CAJ | 학술논문

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10-6、104 mol/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10-mol/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5 mg/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.
목적 탐토잡박삼순대백전풍환자흑소세포급피손주변CD8+세포독T세포(CD8+CTL)증식급기세포인자분비적영향.방법 실험분흑소세포조、CD8+CTL조、흑소세포여CD8+CTL공배양조,세포계수법검측잡박삼순처리전후세포수변화정황,ELISA측정잡박삼순처리전후분비백개소6(IL-6)、종류배사인자α(TNF-α)급간우소γ(IFN-γ)적변화；세포계수법검측잡박삼순처리과적흑소세포여CD8+CTL공배양조재첨가여불첨가항인IL-6항체시,흑소세포급CD8+CTL수량적변화.사선10-6、104 mol/L잡박삼순용우실험.결과 재조망검측중발현,CD8+CTL여흑소세포공배양중흑소세포유현저조망.가잡박삼순후흑소세포조적흑소세포계수여미가약대조조상비,차이무통계학의의(P＞0.05),이흑소세포여CD8+CTL공배양조흑소세포수명현증가(P＜0.05)；CD8+CTL조화흑소세포여CD8+CTL공배양조적CD8+CTL세포수야명현증가(P＜0.05).여흑소세포화CD8+CTL단독배양조상비,흑소세포여CD8+CTL공배양조분비적IL-6、IFN-γ화TNF-α현저감소；경10-mol/L잡박삼순처리후적흑소세포여CD8+CTL공배양조분비적IL-6감소경가명현,여미가약조감소솔비교,차이유통계학의의(t=2.89,P＜0.05),이IFN-γ화TNF-α재가약전후무명현변화(P＞0.05).재잡박삼순처리과적공배양조중첨가5 mg/L항IL-6항체후,흑소세포현저증가,이CD8+CTL감소,기차이유통계학의의(t=3.53,P＜0.05；t=3.15,P＜0.05).결론 백전풍피손처적CD8+CTL대흑소세포유일정적특이성살상작용,잡박삼순가이감소염증세포인자IL-6적분비,종이감소CD8+CTL대흑소세포적살상,가능시잡박삼순치료백전풍적궤제지일.
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-dimethylthiazol-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 supematant 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 108 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 104 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 cocultured 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-γlevels in the supernatant of cocultured melanocytes and CTLs compared with those in the supernatant of melanocytes and CTLs cultured alone,and calcipotriol of 10-9 mol/L intensified the decrease 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-γin the supernatant of the coculture system after treatment with calcipotriol of 104 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.