中创缘表皮干细胞的再分布[J].中华医学杂志,2003,83(3):228-231.
[16] Fu X, Sun X, Li X, et al. Dedifferentiation of epidermal cells to stem cells in vivo [J]. Lancet, 2001,358 (9287):1067-1068.
[17] David J G, Stelios T A. A novel role of fibrin in epidermal healing: plasminogen-mediated migration and selective detachment of differentiated keratinocytes [J]. Invest Dermatol,2003,121(5):1210-1216.
[18] Rabbani P, Takeo M, Chou W,et al.Coordinated activation of wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration [J].Cell,2011, 145(6):941-955.
[19] Li Jianfu,Fu Xiaobing,Shen Zhiyong,et al. The interaction between epidermal growth factor and metalloproteinases induces development of sweat glands in human fetal skin [J]. Surg Res,2002,106(2):258-263.
[20] El Ghalbzouri A, Ponec M. Diffusible factors released by fibroblasts support epidermal morphogenesis and deposition of basement membrane components [J]. Wound Repair Regen,2004,12(3):359-367.
[21] Miller R J,Banisadr G, Bhattacharyya B J.CXCR4 signaling in the regulation of stem cell migration and development [J]. Neuroimmunol,2008,198(1-2):31-38.
[22] Zlotnik A.New insights on the role of CXCR4 in cancer metastasis [J]. J Pathol, 2008,215(3):211-213.
