Regulation of epidermal growth factor receptor expression in human melanocytes

The epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor alpha (TGFalpha), are reportedly involved in autocrine growth of melanoma cells. The signal pathway has also been implicated in early events of transformation, suggesting a function for EGFR in normal cells. This study reports the presence of EGFR in cultured melanocytes and examines some cellular responses to TGFalpha. Western analysis revealed 170 kDa bands in extracts of cultured neonatal human melanocytes, corresponding to the receptor Mr. Protein expression was more pronounced in cells during active growth. EGFR were less evident in cultures populated predominantly by melanized cells, indicating that receptor expression became reduced in differentiating cells. Immunocytochemistry confirmed these observations and also showed that EGFR reactivity was predominantly localized in the cell body but absent from dendrites. Addition of TGFalpha to early cultures induced a rapid increase in phosphotyrosine signal of the 170 kDa protein. Longer treatment (24-48 h) increased the intensity of the EGFR signal, suggesting that receptors had been upregulated. However, inclusion of TGFalpha in cultures did not result in an increase in cell numbers when compared to controls. The observations provide evidence of the existence of a receptor-mediated pathway in melanocytes which has transforming potential in vivo.     

自体表皮与黑素细胞移植治疗白癜风疗效对比

目的:研究和比较自体表皮移植与黑素细胞移植治疗白癜风的临床效果.方法:自体表皮移植采用局部皮肤发疱后直接进行移植;黑素细胞移植是从疱壁七获取黑素细胞、纯黑素细胞培养与增殖、移植区刮除种植法进行自体黑素细胞移植治疗.结果:自体表皮移植与黑素细胞移植治疗白癜风对其皮损的评分分别为(7.97±2.36)分和(11.46±2.57)分,经统计学分析差异有统计学意义(P＜0.01).结论:自体表皮移植操作方法较简单,黑素细胞移植可治疗面积大的皮损,而且色素分布均匀,临床效果更好.     

他卡西醇对人表皮黑素细胞增殖、黏附、迁移及c-kit表达的影响

【摘要】 目的 探讨他卡西醇对体外培养的人表皮黑素细胞增殖、黏附、迁移及c-kit mRNA相对表达的影响。 方法 用不同浓度的他卡西醇干预体外培养的人表皮黑素细胞,采用四唑氮氢氧化物（XTT）法分别检测培养24、48、72 h后黑素细胞的增殖活性;用纤维连接蛋白（FN）包被细胞培养板检测培养72 h后黑素细胞的黏附率;用Transwell微孔膜法检测培养24 h后黑素细胞在FN上的迁移情况;逆转录-聚合酶链反应（RT-PCR）检测培养72 h后黑素细胞c-kit mRNA相对表达量。 采用重复测量方差分析及完全随机设计方差分析进行统计学检验。 结果 重复测量方差分析结果显示,10-10、10-9、10-8、10-7、10-6 mol/L他卡西醇促进黑素细胞增殖作用的差异有统计学意义（F = 9.47,P < 0.01）,上述浓度他卡西醇在培养24、48、72 h后促进黑素细胞增殖作用的差异有统计学意义（F = 14.44,P < 0.01）,药物浓度和培养时间之间存在交互作用（F = 2.47,P < 0.01）,其中10-8 mol/L他卡西醇与黑素细胞共同培养72 h黑素细胞增殖活性最高。10-8 ～ 10-7 mol/L他卡西醇可显著促进黑素细胞在FN上的黏附（均P < 0.01）;10-9 ～ 10-8 mol/L他卡西醇在培养24 h能显著促进黑素细胞迁移（均P < 0.01）;RT-PCR显示10-9 ～ 10-7 mol/L他卡西醇在培养72 h均能显著增加黑素细胞c-kit mRNA的相对表达量（均P < 0.01）。 结论 他卡西醇可促进培养的人表皮黑素细胞增殖及在FN上的黏附、迁移作用,并可上调黑素细胞c-kit mRNA的表达。 【关键词】 他卡西醇; 黑素细胞; 细胞增殖; 细胞运动; 原癌基因蛋白质c-kit     

白癜风表皮黑素细胞超微结构及小眼畸形相关转录因子转录调控研究

目的 研究白癜风黑素细胞超微结构和小眼畸形相关转录因子 （MITF）及其转录调控的酪氨酸酶相关蛋白（TRP）与白癜风临床类型与病程的相关性。方法 选择不同病程的寻常型白癜风（VV）12例和节段型白癜风（SV）8例,分别取白斑区、白斑边缘正常肤色区和远离白斑正常肤色区的表皮片,经组织学确定其表皮的完整性。透射电镜观察10例患者（VV 6例,SV 4例）不同区表皮黑素细胞的超微结构特点。对所有20例远离白斑正常肤色区的表皮片黑素细胞进行培养,应用免疫印迹方法检测 MITF及其转录调控的酪氨酸酶（TYR）、酪氨酸酶相关蛋白1（TYRP1）和酪氨酸酶相关蛋白2（TYRP2）的表达水平。结果 白癜风表皮黑素细胞超微结构病理改变：10例中7例白斑区表皮内未见黑素细胞,1例短病程和2例长病程VV分别可见少量黑素体显著减少或缺失的黑素细胞;白斑边缘正常肤色区,6例VV中,3例病程小于15个月者可见黑素细胞超微结构异常,而4例SV中仅1例异常;远离白斑正常肤色区,10例黑素细胞超微结构均正常。白癜风表皮黑素细胞MITF及其转录调控TRP的表达：VV的MITF表达下调与TYR、TYRP1、TYRP2的表达下调一致;SV存在MITF显著表达下调,而TYR、TYRP1、TYRP2几均正常表达。结论 VV和SV可能存在不同的表皮黑素细胞超微结构病理改变和MITF转录调控机制。     

Transplantation of melanocytes in vitiligo

Summary We report the results of a study on 100 patients (aged 12–68) with vitiligo, who were treated by transplantation of cultured autologous melanocytes to the depigmented areas, after removal of the epidermis at the recipient site by dermabrasion. The melanocytes were cultured from a 2x3 cm² superficial shave biopsy taken from pigmented buttock skin. After 2–3 weeks in culture, 700–1000 cells per mm² were applied on 60–500 cm² dermabraded areas, and occluded for 1 week. The repigmented portion of the total treated area amounted to 95–100% in 40 patients, 65–94% in 32, 20–64% in 22, and 0–19% in six. It was more difficult to achieve complete pigmentation on the fingers, elbows and knees. In the first few months following the procedure, the treated areas were often hypo- or hyperpigmented, but after 6–8 months they had acquired the same colour as the surrounding skin. No scarring or other side-effects occurred. The donor site had repigmented after 3–6 months in all but two patients, who also showed poor pigmentation in the transplanted areas. At follow-up after 1 and 2 years in 50 and 10 patients, respectively, the repigmented areas remained unchanged. The method is time-consuming, but the results obtained indicate that the procedure can be valuable in motivated patients, when the extent of vitiligo does not exceed 30% of the total body area, and when the areas to be treated are not actively extending.     

Retinoic-acid receptor beta expression in melanocytes

Retinoic acid receptors (RAR) and retinoic X receptors (RXR) are critical for skin homeostasis. In epidermis, RXRalpha and RARgamma isoforms are highly expressed but only weak or no expression of RARbeta has been reported. Here, we re-examined RARbeta in situ expression in comparison with that of the RXRalpha, in both normal skin and melanocytic tumours. In normal skin, RXRalpha was localized in epidermis, sebaceous glands and hair follicles, while RARbeta was detectable only in melanocytes and in stratum granulosum. RXRalpha was never detected in melanocytic tumours, neither in nevi, nor in melanomas. RARbeta was also absent from melanoma cells but was present in nevus cells. These results indicate that melanoma are characterised by simultaneous decrease of RARbeta and absence of RXRalpha that may be responsible for the RA-resistance of most melanoma cell lines.     

Subtoxic levels hydrogen peroxide-induced expression of interleukin-6 by epidermal melanocytes

Oxidative stress and autoimmune reaction are involved in the pathogenesis of vitiligo. Levels of hydrogen peroxide (H₂O₂) and interleukin-6 (IL-6), a proinflammation cytokine and a key factor in the pathogenesis of autoimmune diseases, have been reported to be elevated in vitiligo lesions. The objective of this study was to evaluate the effects of subtoxic levels of H₂O₂ on the expression of IL-6 by cultured human epidermal melanocytes and to explore the relevant signal pathways. Cultured human melanocytes were stimulated with of H₂O₂ at subtoxic levels. Levels of IL-6 protein in the medium and IL-6 mRNA in the cells were measured by IL-6 ELISA analysis and RT-PCR, respectively. NF-??B and phosphorylated p38 mitogen-activated protein kinase (MAPK), ERK and JNK in cells cultured with and without H₂O₂ were measured by relevant ELISA kits. In cultured melanocytes, subtoxic levels of H₂O₂ (30?C300???M) significantly increased the IL-6 mRNA and protein levels in a dose-dependent manner. NF-??B in nuclear extracts and phosphorylated p38 MAPK levels in cell lysates were significantly increased in H₂O₂ treated cells. Pretreatment of cells with inhibitors of p38 MAPK (SB203580) and NF-??B (BAY11-7082), but not inhibitors of ERK (UO1026) and JNK (SP600125), abolished H₂O₂-induced expression of IL-6. H₂O₂-induced overexpression of IL-6 by melanocytes may be a molecular linkage for the oxidative stress and inflammatory/autoimmune reactions in vitiligo and may provide a novel target for the treatment of vitiligo.     

Expansion of vitiligo lesions is associated with reduced epidermal CDw60 expression and increased expression of HLA-DR in perilesional skin

BACKGROUND: Detection of CDw60 in skin is representative of ganglioside D3 expression. This ganglioside is expressed primarily by melanocytes, and is of interest as a membrane antigen targeted by immunotherapy for melanoma patients. Expression of CDw60 by keratinocytes is defined by the presence of T-helper cell (Th)1 vs. Th2 cytokines, and can serve as a sentinel molecule to characterize an ongoing skin immune response. OBJECTIVES: These immunobiological characteristics have provided the incentive to study the expression of CDw60 in the context of progressive vitiligo. METHODS: Frozen sections were obtained from control skin and from vitiligo lesions and immunostained to show CDw60. Cells were cultured, their CDw60 expression studied and ribonuclease protection assays run to detect cytokine mRNA. RESULTS: Resistance to cytokine-mediated regulation of CDw60 expression was demonstrated in vitro by melanocytes, which appeared capable of generating autocrine and paracrine regulatory molecules supporting CDw60 expression. Induction of CDw60 expression was inhibited by antibodies to interleukin (IL)-4, suggesting that this cytokine was responsible, at least in part, for melanocyte-induced CDw60 expression. Marginal skin from patients with progressive generalized vitiligo consistently showed a reduction in epidermal CDw60 expression alongside elevated human leucocyte associated antigen (HLA)-DR expression at the margin. It thus appears that inflammatory infiltrates present in marginal skin generate type 1 rather than type 2 cytokines, supportive of a cell-mediated autoimmune response. CONCLUSIONS: These results support an active role of melanocytes within the skin immune system, and associate their loss in generalized vitiligo with a cell-mediated immune response mediated by type 1 cytokines.     

白癜风免疫微环境对黑素细胞CXCL10表达的影响

目的:明确白癜风免疫微环境对黑素细胞CXCL10表达的影响。方法:采用qRT-PCR检测进展期白癜风患者皮损组织中的细胞因子;采用qRT-PCR及ELISA检测白癜风免疫微环境中上调的细胞因子对黑素细胞系PIG1 CXCL10表达和分泌。结果:进展期白癜风患者皮损组织中IFN-γ、TNF-α和IL-1βmRNA表达上调;三者联合刺激显著上调PIG1细胞CXCL10 mRNA的表达以及蛋白分泌,并呈现时间依赖性。结论:白癜风局部免疫微环境促进黑素细胞表达及分泌CXCL10,可能参与白癜风发病。     

Keratin 16 expression in epidermal melanocytes of normal human skin

Although the prevailing dogma states that keratin filaments are the hallmark of keratinocytes and other epithelial cells, recent publications suggest that they may be expressed by a variety of normal and malignant cells of different embryonic origin. Keratin expression has been reported in fibroblasts and endothelial cells as well as in various sarcomas. Also, some human melanomas express keratins in addition to the traditional diagnostic markers of differentiation, such as S-100 and melanocyte-specific antigens. Many studies have shown that cultured cells obtained from various melanomas express keratin. Most recently, keratin expression has also been shown in cultured melanocytes of normal skin. We now report that normal human melanocytes in vivo express keratin 16 (K16) but not keratins 1, 5, 8, 10, 14, or even keratin 6, the type II partner that is normally expressed with K16 in keratinocytes. Similarly, melanocytes in vitro express K16 but not K6. Keratin 16 expression in vivo was present in basal melanocytes in specimens derived from donors (0-77 years) and from different anatomic locations, suggesting that keratin 16 is constitutively expressed by all melanocytes. It appears that keratin expression may be more prevalent than previously assumed, and that these cytoskeletal filaments may play important roles in tissues and cells other than epithelia.     

Detection of antibodies to melanocytes in vitiligo by specific immunoprecipitation

Immunoprecipitation was used to assay for antibodies to normal human melanocytes in the sera of 12 patients with common vitiligo and 12 normal individuals. The procedure is based on the specific immunoprecipitation using protein A-sepharose of antibodies binding to detergent-soluble, radioiodinated macromolecules of normal human melanocytes grown in culture. Antibodies to melanocytes were found in all 12 patients with vitiligo but in none of the normal sera. None of the sera reacted specifically to normal human fibroblasts or to human melanoma cells radioiodinated in a similar manner. These observations suggest that antibodies to melanocyte-associated antigens are present in common vitiligo.     

The hair follicle melanocytes in vitiligo in relation to disease duration

Background and aims  Vitiligo is an acquired pigmentary disorder of skin and hair. Active melanocytes in hair follicles can be detected by DOPA and immunohistochemical staining, while amelanotic melanocytes can only be detected by the latter. None of the studies on hair melanocytes in vitiligo discussed the effect of disease duration on these melanocytes.
Here, we study the presence of melanotic and amelanotic melanocytes in vitiligo hair follicles and statistically correlating their presence with the disease duration.
Methods  This study was conducted on 30 patients with vitiligo and 10 normal volunteers. Three biopsies were taken from each patient: two from black and white hairs from vitiliginous areas and the third from apparently normal skin of the same patients. Sections were stained by DOPA reaction and NKI/beteb then examined for the presence of melanocytes. The presence of melanocytes and the disease duration were correlated statistically using the t -test.
Results  Active melanocytes were detected in black hairs of 6.7% of vitiligo patients and in 100% of apparently normal skin of the same patients and controls. On examining black hairs of the 28 vitiligo patients with negative DOPA reaction, 19 of them (67.9%) showed positive NKI/beteb stain. Disease duration was inversely correlated with the melanocytes' presence within hair follicles. Melanocytes were absent from 100% of white hairs.
Conclusions  The melanotic melanocytes were the first target of the disease process followed by the amelanotic melanocytes. Since the disappearance of the latter was inversely correlated with the disease duration, early treatment in vitiligo is advised.

Conflicts of interest

None declared.     

姜黄素对人表皮黑素细胞活性、迁移及c-kit mRNA表达量的影响

目的 探讨姜黄素对人表皮黑素细胞活性、黑素细胞迁移及c-kit mRNA相对表达量的影响。方法 不同浓度（5、10、20、30 μmol/L）姜黄素对人表皮黑素细胞活性影响实验,分为阴性对照组（添加MelM-2完全培养基 + 细胞）、药物对照组（MelM-2完全培养基 + 姜黄素）、空白对照组（仅添加MelM-2完全培养基）及实验组（MelM-2完全培养基 + 姜黄素 + 细胞）,用MTS法分别检测培养24 h、48 h后细胞活性。划痕实验检测黑素细胞迁移的情况,实时荧光定量PCR检测黑素细胞c-kit mRNA相对表达量。实验分为对照组（仅添加MelM-2完全培养基 + 细胞）及3个浓度（5、10、20 μmol/L）实验组。 结果 不同浓度（5、10、20、30 μmol/L）姜黄素培养基培养黑素细胞24 h、48 h时,与对照组相比,30 μmol/L组均可明显抑制黑素细胞的增殖（P < 0.05）,其他浓度差异无统计学意义（均P > 0.05）。划痕实验结果显示,与对照组相比,在培养48 h时,5、10、20 μmol/L姜黄素均能显著抑制黑素细胞迁移（均P < 0.05）。实时荧光定量PCR检测结果显示,与对照组相比,经各浓度（5、10、20 μmol/L）姜黄素作用48 h后,均可明显抑制黑素细胞c-kit mRNA相对表达量（均P < 0.05）。 结论 低浓度的姜黄素（≤ 20 μmol/L）对黑素细胞无明显细胞毒性,30 μmol/L的姜黄素能够促进黑素细胞的凋亡。姜黄素（≤ 20 μmol/L）可抑制黑素细胞的迁移,并下调人表皮黑素细胞c-kit mRNA的相对表达量。     

体外培养自体黑素细胞移植治疗白癜风78例疗效观察

目的评价自体黑素细胞培养移植治疗白癜风的疗效和影响因素。方法临床选择稳定期白癜风患者78例,采用自体黑素细胞培养与移植治疗方法,记录疗效和并发症,分析可能影响因素。结果 78例患者总计189处皮损,98处皮损(51.85%)获得了90%以上的复色,53处皮损(28.04%)获得了50%~90%以上的复色,总有效率达到79.89%;节段型白癜风患者有效率为86.04%,优于局限型白癜风患者(83.54%)和泛发型白癜风患者(71.64%)的治疗效果;面颈部位的皮损治疗效果最佳(有效率87.80%),要优于躯干部(83.67%)和四肢部位(80.39%),手足部位治疗效果最差(有效率68.75%),黑素细胞分裂时间≤3.5 d的移植有效率明显高于3.5 d者(P0.05)。结论自体培养黑素细胞移植是治疗稳定期白癜风的一种有效方法,其疗效与白癜风的分型、皮损的部位和黑素细胞的生物活性有关。     

Increased sensitivity of melanocytes to oxidative stress and abnormal expression of tyrosinase-related protein in vitiligo

BACKGROUND: Vitiligo is a depigmenting disease of the skin, which may derive from programmed melanocyte death or destruction due to inherent sensitivity to oxidative stress arising from either toxic intermediates of melanin, a melanocyte-specific protein, or other sources. Tyrosinase-related protein (TRP) -1 has been shown to be involved not only in melanin biosynthesis but also in the prevention of premature melanocyte death in animals. OBJECTIVES: To clarify the biological role of human TRP-1 in melanocyte survival. METHODS: Cultured melanocyte strains from an active advancing border of vitiligo were established and studied. RESULTS: The established 'vitiligo melanocytes' showed large perikaryon and stubby dendrites. They showed early cell death when exposed to oxidative stress (ultraviolet B) and increased and abnormal immunostaining and immunoprecipitation by antibodies against human and mouse TRP-1, indicating an altered synthesis and processing of TRP-1. In pulse-chase and sequential immunoprecipitation experiments, vitiligo melanocytes revealed abnormal protein-protein interaction with calnexin, a melanogenesis-associated chaperone, suggesting altered folding and maturation of nascent TRP-1 polypeptides. Northern blot analysis indicated a decreased expression of TRP-1 mRNA, but heteroduplex analysis and verification of the mutation at the carboxy terminus of TRP-1 by restriction enzyme analysis did not show any abnormality. CONCLUSIONS: Our study suggests that the early cell death of vitiligo melanocytes is related to their increased sensitivity to oxidative stress, which may arise from complex processes of abnormal synthesis and processing of TRP-1 and its interaction with calnexin.