卡泊三醇对白癜风黑素细胞和CD8＋细胞毒T细胞的影响

Effects of calcipotriol on melanocytes and CD8＋ cytotoxic T lymphocytes from patients with vitiligo

目的探讨卡泊三醇对白癜风患者黑素细胞及皮损周边CD8＋细胞毒T细胞（CD8＋CTL）增殖及其细胞因子分泌的影响。方法实验分黑素细胞组、CD8＋CTL组、黑素细胞与CD8＋CTL共培养组，细胞计数法检测卡泊三醇处理前后细胞数变化情况，ELISA测定卡泊三醇处理前后分泌白介素6（IL-6）、肿瘤坏死因子α（TNF-α）及干扰素γ（IFN-γ）的变化；细胞计数法检测卡泊三醇处理过的黑素细胞与CD8＋CTL共培养组在添加与不添加抗人IL-6抗体时，黑素细胞及CD8＋CTL数量的变化。筛选10^-8、10^-9mol／L卡泊三醇用于实验。结果在凋亡检测中发现，CD8＋CTL与黑素细胞共培养中黑素细胞有显著凋亡。加卡泊三醇后黑素细胞组的黑素细胞计数与未加药对照组相比，差异无统计学意义（P〉0．05），而黑素细胞与CD8＋CTL共培养组黑素细胞数明显增加（P〈0．05）；CD8＋CTL组和黑素细胞与CD8＋CTL共培养组的CD8＋CTL细胞数也明显增加（P〈0．05）。与黑素细胞和CD8＋CTL单独培养组相比，黑素细胞与CD8＋CTL共培养组分泌的IL-6、IFN-γ和TNF-α显著减少；经104mol／L卡泊三醇处理后的黑素细胞与CD8＋CTL共培养组分泌的IL-6减少更加明显，与未加药组减少率比较，差异有统计学意义（t=2．89，P〈0．05），而IFN-γ和TNF-α在加药前后无明显变化（P〉0．05）。在卡泊三醇处理过的共培养组中添加5mg／L抗IL-6抗体后，黑素细胞显著增加，而CD8＋CTL减少，其差异有统计学意义（t=3．53，P〈0．05；t=3．15，P〈0．05）。结论白癜风皮损处的CD8＋CTL对黑素细胞有一定的特异性杀伤作用，卡泊三醇可以减少炎症细胞因子IL-6的分泌，从而减少CD8＋CTL对黑素细胞的杀伤，可能是卡泊三醇治疗白癜风的机制之一。

Objective To evaluate the effect of calcipotriol on the proliferation of and cytokine secretion by melanocytes and perilesional CD8＋ cytotoxic T lymphocytes （CTLs） from patients with vitiligo. Methods Melanocytes isolated from abdominal skin and CD8＋ CTLs from perilesional skin of patients with vitiligo were subjected to successive culture in vitro. After several passages, the melanocytes and CD8＋ CTLs were cultured alone or in combination with or without the presence of various concentrations of calcipotriol for 24 to 48 hours. MTS （3-（4,5-dimethyhhiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfopheny）-2H-tetrazolium, inner salt） method was used to evaluate the proliferative activity of cells, enzyme-linked immunosorbent assay to determine the levels of interleukin （IL）-6, tumor necrosis factor （TNF）-α and interferon （IFN）-γ in the culture supernatant of cells, flow cytometry to detect cell apoptosis. Some co-cultured melanocytes and CTLs were treated with calcipotriol of 10^-8 mol/L and anti-IL-6 antibody of various concentrations （0, 1, 2, 2.5, 5, 10 mg/L） for two days followed by enumeration of cells. The concentrations of 10^-8 and 10^-9 mol/L （calcipotriol） were chosen for relevant tests. Results There was a marked apoptosis in MCs after coculture with CD8＋ CTLs. The 24-hour treatment with calcipotriol of 10^-8 and 10^-9 mol/L had no obvious effect on the proliferation of melanocytes cultured alone （both P 〉 0.05）, but accelerated the proliferation of melanocytes cocuhured with CTLs （both P 〈 0.05） as well as that of CD8＋ CTLs cultured alone or in combination with melanocytes （all P 〈 0.05）. A statistical decrease was observed in IL-6, TNF-α and IFN-γ/levels in the supernatant of cocuhured melanocytes and CTLs compared with those in the supernatant of melanocytes and CTLs cultured alone, and calcipotriol of 10^-9 mol/L intensified thedecrease in supernatant IL-6 level （t - 2.89, P 〈 0.05）, but no statistical changes were noted for the level of TNF-α or IFN-γ, in the supernatant of the cocuhure system after treatment with calcipotriol of 10^-8 or 104 mol/L compared with that before treatment （both P 〉 0.05）. In the coculture system pretreated with calcipotriol of 10^-8 mol/L, the number of CD8＋ CTLs significantly decreased （t = 3.15, P 〈 0.05）, whereas that of melanocytes significantly increased （t = 3.53, P 〈 0.05） after the treatment with anti-IL-6 antibody of 5 mg/L. Conclusions Perilesional CD8＋ CTLs have a specific cytotoxic effect on melanocytes, and calcipotriol may inhibit the cytotoxic effect of CD8＋ CTLs by suppressing the secretion of IL-6, which may partly explain the therapeutic mechanism of calcipotriol for vitiligo.