丹皮酚在体外对人黑素细胞酪氨酸酶活性及黑素生成的影响

目的探讨丹皮酚对人表皮黑素细胞与角质形成细胞共培养体系酪氨酸酶活性及黑素生成的影响。方法培养人表皮黑素细胞及人表皮黑素细胞与角质形成细胞共培养体系,利用四甲基偶氮唑蓝(MTT)还原法测定细胞活力,采用酶学方法测定酪氨酸酶活性,475nm比色法测定黑素含量。结果50,100和200μmol/L丹皮酚对于黑素细胞及共培养细胞酪氨酸酶活性及黑素生成均有较强的剂量相关性抑制作用,100及200μmol/L丹皮酚与对照组相比差异有统计学意义(P<0.01)。结论丹皮酚对于共培养体系酪氨酸酶活性及黑素生成有较强的剂量相关性抑制作用。     

Paracrine crosstalk between endothelial cells and melanocytes through clusterin to inhibit pigmentation

Cutaneous vasculature systems play a role in regulating skin pigmentation. We analysed RNA sequencing data to identify novel antimelanogenic factors secreted from endothelial cells and found that one of the secreted factors, clusterin, is highly expressed by HDMECs. To investigate the paracrine effect of clusterin from HDMECs on the regulation of melanogenesis, HDMECs were infected with clusterin or sh‐clusterin lentivirus and the HDMEC‐derived conditioned media were used to treat normal human melanocytes. It was found that HDMEC‐derived clusterin inhibits melanogenesis through MITF/tyrosinase downregulation. The findings here suggest that HDMECs secrete copious amounts of clusterin and that the clusterin is a factor contributing to the inhibitory effect of endothelial cells on melanogenesis via paracrine crosstalk between endothelial cells and melanocytes.     

Growth and differentiation of normal human melanocytes in a TPA-free,cholera toxin-free,low-serum medium and influence of keratinocytes

Melanocyte cultures were obtained from a modification of the keratinocyte culture system MCDB153. Either promelanocytes or mature melanocytes were selected from epidermal cell primary cultures. Pure subcultures of actively dividing melanocytes of both types were grown in a low-serum medium totally deprived of TPA and cholera toxin called melanocyte growth medium (MGM). Early passaged cells from MGM primary cocultures were similar to normal adult human melanocytes in vivo, exhibiting numerous melanosomes, strong dopa positivity and a high dendricity. The ability of MGM to support melanocyte growth was mainly a consequence of its basic composition, combined with a low serum concentration. Bovine pituitary extract significantly enhanced melanocyte growth. Using complete MGM, in the absence of mitogens and keratinocytes, cell growth was maintained, but the differentiation of melanocytes decreased. The presence of keratinocytes was found to promote melanocyte growth. The coculture system used strongly suggests the action of soluble keratinocyte-derived factors. Keratinocyte contact was necessary to sustain melanocyte dendricity and melanization. Melanization and dendricity behaved mostly as independent features when keratinocyte influence was withheld. Our results underline the essential role of keratinocytes in the regulation of melanocyte growth and differentiation in a physiological culture system.Read in part before the European Society for Dermatological Research, Turin, 9–12 June 1990Supported by grants from Laboratoires Pierre Fabre, Castanet Tolosan, France, Laboratoires Roc, Paris, to A.T.     

Pure melanocyte cultures: Differential serum requirements of guinea pig keratinocytes and melanocytes in primary epidermal cell cultures

Summary Contrary to melanocytes guinea pig keratinocytes do not attach and grow in primary epidermal cell cultures if plated in media containing guinea pig serum. This phenomenon is based on the low content or lack of guinea pig serum of a factor(s) promoting keratinocyte attachment. This factor, which is contained in fetal calf serum, binds to the keratinocyte cell surface and can be removed by trypsin. Guinea pig epidermal cell cultures plated in medium containing guinea pig serum therefore lead to pure or almost pure melanocyte cultures.Presented in part at the 5th Annual Meeting of the Arbeitsgemeinschaft Dermatologische Forschung, Nov. 18–20, 1977, Frankfurt/Main     

人黑素细胞体外培养条件的研究进展

从培养人黑素细胞的基本培养液、添加剂、紫外线以及共培养系统等方面综述安全、有效的黑素细胞体外培养条件的研究进展;同时总结了人毛囊无色素性黑素细胞、永生化黑素细胞的培养条件,对色素疾病的基础研究及临床治疗有重要意义。     

Variations in melanin formation by cultured melanocytes from different skin types

Abstract In many laboratories, culturing skin melanocytes has become a routine research activity. However, recent investigations have revealed that the quality and quantity of the pigment formed in the cultured cells may differ significantly from those of the original skin pigment cells. To shed more light on this issue, we examined the influence of different culture media on pigment production. We showed that there were notable passage-to-passage variations in the synthesis of melanin. This was particularly true for phaeomelanin. It is therefore advisable to analyse the melanin in the cells before the start of experiments. In spite of the variations, basic differences in the pigmentation pattern between melanocytes isolated from light-skinned and dark-skinned individuals remained preserved in the corresponding cultures as observed by electron microscopy. Also, the total melanin content was higher in a skin type VI melanocyte culture than in skin type I and II melanocyte cultures. In contrast to total melanin, the phaeomelanin concentration of skin type VI cells was similar to that of the skin type I melanocytes. With higher l-tyrosine concentrations in the medium, as well as increased eumelanin synthesis, phaeomelanogenesis was also stimulated in all cultures tested. This stimulation was particularly prominent in skin type I melanocytes. Our preliminary experiments also showed that a melanocyte culture from atypical naevus cells exhibited a similar preference for phaeomelanogenesis when pigmentation was stimulated. Received: 8 August 1997     

Mode of release of interleukin-8 from proliferating human epidermal keratinocytes in vitro

Abstract Keratinocytes have been shown to express interleukin-8 (IL-8) mRNA on stimulation with IL-1 and other substances. This has been assumed to account for the large amount of this neutrophil chemotaclic cytokine in psoriasis. We found that, without any added agents, commercially available normal human epidermal keratinocytes proliferating in Keratinocyte Growth Medium® (KGM) released a chemotactic peptide extracellularly, which was confirmed to be IL-8. To determine whether most of the IL-8 is secreted extracellularly from proliferating keratinocytes or is mainly stored to be released only on stimulation. We quantified cell-associated and released immunoreactive IL-8 from keratinocytes cultured in KGM for up to 11 days at the peptide level. The keratinocytes proliferated, taking a sigmoid growth curve, to reach a plateau at day 7. We found that the amounts of immunoreactive IL-8 gradually increased in the culture supernatant with cell growth but its prominent release took place only after the cell growth reached a plateau. The cell-associated IL-8 was much smaller in amount than that noted in the supernatant. These results suggest that the IL-8 constitutively produced by keratinocytes was mostly released extracellularly but that the production by actively proliferating cells seems to be far less than that by non-proliferating cells that probably occurred in an autocrine fashion under the stimulation of keratinocyte-derived cytokines accumulated in the culture medium. Neutrophil chemotactic activity assayed concomitantly showed a consistent increase during the culture period, indicating that, with their growth, the keratinocytes release substances other than IL-8 that exert an influence on neutrophil chemotactic functions.     

卡泊三醇对银屑病皮损中角质形成细胞凋亡的影响

从细胞凋亡角度探讨卡泊三醇(CPT)治疗银屑病(PS)的药理机制。采用末端标记(TUNEL)技术 ,分别检测了30例CPT治疗前后的PS患者和10名正常人皮肤标本的角质形成细胞凋亡。经CPT治疗后的PS皮损中的角质形成细胞凋亡数较治疗前明显增加(P<0.05)。CPT治疗PS的疗效可能与其增加角质形成细胞的凋亡有关。     

体外培养人黑素细胞HLA—DR抗原的表达

为了解正常人表皮黑素细胞是否表达ＨＬＡ－ＤＲ抗原,黑素细胞经体外培养后,是否因细胞增减的添加或培养时间的长短而影响ＬＨＡ－ＤＲ抗原表达而应用免疫酶染色法进行检测。结果发现表皮基底层的黑素细胞不表达ＨＬＡ－ＤＲ抗原;接种于含ＴＰＡ增减液的黑素细胞,在传代后２周,黑素细胞表达ＨＬＡ－ＤＲ抗原,７周后ＨＬＡ－ＤＲ抗原失表达;接种于不含ＴＰＡ培养液的黑素细胞,在传代后２周和７周时均不表达ＨＬＡ－ＤＲ抗原是     

玻璃酸钠对黑素细胞生物学活性的影响

目的 观察玻璃酸钠对黑素细胞增殖及酪氨酸酶活性的影响。方法 噻唑蓝法和酪氨酸酶活性测定法分别检测不同浓度玻璃酸钠对正常人黑素细胞生长和黑素合成能力的影响。结果 玻璃酸钠质量浓度为0、0.008、0.016、0.313、0.625、1.250、2.500、5.000、7.500、10.000 g/L时,黑素细胞增殖活性（A490）分别为0.14 ± 0.02、0.37 ± 0.08、0.45 ± 0.11、0.49 ± 0.07、0.55 ± 0.12、0.52 ± 0.11、0.49 ± 0.07、0.39 ± 0.05、0.19 ± 0.03、0.01 ± 0.01,玻璃酸钠质量浓度在0. 08 ~ 5 g/L范围内时,对黑素细胞的增殖有明显促进作用,达10 g/L时会抑制黑素细胞的增长。玻璃酸钠质量浓度在0.2 ~ 5 g/L时可明显促进酪氨酸酶活性,到10 g/L时酪氨酸酶活性反而下降。在接种黑素细胞同时加入玻璃酸钠的黑素细胞增殖作用明显快于接种后8 h再加入玻璃酸钠组。结论 玻璃酸钠在浓度0.2 ~ 5 g/L时既能促进黑素细胞的增殖,又能明显提高黑素细胞的酪氨酸酶活性。     

白癜风患者血清中抗黑素细胞IgG,IgM抗体的检测及补体介导的血清细胞毒反应

为探讨白癜风血清中的抗黑素细胞抗体类型和反应模式以及对黑素细胞的损伤作用 ,以免疫组化法检测了白癜风患者血清中抗黑素细胞IgG、IgM抗体 ,并以吖啶橙 /溴化乙锭细胞染色法观察补体介导的白癜风血清对黑素细胞损伤作用。结果发现 2 5例活动性寻常型白癜风患者血清中有 19例检出IgG型抗体 ,2 1例检出IgM型抗体 ,明显高于稳定期和正常对照组血清 ;2 5例活动期血清 9例出现对黑素细胞的明显细胞毒作用 ,高于正常人血清 ,对照的成纤维细胞未出现明显的细胞毒作用。结果提示白癜风患者血清内有抗黑素细胞IgG、IgM抗体 ,这些抗体可以通过补体选择性的损伤体外培养的黑素细胞 ,支持白癜风是一种自身免疫性疾病的假设。     

黑素细胞原代培养自体移植治疗节段型白癜风

采用ＭＣＤＢ－１５３培养基添加ｂＦＧＦ、ｗＢＰＥ及ＴＰＡ等促黑素细胞（ＭＣ）增殖的试剂，原代培养ＭＣ３～６周后，胰酶消化培养细胞制成细胞悬液，自体移植于白斑区人工诱发的水疱内，治疗１６例稳定期节段型白癜风患者，１５例取得较为满意的疗效，３个月后平均色素恢复率大于８０％。对其中３例随访１年，色素保持良好。     

赤芍等5种上调酷氨酸酶活性中药乙醇提取液对棕黄色豚鼠背部皮肤的增色作用

为了研究中药对黑素细胞生物学特性的影响,筛选有增色作用的中药,为应用中药治疗白癜风等色素减退性皮肤病提供实验诊据。选定体外对酷氨酸酶活性有显著激活作用的中药5味,用中药乙醇提取液外涂棕黄色豚鼠背部皮肤,然后取材,进行HE、Schmorl、Imokawa等方面染色,观察外用中药后黑素细胞数量和形态的改变。结果,赤芍、川芎、菟丝子、补骨脂、刺蒺藜等中药组多巴阳性黑素细胞数、含黑素颗粒细胞数及黑素含量指数均较空白对照组明显增多（P＜0.01或P＜0.05),其中菟丝子、赤芍组与阳性对照组0.1%8-MOP酊外用无显著性差异（P＞0.05)。提示赤芍、川芎、菟丝子、补骨脂、刺蒺藜等中药具有增色作用,临床上可试用于白癜风等色素减退性皮肤病。     

Expression of different immunological markers by cultured human melanocytes

The expression of different immunological markers by cultured human melanocytes (MC) in relation to immune phenomena, were investigated on ten different MC cell lines from early (1st) to late (22nd) passage. Four melanocyte lines (MC-a) which had undergone changes in growth behaviour during prolonged culture were included in the study, together with two melanoma lines. Cytospin preparations of the cells were stained for the presence of a set of different immunological markers and a melanoma-associated antigen (MAA). All MC lines, including the MC-a and the melanoma lines, showed expression of MHC class I, IL-1, IL-2, ICAM-1 and the MAA, NKI-Beteb, during all passages tested. Interestingly, four of the MC lines showed staining for the Fc receptor. A tendency towards a stronger expression of ICAM-1 on a higher percentage of cells was observed on MC with increasing passage number, the MC-a and the melanoma lines. Expression of the MAA was strongly reduced for the MC-a lines in comparison with the MC and the M14 melanoma lines. Positive staining for the HLA class II molecules was obtained on MC of intermediate and late passages, and on the MC-a and the melanoma lines in the decreasing order HLA-DR, DP and DQ. Additionally, we carried out a preliminary study showing that cultured MC also produce IL-1 and IL-6. However, we were not able to show the production of biologically active IL-2 testing several cultured MC lines. Nevertheless, the overall results taken together suggest that MC are immunologically important cells that are susceptible to changes during long-term culture.Part of this work was presented at the third ESPCR Meeting, Amsterdam, the Netherlands in 1991 and at the 53rd Annual Meeting of the SID in Baltimore, Maryland, USA, in 1992     

个体化培养自体黑素细胞移植治疗白癜风

目的 探讨使用个体化培养基进行自体黑素细胞培养移植治疗白癜风的疗效。方法 负压吸疱获取患者正常表皮片,制成细胞悬液,在Hu16黑素细胞选择性培养基中培养。检测黑素细胞分裂时间（DOT）和黑素含量,根据DOT的大小、黑素含量和细胞形态,调整血清、细胞因子浓度及补充内皮素-1,进行个体化黑素细胞培养。经2 ~ 5次传代后收集黑素细胞,白斑区用超脉冲CO2激光磨削后进行黑素细胞移植,随访观察复色效果。结果 共治疗155例稳定期白癜风患者的204处皮损,进行1次移植119例,进行2 ~ 4次移植36例。应用个体化黑素细胞培养后细胞扩增可达50 ~ 80倍。84.80%的皮损复色面积超过50%,其中52.94%的皮损复色面积超过90%,且复色均匀,未见瘢痕及其他不良反应。性别、年龄、病程长短和皮损面积大小对疗效没有影响。节段型白癜风移植疗效好于寻常型白癜风,两组有效率分别为93.62%和82.16%,痊愈率分别为65.96%和49.04%。手臂和腿部的皮损（不包括肘部和膝盖）移植后痊愈率达73.08%,疗效好于躯干、面颈;肢端皮损疗效最差,痊愈率仅为25.93%。结论 个体化培养技术能提高白癜风患者黑素细胞的培养成功率与细胞扩增倍数。体外培养的自体黑素细胞移植治疗稳定期白癜风疗效肯定,用少量供皮区即可治疗大面积皮损,值得临床应用。     

内皮缩血管肽1和促肾上腺皮质激素对体外培养的正常人黑素细胞增殖的影响

目的 了解内皮缩血管肽1及促肾上腺皮质激素(ACTH)对体外培养的正常人黑素细胞增殖的影响.方法 采用体外黑素细胞培养技术、四甲基偶氮唑蓝比色法(MTT)研究不同浓度内皮缩血管肽1、ACTH对黑素细胞增殖的影响.结果 从0.1~1000nmol/L浓度的内皮缩血管肽1均可不同程度地促进正常人黑素细胞的增殖,低浓度0.1nmol/L内皮缩血管肽1具有很好的促增殖作用.10^-13~10^-9mol/L的ACTH也可不同程度地促进正常人黑素细胞的增殖,与对照组相比,P值均<0.05,其中又以高浓度10^-9mol/LACTH促增殖能力最强,10^-12mol/L、10^-11mol/L、10^-10mol/L 3个浓度间差异无统计学意义.结论 内皮缩血管肽1及ACTH具有促进体外培养的正常人黑素细胞增殖的作用,较低浓度内皮缩血管肽1即具有很好的促增殖作用,正常人生理水平(10^-12~10^-11mol/L)及更高浓度ACTH均有较强的促增殖作用.