[摘要]目的：探讨Notch信号对角质形成细胞分泌功能的影响及意义。方法：采用角质形成细胞的血清刺激模型研究Notch信号对其分泌功能的影响。结果：血清刺激可以明显促进Notch受体，配体及下游基因的表达，其中Notch1，Jagged1的表达明显上升并具有时间依赖性，其下游蛋白P21表达升高，并在血清刺激后6h达到高峰，P63表达下降，并在血清刺激后6h，下降幅度最大。此外给予血清刺激后，纤维化相关因子（包括TGF-β1，TGF-β2，IGF-1，CTGF，VEGF及EGF）的表达明显升高，并在12h达到高峰。给予DAPT（N-[N-(3,5-Difluorophenancetyl-L-alanyl)]-S-phenylglycinet-butyl ester，γ分泌酶抑制剂)进行Notch信号阻断后，明显抑制其下游蛋白P21的表达，提高的P63的表达水平，同时抑制了纤维化相关因子的表达。结论：阻断Notch信号能够明显抑制角质形成细胞分泌纤维化相关因子。
[关键词]瘢痕；Notch信号；角质形成细胞；血清刺激模型；纤维化相关因子
[中图分类号]Q813.1 [文献标识码]A [文章编号]1008-6455（2012）04-0576-04
Blocking Notch signal pathway suppress profibrotic growth factor production in keratinocyte
LI Bing,DIAO Jian-sheng,CHU Fei-fei,WANG Da-lei, GUO Shu-zhong, XIA Wei
(Institute of Plastic Surgery, Xijing Hospital,The Fourth Military Medical University,Xi"an 710032,Shaanxi,China)
Abstract: Objective To study the impact of Notch signaling on the profibrotic growth factor production in keratinocyte. Methods To mimic the components of the acute wound microenvironment, we used serum stimulation model in vitro. Results We found that expression of Notch1 and Jagged-1 in keratinocytes was significantly higher after serum stimulation, and shown time independent. After keratinocytes serum stimulation, the expression of P21 was significantly up-regulated and had a peak at 6h, while the expression of P63 was down-regulated with the biggest fall at 6h. Besides, the result of real time PCR suggested that nearly all the detected profibrotic growth factors had a peak at 12h after serum stimulation, in absence of DAPT. DAPT decreased the expression of profibrotic growth factor production at all the indicated time, and had the most potent effect at 12h. But in presence and absence DAPT, the variation tendency of TGF-β1 expression was nearly the same, so was VEGF. Maybe this is related to the low dose of DAPT(10μM). Conclusion Blocking Notch signaling pathway can suppress probrotic growth factor production in keratinocyte.
Key words:cicatrix; Notch signal pathway; keratinocyte; serum stimulation model; profibrotic growth factor
皮肤创伤后过度愈合常常导致瘢痕增生，尤其是大面积烧伤后常形成增生性瘢痕，不仅影响外表，而且常会给患者带来功能障碍，影响患者身心健康。但其发病机制目前尚未明确阐明。表皮异常的旁分泌功能在瘢痕增生中发挥着重要作用[1-3]。在本课题组前期的研究中发现，在增生性瘢痕的表皮增厚，但其基底细胞减少，上层细胞增多，即表现为增殖减低，过度分化状态，同时发现Notch信号相关分子在增生性瘢痕表皮过量表达及活化[4]。此外，本课题组前期利用DAPT局部注射能够明显抑制兔耳瘢痕增生[5]，同时增生性瘢痕表皮的过度分化状态也得到了逆转。Notch信号是调节表皮增殖分化的重要信号途径，因此我们推测，创伤后，可能由于Notch信号相关分子的大量表达及过度活化导致表皮的增殖分化异常[6-7]，进而产生大量的纤维化相关因子，导致瘢痕增生。本研究即通过血清刺激模型，体外模拟体内急性创伤，来研究Notch信号对角质形成细胞分泌功能的影响，为Notch信号在瘢痕增生中促进作用提供依据。
1 材料和方法
1.1 材料
1.1.1主要试剂及材料：角质形成细胞购买于美国ATCC公司，无血清KGM（Serum free basal keratinocyte growth medium，无血清角质形成细胞基础培养基）及添加剂购买与美国ATCC公司；胰酶及EDTA（Ethylene Diamine Tetraacetie Acid，乙二胺四乙酸）产于英国Sigma公司，购买于西安沃尔森生物技术公司；DAPT（N-[N-(3,5-Difluo rophenancetyl-L-alanyl)]-S-phenylglycinet-butylester，γ分泌酶抑制剂)产于美国ALEXIS公司，购买于西安沃尔森生物技术公司；FCS（（fetal calf serum，胎牛血清）产于美国Hyclone公司，购买于西安沃尔森生物技术公司；TriPure分离试剂产于德国Roche公司，购买于西安沃尔森生物技术公司；反转录试剂盒及SYBR GreenⅠ实时荧光定量试剂盒产于并购买于大连宝生物工程有限公司；引物由上海生工生物工程有限公司合成。
1.2 方法
1.2.1 角质形成细胞培养： 冻存的角质形成细胞以1 000～5 000细胞/cm2密度复苏后培养于无血清的KGM完全培养基中, 当细胞达到70%～80%融合，利用0.02%胰酶和0.05%EDTA进行消化传代，直到第四代细胞用于血清刺激模型建立。
1.2.2 血清刺激模型建立及DAPT处理： 将角质形成细胞在KGM成基础培养基中静置17h，加入或不加10μM DAPT，孵育2h后，加入10%FCS，于血清刺激后0h、1h、6h、12h及24h收集细胞及培养上清。
1.2.3 Real-time PCR检测：采用TriPure试剂按说明书进行RNA提取。DNase I 37℃，10～3Omin，以去除基因组DNA污染。RNA沉淀，重悬后进行定量。利用反转录试剂盒按照说明书对RNA进行反转录。采用SYBR Green I染料在MJ Research 0ption 2荧光定量PCR仪上检测目的基因的表达。PCR反应体系20μl。PCR反应条件：激活95℃，10 min；40个循环，变性95℃，15 s；退火55℃，30 s，延伸72℃，30 s；分别在78℃、80℃、82℃和85℃时检测荧光。扩增完毕后，进行熔解曲线分析，95℃，1 min，65℃，l min，以0.10℃/s的速度升温到95℃，连续监测荧光。扩增结束后分析熔解曲线，在熔解曲线上以具有单峰判断PCR扩增的单一性。同时设无模板对照，每份标本行3次Rea1-time PCR检测，取平均值。对cDNA模板进行倍比稀释，以1.0×、0.5×、0.25×、0.125×四个浓度梯度的cDNA模板进行Rea1-time PCR扩增。将不同浓度模板的对数和相应到达阈值的循环数(ct值)作图，做标准曲线。基因mRNA表达采用直线相关回归分析，管家基因GAPDH的表达量作为内参照，分析目的基因的相对表达量。
1.2.4统计学处理：应用STAT4．0软件进行统计分析，用方差分析处理数据。
2 结果
2.1 血清可以明显刺激角质形成细胞Notch信号相关分子的表达与活化。血清刺激后，Notch1及Jagged1表达明显上升，并呈现时间依赖性。此外，Notch下游分子，P21表达明显升高，并在血清刺激后6h达到高峰（P=0.0004）；P63表达下降，并在血清刺激后6h下降幅度最大（P [2]Corrie L,Gallant-Behm,PhD and Thomas A Mustoe,MD. Occlusion regulates epidermal cytokine production and inhibits scar formation[J].Wound Repair Regen,2010,18(2): 235-244.
[3]Tandara AA,Kloeters O,Mogford JE,et al.Hydrated keratinocytes reduce collagen synthesis by fibroblasts via paracrine mechanisms[J].Wound Repair Regen, 2007,15(4):497-504.
[4]夏炜,潘宝华,刘蓓,等.Notch信号在增生性瘢痕表皮中的表达[J].中华整形外科杂志,2009,25(1):41-45.
[5]刁建生,张曦,夏炜,等.阻断Notch信号对兔耳增生性瘢痕形成的影响及作用机制[J].中华整形外科杂志,2009,89(16):1088-1092.
[6]Ryuhei Okuyama,Hachiro Tagami,Setsuya Aiba.Notch signaling: Its role in epidermal homeostasis and in the pathogenesis of skin diseases[J].J Dermatol Sci,2008,49:187-194.
[7]Lowell S,Jones PH,LeRoux I,et al. Stimulation of human epidermal differentiation by Delta-Notch signaling at the boundaries of stem cell clusters[J].Curr Biol,2000,10:491-500.
[8]Okuyama R,Nguyen BC,Talora C,et al.High commitment of embryonic keratinocytes to terminal differentiation through a Notch-1-caspase 3 regulatory mechanism[J].Dev Cell,2004,6:549-562.
[9]Rangarajan A,Talora C,Okuyama R,et al.Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation[J].EMBO J,2001,20:3225-3234.
[10]Nickoloff BJ,Qin JZ,Chaturvedi V,et al. Jagged-1 mediated activation of notch signaling induces complete maturation of human keratinocytes through NF-kappaB and PPARgamma[J].Cell Death Differ,2002,9:840-855.
[11]Okuyama R,Ogawa E,Nagoshi H,et al.p53 homologue, p51/ P63, maintains the immaturity of keratinocyte stem cells by inhibiting Notch1 activity[J].Oncogene, 2007,26 (31):4478-4488.
[12]Corrie L,Gallant-Behm,Pan Du,Simon M. Lin, et al. Epithelial Regulation of Mesenchymal Tissue Behavior[J].J Invest Dermatol,2011,131:892-899.
[13]Corrie L,Gallant-Behm,PhD and Thomas A Mustoe,MD. Occlusion Regulates Epidermal Cytokine Production and Inhibits Scar Formation[J].Wound Repair Regen,2010,18(2): 235-244.
[14]Tandara AA, Kloeters O, Mogford JE,et al. Hydrated keratinocytes reduce collagen synthesis by fibroblasts via paracrine mechanisms[J].Wound Repair Regen, 2007,15(4):497-504.
[15]Gianni-Barrera,Roberto,et.al. To sprout or to split? VEGF, Notch and vascular morphogenesis[J].Biochemical Society Transactions,2011,39(6):1644-1648.
[16]Chunli Yan, Wesley A. Grimm, et al. Epithelial to mesenchymal transition in human skin wound healing is induced by tumor necrosis factor-γ through bone morphogenic protein-2[J].Am J Pathol,2010,176(5): 2247-2258.
[17]Radisky DC, Kenny PA, Bissell MJ. Fibrosis and cancer: do myofibroblasts come also from epithelial cells via EMT[J]? J Cell Biochem,2007,101:830-839.
[收稿日期]2011-12-26 [修回日期]2012-02-16
编辑/张惠娟