他克莫司对HaCaT细胞干细胞因子mRNA及小鼠B16黑素瘤细胞c-kit mRNA表达的影响

目的 探讨他克莫司对角质形成细胞干细胞因子（SCF）mRNA及黑素细胞c-kit mRNA表达水平的影响。方法 分别培养人永生化角质形成细胞（HaCaT）和小鼠B16黑素瘤细胞,经他克莫司作用48 h后,采用实时荧光定量PCR法检测SCF mRNA及c-kit mRNA的表达。结果 他克莫司作用后,HaCaT细胞SCF mRNA、B16黑素瘤细胞c-kit mRNA的相对表达量均升高,与空白对照组比较,差异均有统计学意义（P均 ＜ 0.05）。结论 他克莫司可以上调HaCaT细胞SCF mRNA及B16黑素瘤细胞c-kit mRNA的表达量。     

干细胞因子及其受体在寻常型白癜风表皮中的表达

目的 探讨SCF/c-kit信号通路在白癜风发病中的作用。方法 采用免疫组化和RT-PCR法检测17例寻常型稳定期白癜风患者和10例正常对照标本中表皮角质形成细胞的干细胞因子表达及基底层黑素细胞c-kit的表达情况。结果 白癜风非皮损区干细胞因子、c-kit蛋白表达与正常对照无明显差异(P>0.05),皮损区干细胞因子表达显著高于正常对照皮肤(P<0.05),而c-kit表达显著低于正常对照皮肤(P<0.05)。白癜风非皮损区表皮干细胞因子、c-kit mRNA表达平均水平与正常对照近似(P>0.05);皮损区干细胞因子mRNA表达水平高于非皮损区及正常对照组差异有统计学意义(P<0.05);皮损区c-kit mRNA表达水平显著低于非皮损区及正常对照组(P<0.05)。结论 SCF/c-kit的异常表达可能与白癜风的发病有关。     

补骨脂对人黑素细胞酪氨酸酶活性及黑素合成的影响

目的观察补骨脂对人黑素细胞酪氨酸酶活性及黑素合成的影响。方法给已经稳定传代的黑素细胞、角质形成细胞添加高、中、低三种浓度的补骨脂培养,用MTT法测细胞增殖、多巴氧化法测酪氨酸酶的活性、NaOH裂解法测黑素含量。结果与对照组相比,三种浓度补骨脂均能促进黑素细胞与角质形成细胞增殖,增加酪氨酸酶活性及黑素合成,有统计学意义(P 0.05),以中浓度组效果最佳(P 0.01)。结论补骨脂从多个环节促进黑素的合成,且适宜的浓度效果最佳,对于临床治疗白癜风有着指导意义。     

马拉色菌与人角质形成细胞株共同培养液对培养人黑素细胞生长及酪氨酸酶mRNA表达的影响

目的:观察不同马拉色菌与人角质形成细胞株共同培养液对培养人黑素细胞生长及酪氨酸酶mRNA表达的影响,探讨花斑癣的色素改变是否与马拉色菌的不同菌种有关。方法:将球形、钝形、糠秕、合轴马拉色菌分别与人角质形成细胞株共同培养,24 h后收集上清液,按一定比例加入黑素细胞培养体系中继续培养24 h:①用四甲基偶氮唑兰比色法观察对黑素细胞生长的影响;②用实时荧光定量PCR法测定黑素细胞酪氨酸酶mRNA的表达。结果:各共同培养液对黑素细胞生长无明显影响,但除球形马拉色菌与人角质形成细胞共同培养液可上调黑素细胞酪氨酸酶mRNA的表达(P<0.05)外,未见其它种马拉色菌与人角质形成细胞株共同培养液对黑素细胞酪氨酸酶mRNA的表达有影响。结论:球形马拉色菌与人角质形成细胞株共同培养液刺激黑素细胞后,能提高酪氨酸酶mR-NA的表达,该菌可能与花斑癣的色素沉着有关。     

人参皂苷Rb1对人表皮黑素细胞黑素生成的影响

目的 探讨人参皂苷Rb1对体外培养的人黑素细胞黑素生成的影响及其作用机制。方法 人表皮黑素细胞取自小儿包皮环切术标本,取第2 ～ 5代细胞进行实验。采用MTT法测定体外培养的人黑素细胞增殖情况,分光光度计法测定酪氨酸酶的多巴氧化酶活性,氢氧化钠裂解法测定黑素含量,免疫蛋白印记法测定黑素细胞酪氨酸酶、小眼畸形相关转录因子（MITF）的表达及cAMP应答元件结合蛋白（CREB）的磷酸化。结果 体外培养的人黑素细胞分别加入25、50、100 μmol/L人参皂苷Rb1 作用72 h后,黑素细胞存活率3组间差异无统计学意义（P ＞ 0.05）,黑素含量呈浓度依赖性增加（与溶剂对照组相比的相对黑素含量分别为112.4% ± 5.7%、155.7% ± 6.3%、217.2% ± 11.7%）,酪氨酸酶活性增加（相对酪氨酸酶活性分别为117.9% ± 5.7%、158.2% ± 9.6%、182.6% ± 10.0%）。100 μmol/L人参皂苷Rb1作用72 h,黑素细胞酪氨酸酶（225.4 %± 12.8%）、MITF（313.5% ± 16.7%）蛋白表达明显升高（与溶剂对照组相比,P值均 < 0.01）,CREB磷酸化明显增加（322.5% ± 21.1%）（与溶剂对照组相比,P < 0.01）。100 μmol/L人参皂苷Rb1 作用黑素细胞8 h,MITF蛋白表达无明显变化;作用24 h后,MITF表达明显增加,与0时间点相比,P < 0.01。10 μmol/L的蛋白激酶A（PKA）抑制剂H-89预处理细胞,可明显抑制100 μmol/L人参皂苷Rb1引起的CREB激活、酪氨酸酶及MITF蛋白表达的增加,与100 μmol/L人参皂苷Rb1作用72 h比较,P值均 < 0.01,进而抑制人参皂苷Rb1引起的酪氨酸酶活性作用增强及黑素合成增加。 结论 人参皂苷Rb1可促进正常人黑素细胞的黑素合成和酪氨酸酶活性。PKA/CREB/MITF/酪氨酸酶信号通路可能参与了人参皂苷Rb1的促黑素生成机制。     

干细胞因子及受体c-kit对A375细胞增殖和凋亡影响的体外研究

目的利用基因工程方法建立稳定表达干细胞因子受体(SCF/c-kit)的恶性黑素瘤A375细胞株,再给予外源性SCF/c-kit,观察其对A375细胞增殖、凋亡的影响。方法通过脂质体转染技术构建稳定表达c-kit的黑素瘤细胞株(hc-kit/A375),给予A375细胞和hc-kit/A375细胞外源性SCF,将所有的细胞分为A375组、A375-SCF组、hc-kit/A375组和hc-kit/A375-SCF组。应用MTT法及流式细胞仪分析细胞的增殖和凋亡,并应用酶联免疫吸附法(ELISA)和免疫组化方法检测肿瘤细胞血管内皮生长因子(VEGF),基质金属蛋白酶(MMP)-9及肿瘤坏死因子(TNF)-α的表达。结果①通过脂质体介导能将c-kit-pcDNA3.1neo重组质粒成功转染入黑素瘤细胞,建立稳定表达c-kit的黑素瘤细胞株hc-kit/A375,hc-kit/A375细胞中c-kit表达明显增加。②给予外源性SCF后,细胞的生长受到抑制,以培养后第5天尤为显著;细胞的凋亡率明显增加,S期比例降低,且hc-kit/A375-SCF组尤明显。③转染后细胞及加入SCF干预组VEGF、MMP-9分泌减少,而TNF-α的分泌增加。结论通过脂质体转染和G418筛选成功构建稳定表达c-kit的A375细胞株;通过增加A375细胞中SCF/c-kit的表达能改变肿瘤细胞对VEGF、MMP-9、TNF-α的分泌,抑制肿瘤细胞的增殖,促进其凋亡。     

葛根素促进黑素细胞黑素合成及其机制的初步探讨

目的 探讨葛根素对正常人黑素细胞黑素合成的影响和可能机制。 方法 用噻唑蓝（MTT）法、NaOH裂解法观察葛根素对黑素细胞增殖及黑素合成作用,RT-PCR及Western印迹法检测葛根素对黑素细胞小眼畸形相关转录因子（MITF）、酪氨酸酶（TYR）、酪氨酸酶相关蛋白1（TRP-1）基因转录和蛋白表达水平的影响。 结果 1 ~ 40 μmol/L浓度范围内葛根素对体外培养的黑素细胞增殖影响与正常对照组相比,差异无统计学意义（P > 0.05）。与正常对照组相比,40 μmol/L葛根素可显著促进黑素细胞的黑素合成（P < 0.05）,并可显著增加MITF、TYR、TRP-1 mRNA及蛋白的表达（P < 0.05）。40 μmol/L葛根素使MITF、TYR、TRP-1的蛋白表达量分别比正常对照组增加8.69%,10.28%和10.58%（P < 0.05）;并使MITF、TYR、TRP-1的mRNA表达量分别比正常对照组增加2.48倍,1.91倍和1.63倍（P < 0.05）。 结论 葛根素能增加MITF、TYR、TRP-1 mRNA及蛋白的表达水平,促进黑素合成。     

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。     

不同肤色个体表皮角质形成细胞Caspase14表达的研究

目的 探讨不同肤色人群角质形成细胞中Caspase14表达变化,明确黑素细胞和（或）黑素对其表达的影响。 方法 以Western印迹检测不同肤色个体原代角质形成细胞Caspase14的表达。取体外培养不同肤色的2代角质形成细胞,分别加入不同肤色的黑素细胞在分化培养基下共同培养24 h,以不加黑素细胞组为对照,Western 印迹检测Caspase14的表达。结果 浅、深肤色个体包皮原代角质形成细胞Caspase14的表达存在显著差异,深肤色者（Fitzpatrick Ⅳ/Ⅴ）原代角质形成细胞中Caspase14的表达显著高于浅肤色者（FitzpatrickⅠ/Ⅱ）（P ＜ 0.01）;深、浅肤色个体的角质形成细胞在与不同肤色个体的黑素细胞在分化培养基中共培养后24 h后,检测发现,与对照组相比,浅肤色个体角质形成细胞Caspase14表达的增加,差异无统计学意义（P ＞ 0.05）,而深肤色个体角质形成细胞Caspase14的表达则显著上调（P ＜ 0.05）。结论 深肤色个体角质形成细胞Caspase14表达显著高于浅肤色个体者。黑素细胞显著增加深肤色者角质形成细胞中Caspase14水平的表达。     

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。     

Less keratinocyte-derived factors related to more keratinocyte apoptosis in depigmented than normally pigmented suction-blistered epidermis may cause passive melanocyte death in vitiligo

Stem cell factor (SCF) of keratinocyte origin regulates melanocyte growth and survival. Deprivation of survival factors causes the apoptosis of melanocytes. Vitiligo often develops following physical trauma, even if this is minor. The exact mechanism of the Koebner phenomenon in vitiligo is unclear. Apoptosis of keratinocytes, which occurs more in depigmented suction-blistered epidermis than in the normally pigmented counterpart, could reduce levels of keratinocyte-derived factors such as SCF and basic fibroblast growth factor (bFGF). Levels of SCF expression were examined in the depigmented and normally pigmented paired epidermis of 19 patients with vitiligo, and bFGF expression in six patients. The expression of SCF (p<0.001) and bFGF was usually reduced in the depigmented compared with the normally pigmented epidermis. Apoptosis of cultured normal human keratinocytes, which was induced by staurosporine, resulted in a concentration-dependent decrease in levels of SCF mRNA and protein. Normal human melanocytes proliferated more in medium containing SCF or keratinocyte (XB-2) feeder than in medium with neither. Deprivation of SCF or keratinocyte feeder in the culture medium induced a marked decrease in melanocytes as a result of apoptosis. Therefore, lower expression of keratinocyte-derived factors, including SCF, in vitiliginous keratinocytes, which could result from keratinocyte apoptosis, might be responsible for passive melanocyte death and may explain the Koebner phenomenon.     

人表皮黑素细胞与角质形成细胞共培养体外模型中黑素小体转运的观察

目的:建立人表皮黑素细胞与角质形成细胞共培养的体外模型,并且在模型中观察黑素小体的转运。方法:分别培养人表皮黑素细胞和角质形成细胞,用二乙酰羧基荧光素-琥珀酰亚胺酯(CFDA-SE)标记黑素细胞内黑素小体,然后将两种细胞共培养,并在倒置显微镜和共聚焦显微镜下观察共培养模型中黑素小体由黑素细胞向角质形成细胞的转运。结果:培养至第3天和第7天可在倒置显微镜下观察到黑素细胞与角质形成细胞共同存活,并通过树突发生联系,在共聚焦显微镜下可直接观察到黑素小体由黑素细胞向角质形成细胞的转运。结论:成功建立黑素细胞与角质形成细胞共培养的体外模型,并利用CFDA-SE标记,在共聚焦显微镜下直接观察到黑素向角质形成细胞的转运。     

Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes

Background: There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required. Aim: In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients. Methods: The skin grafts were taken from control and patients of vitiligo. In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes. Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase assay, MTT assay and melanin content assay were performed. Results: Melanocytes and keratinocytes were successfully cultured from control and vitiligo patients and after that co-culture models were prepared. After treatment of co-culture model with melanogenic stimulator we found that tyrosinase activity, cell proliferation and melanin content increased whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and melanin content decreased. We also found some differences in the control co-culture model and vitiligo co-culture model. Conclusion: We successfully constructed in vitro co-culture pigmentation model for control and vitiligo patients using primary cultured melanocytes and keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over the use of transformed cells. The only limitation of these models is that these can be used for screening small numbers of compounds.     

CRISPR-Cas9诱导KRT5基因突变的HaCaT细胞对共培养人黑素细胞的影响研究

【摘要】 目的 探究敲减角质形成细胞KRT5基因对共培养黑素细胞黑素含量的影响,以解释屈侧网状色素沉着症（DDD）的色素增加性皮损形成的机制。方法 通过成簇的规律间隔的短回文重复序列-相关蛋白9（CRISPR-Cas9）技术获得KRT5基因杂合突变的HaCaT细胞,将转染非靶向单向导RNA：Cas9蛋白复合物的HaCaT细胞设为对照组,分别与人原代黑素细胞HEMn体外共培养。免疫荧光观察共培养细胞中细胞角蛋白及黑素小体表达;差异胰酶消化获得不同共培养组中黑素细胞,检测细胞内黑素含量。免疫组化检测1例KRT5突变DDD患者皮损和正常对照皮肤中黑素细胞特异性前黑素小体蛋白17（Pmel17）的表达改变。结果 Sanger测序显示,实验组HaCaT细胞KRT5基因第1外显子起始密码子发生杂合突变（c.1delA）,对照组HaCaT细胞KRT5基因未发生突变。Western印迹显示,实验组HaCaT细胞KRT5蛋白表达水平（0.60 ± 0.05）显著低于对照组（1.00 ± 0.00,t = 32.38,P = 0.001）。HEMn细胞与实验组HaCaT细胞共培养体系中Pmel17标记的黑素小体较与对照组共培养体系增多,且细胞内黑素含量增加52.5%,差异具有统计学意义（t = -3.48,P = 0.025）。KRT5突变DDD患者皮损组织中Pmel17表达量较正常对照皮肤增加。结论 本研究通过CRISPR-Cas9诱导KRT5基因突变的HaCaT细胞,与黑素细胞在体外建立共培养细胞模型,验证了其对共培养黑素细胞的影响,为进一步研究角质形成细胞与黑素细胞相互作用机制以及皮肤色素异常的发病机制提供了初级研究模型。     

16-Kauren-2-beta-18,19-triol inhibits melanosome transport in melanocytes by down-regulation of melanophilin expression

BackgroundRab27a, Mlph, and MyoVa form a tripartite complex and relate to melanosome distribution. Melanophilin (Mlph) acts as a linker protein between Rab27a and MyoVa. The biological activity and function of 16-kauren on the expression of Mlph has not yet been studied.ObjectiveWe examined the effect of 16-kauren on melanosome transport and skin pigmentation.MethodsMurine Melan-a melanocytes and SP-1 keratinocytes were used for in vitro analysis. Western blot analysis, quantitative real-time polymerase chain reaction, luciferase assay and immunohistochemical staining in 3D pigmented human skin model were performed.ResultsWe found that 16-kauren inhibits melanosome transport in Melan-a melanocytes without affecting melanin synthesis. Treatment with 16-kauren reduced melanophilin (Mlph), a key protein in melanosome transport, in Melan-a melanocytes, at both the protein and mRNA levels while it did not affect the expression of Rab27a and MyoVa, the other two key proteins for melanosome transport. Notably, the expression of melanogenic proteins, including tyrosinase, trp1, trp2, and MITF, was not affected by 16-kauren. However, 16-kauren attenuated melanosome distribution in co-culture of Melan-a melanocytes and SP-1 keratinocytes as well as in Melan-a monolayer culture. In further confirmation of the depigmenting effects of 16-kauren on Melanoderm™, a 3D pigmented human skin model, treatment with 16-kauren for 12 days increased the brightness of the tissue as determined by lightness value and reduced the distribution of melanosomes as shown in histological examination.ConclusionThese results demonstrated that 16-kauren is a selective modulator of a melangenic target, Mlph expression, and can be employed as a new depigmenting strategy.