克白合剂通过SCF/Kit/MITF信号通路治疗白癜风的作用机制研究

Study on Mechanism of Kebai Mixture in Treating Vitiligo by SCF/Kit/MITF Signal Pathway

关键词:  克白合剂  黑素细胞  酪氨酸酶  白癜风

Key words:  Kebai mixture  melanocytes  tyrosinase  vitiligo

摘要:

目的 研究克白合剂治疗白癜风的作用机制。方法 建立正常人表皮黑素细胞（melanocytes，MC）培养体系，加入克白合剂并作用后采用MTT法、L-DOPA法、NaOH裂解法等分别测定MC细胞增殖活性、酪氨酸酶（tyrosinase，Tyr）活性、黑素含量等；测定克白合剂处理后角质形成细胞（keratinocytes，KC）上清对MC细胞的影响；采用特异性抗体ACK2阻断Kit受体后，检测克白合剂处理KC细胞后上清对MC细胞MITF、Tyr表达的影响。结果 克白合剂2，4 mg·mL^-1无明显细胞毒性。与对照组比，克白合剂组48 h时MC细胞增殖作用明显升高（P<0.01），Tyr活性明显升高（P<0.01）；56 h时，2组黑素含量无显著性差异。克白合剂组对MC细胞c-kit的mRNA表达为对照组的4.43倍；MITF的mRNA表达为对照组的2.98倍；Tyr表达为对照组的2.29倍。加克白合剂后KC细胞上清中干细胞因子（stem cell factor，SCF）量为（52.78±11.24）ng·mL^-1，对照组为（38.43±10.87）ng·mL^-1，SCF的mRNA表达量为对照组的3.63倍，MC细胞c-kit的mRNA表达为对照组的4.45倍；MITF的mRNA表达为对照组的4.12倍，Tyr表达为对照组的3.78倍，MC细胞增殖增加1.89倍，黑素合成量增加35%。用特异性抗体ACK2阻断Kit受体后，MC细胞的MITF、Tyr表达明显下降，均不及空白组。结论 克白合剂具有促进MC细胞活力、增加Tyr活性的作用，可能是通过SCF/Kit/MITF信号通路发挥疗效。

Abstract:

OBJECTIVE Study on the mechanism of Kebai mixture in the treatment of vitiligo. METHODS This study established a normal human epidermal melanocytes culture system, added Kebai mixture and applied for a certain period of time. To determine the influence of Kebai mixture recipe on melanocyte proliferation, tyrosinase(Tyr) activity, melanocyte content by MTT method, L-DOPA method, NaOH decomposition method, respectively. The effect of the keratinocytes supernatant on the melanocytes was determined after the treatment with Kebai mixture. After blocking the Kit receptor with the specific antibody ACK2, the supernatant was treated with Kebai mixture to treat the melanocytes MITF, Tyr effect of expression. RESULTS Kebai mixture of the concentration 2, 4 mg·mL^-1 showed no obvious cytotoxicity. Compared with the control group, the proliferation of melanocytes was significantly increased at 48 h(P<0.01), and the Tyr activity was significantly increased (P<0.01). At 56 h, there was no significant difference in melanin content between the two groups. The mRNA expression of c-kit in melanocytes was 4.43 times that of the control group; the mRNA expression of MITF was 2.98 times that of the control group; the expression of Tyr was 2.29 times that of the control group. The amount of stem cell factor(SCF) in the keratinocyte's supernatant of the Kebai mixture group was (52.78±11.24)ng·mL^-1, the control group was (38.43±10.87)ng·mL^-1, and the expression of SCF mRNA was 3.63 times that of the control group. The mRNA expression of c-kit was 4.45 times; the mRNA expression of MITF was 4.12 times, the expression of Tyr was 3.78 times; the proliferation of melanocytes was 1.89 times that of the control group, respectively. The amount of melanin synthesis was increased by 35%. After blocking the Kit receptor with the specific antibody ACK2, the expression of MITF and Tyr in melanocytes was significantly decreased, which was inferior to the blank group. CONCLUSION Kebai mixture can promote the activity of melanocytes and increase the activity of Tyr. It may be through SCF/Kit/MITF signaling pathway.