三种人工合成寡核苷酸序列对体外培养的毛囊黑素细胞的影响(1)
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[摘要]目的：研究三种人工合成寡核苷酸序列对体外培养的毛囊黑素细胞形态及酪氨酸酶活性的影响。方法：采用胶原酶和胰酶两步消化法，分离培养毛囊内黑素细胞。多巴氧化法测酪氨酸酶活性，硝酸银染色观察细胞形态的变化。结果：三种人工合成寡核苷酸序列可以显著增强酪氨酸酶活性(P<0.05)；银染色细胞显示CpG-1826和CpG-2006组胞体变得肥大，黑素化明显，树突增多，与对照组相比有显著差异。结论：部分人工合成寡核苷酸序列可促进毛囊黑素细胞的酪氨酸酶活性和树突增加，为白癜风皮损复色的研究奠定基础。
[关键词]寡核苷酸序列；毛囊；黑素细胞
[中图分类号]Q813.1[文献标识码]A[文章编号]1008-6455(2011)01-0065-03
Effects of three synthetie oligonucleotide sequence to melanocytes from human hair follicle
LI Hong-zhou1,2, XUE Dong-zhang3, ZHANG Ru-zhi1
(1. Department of Dermatology,The Affiliated Hospital of Bengbu Medical University, Bengbu 233004,Anhui,China; 2. Department of Pharmacology, Bengbu Medical College;3.The 123th Hospital of PLA.)
Abstract:ObjectiveTo investigate effects of three synthetic oligonucleotides on tyrosinase activity and morphology of the melanocytes from fetal scalp hair follicles.MethodsThe follicle melanocytes were isolated and cultured by two steps digestion of collagenase and trypsin. Tyrosinase activity was measured by Dopa oxidation. Cell morphology was detected by silver nitrate staining.ResultsMelanocyte tyrosinase activity was promoted significantly by three synthetic oligonucleotides (P<0.05). The significant changes werer detected in cell morphology in group CpG2006 and CpG1668, with big cell body, obvious pigmentation, and many dendrites. Conclusions Part of the synthetic oligonucleotides is able to promote tyrosinase activity and increase cell dendrite. The research is the basis of studying vitiligo repigmentation.
Key words:oligonucleotide sequence;hair follicles;melanocytes
研究表明毛囊内不成熟黑素细胞是表皮黑素细胞的干细胞，为黑素细胞的储备库[1]。在特定阶段或白癜风治疗后，这些不成熟的黑素细胞活化，并逐渐向角质形成细胞转移，使白癜风的脱色区复色[2]。临床上常见的炎症、创伤后色素沉着通常与炎症介质的局部释放有关[3]。据报道细菌基因组中未甲基化的CpG基序寡核苷酸(CpG motif containing oligonucleotides,CpG-ODN)参与这一过程，并且具有强大的促树突状细胞 (dendritic cell, DC)成熟作用[4]。本实验首次以胎儿头皮毛囊黑素细胞为模型，探讨三种人工合成寡核苷酸序列对黑素细胞酪氨酸酶活性及细胞形态的影响。这一研究对白癜风复色的研究有着重要的意义。
1材料和方法
1. 1材料：Non- CpG-ODN:5′-TGC TGC TTC CCC CCC CCC CCC- 3′；CPG1826:5′-TCC ATG ACG TTC CTG ACG TT- 3′；CPG2006：5′-TCG TCG TTT TGT CGT TTT GTC GTT- 3′；CpG-ODN 1668：5′-TCC ATG ACG TTC CTG ATG CT- 3′。全硫代修饰,上海细胞研究所合成。左旋多巴 (L-Dopa)、α-MSH、和TritonX-100为 Sigma公司产品，M254培养基(北京清源浩)，氨银，氯化金，硫代硫酸钠，核固红(合肥志宏)。
1.2 方法
1.2.1 毛囊黑素细胞培养：获取胎儿头皮标本(胎龄6月畸胎引产)，聚氯酮碘洗涤 ......
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