不同培养条件对皮肤角质形成细胞和成纤维细胞的活性增殖影响

目的 观察含不同血清浓度培养基对皮肤角质形成细胞和成纤维细胞体外活性的影响. 方法 体外培养、扩增皮肤角质形成细胞和成纤维细胞,按3×105/ ml的密度接种于96孔板,加入含不同血清浓度的K-SFM培养基培养.并用MTT比色法观察细胞活性. 结果 应用K-SFM+10%FBS组培养基培养成纤维细胞与DMEM+10%FBS为对照组培养细胞的活性影响相同(P＞0.05);应用K-SFM+1%FBS培养基培养角质形成细胞与K-SFM为对照组培养细胞的活性影响相同(P＞0.05); 结论 在构建人工皮肤体外模型时,角质形成细胞和成纤维细胞可分别加用含1%FBS、10%FBS的K-SFM培养基进行培养.     

正常新生小鼠角质形成细胞和成纤维细胞Fas和Fas配体的表达

Ｆａｓ和Ｆａｓ配体（ＦａｓＬ）结合可导致表达Ｆａｓ的细胞凋亡,是细胞凋亡的一个主要途径［１］。已经证明Ｆａｓ表达于正常角质形成细胞和成纤维细胞中,但是到目前为止还没有ＦａｓＬｍＲＮＡ表达在正常角质形成细胞中的直接证据。我们采用高敏感的ＲＮＡ酶保护法,半定量地检测了正常新生小鼠角质形成细胞和成纤维细胞的Ｆａｓ和ＦａｓＬｍＲＮＡ,并用流式细胞仪检测了细胞表面表达Ｆａｓ和ＦａｓＬ蛋白的阳性细胞数,以阐明Ｆａｓ及ＦａｓＬ在角质形成细胞和成纤维细胞凋亡中的作用,为研究正常皮肤生长、凋亡及某些皮肤病的发病机理提供…     

茶多酚对紫外线辐射后的角质形成细胞和成纤维细胞增生影响的保护作用

紫外线辐射(UVR)是皮肤生物性损伤的主要因素,而皮肤角质形成细胞和成纤维细胞是此生物损伤过程中受到影响的主要细胞[1]。本实验通过MTT法(四甲基偶氮唑盐)研究不同剂量的中波紫外线(UVB)和长波紫外线(UVA),对体外培养的人角质形成细胞株HaCaT和皮肤成纤维细胞增生的影响,并探讨具有抗氧化作用的茶多酚(teapolyphenol,TPP)对此影响的保护作用。1材料和方法1.1主要仪器和试剂酶联检测仪(Clinibio公司),紫外线辐照仪(Sigma公司,UVB光谱峰值为310～315nm,UVA为365nm),96孔培养板(Costar公司),DMEM培养基(Gibeco公司),小牛血清(杭…     

不同培养条件对皮肤角质形成细胞和成纤维细胞增殖的影响

目的观察含不同血清浓度培养基对皮肤角质形成细胞和成纤维细胞体外活性的影响。方法体外培养、扩增皮肤角质形成细胞和成纤维细胞,按3×10^5／ml的密度接种于96孔板,加入含不同血清浓度的K-SFM培养基培养。并用MTY比色法观察细胞活性。结果应用K—SFM＋10％FBS组培养基培养成纤维细胞与DMEM＋10％FBS为对照组培养细胞的活性影响相同（P〉0．05）;应用K-SFM＋1％FBS培养基培养角质形成细胞与K—SFM为对照组培养细胞的活性影响相同（P〉0．05）;结论在构建人工皮肤体外模型时,角质形成细胞和成纤维细胞可分别加用含1％FBS、10％FBS的K—SFM培养基进行培养。     

用同一皮肤标本培养、纯化人正常黑素细胞、角质形成细胞及成纤维细胞的观察

目的建立从同一皮肤标本同步培养人正常黑素细胞、角质形成细胞和成纤维细胞的技术。方法利用同一供体的包皮,表真皮分离后,置37℃,5%CO2的孵箱中培养,根据细胞生长状况进行相应的传代和纯化处理。结果获得大量纯化、生长良好的人正常黑素细胞、角质形成细胞和成纤维细胞。结论实现了对同一组织来源的三种细胞在不同培养基中生长状况的同步观察。     

紫外线对皮肤角质形成细胞和成纤维细胞产生MMP-1和MMP-3的影响

目的:研究中波紫外线辐射对体外培养的表皮角质形成细胞和真皮成纤维细胞产生基质金属蛋白酶-1(MMP-1)和MMP-3的直接和间接影响,研究绿茶中的主要活性成分表没食子儿茶酚没食子酸酯(EGCG)对此影响的保护作用。方法:体外培养角质形成细胞株HaCaT和真皮成纤维细胞,中波紫外线辐射、不同浓度IL-6刺激及EGCG处理后,ELISA方法测定上清液中Pro-MMP-1和MMP-3蛋白含量,半定量RT-PCR方法测细胞中mRNA含量。结果:UVB30mJ/cm2辐射后角质形成细胞分泌的pro-MMP-1和MMP-3并未增加(P>0.05),真皮成纤维细胞合成和分泌MMP-1和MMP-3mRNA含量和蛋白水平均显著增加(P<0.05),IL-6(8、16、24pg/mL)可显著增加成纤维细胞产生MMP-1和MMP-3(P<0.05)。EGCG(0.15、0.3mM)能够显著抑制紫外线诱导成纤维细胞产生MMP含量的增加(P<0.05),但IL-6刺激成纤维细胞所产生的MMP-1和MMP-3的增加不受EGCG的影响(P>0.05)。结论:中波紫外线辐射并不能直接导致角质形成细胞分泌MMP-1和MMP-3增加,但紫外线辐射后角质形成细胞分泌的IL-6可促进成纤维细胞产生MMP。EGCG对IL-6刺激成纤维细胞产生MMP增加没有影响,但它可以显著抑制紫外线辐射直接导致的成纤维细胞产生MMP-1和MMP-3的增加,对防治皮肤光老化可能有一定作用。     

五倍子对角质形成细胞、黑素细胞和成纤维细胞体外增殖的影响

目的 了解中药五倍子治疗瘢痕疙瘩的药用成分和药用机制以及可能存在的不良反应。方法 用MTT比色试验研究五倍子单宁酸对正常人皮肤中的角质形成细胞、黑素细胞和成纤维细胞体外增殖的影响。并与秋水仙碱及红花和芦荟进行了比较。结果 ①五倍子单宁酸与五倍子生药醇提物对人成纤维细胞的增殖具有相似的抑制作用。在20μg/mL及以上质量浓度时,对人皮肤黑素细胞的抑制作用较大,其细胞增殖率是不加药对照组的73.6%～68%。在50μg/mL以下的质量浓度对人角质形成细胞的体外增殖有轻微的促进作用(111%～112%)。②红花生药醇提物和芦荟水提液在实验浓度下均可促进人角质形成细胞的体外增殖,芦荟水提液还可促进人成纤维细胞的体外增殖。结论 单宁酸是五倍子醇提物中的主要活性物质,其对黑素细胞的抑制作用提示五倍子单宁酸可能存在毒副作用。     

人表皮角质形成细胞和真皮成纤维细胞对蛋白激酶C激活的不同生物学效应

细胞信号传导调节通路主要包括蛋白激酶A(PKA)、蛋白激酶C(PKC)、酪氨酸激酶等酶系统。它们广泛参与细胞的各种生物学过程如增殖、分化、基因的表达和功能蛋白的合成等^[1,2]。PKC是其中重要的调节通路。已发现人角质形成细胞和真皮成纤维细胞中均有PKC的多种亚型,但是对这两种细胞在PKC激活后生物学效应的比较研究甚少。我们通过体外实验,观察了PKC激活剂乙酸肉寇佛泊酯(PMA)刺激后角质形成细胞和真皮成纤维细胞在形态学、细胞增殖以及细胞因子产生等方面的变化,发现这两种细胞在PKC激活后生物学效应存在差异。     

银屑病成纤维细胞培育上清对角质形成细胞增殖的影响

目的 探讨银屑病成纤维细胞对角质形成细胞增殖的作用。方法 原代培养银屑病成纤维细胞、正常人成纤维细胞，无血清培养的角质形成细胞，四甲基偶氮唑蓝比色法(MTT法)检测银屑病成纤维细胞、正常人成纤维细胞培养上清对角质形成细胞增殖的影响。结果 银屑病成纤维细胞培养上清培养的角质形成细胞的增殖活性高于正常人成纤维细胞培养上清组和无血清培养的角质形成细胞。结论 银屑病成纤维细胞可能通过释放某些可溶性细胞因子促进角质形成细胞的增殖，维持银屑病皮损表皮的过度增殖状态。     

在Ⅳ型皮肤的皮肤类似物中成纤维细胞对角质形成细胞起增殖作用而对黑素细胞的生长和色素沉着起抑制作用

利用去表皮的真皮和成纤维细胞基质构建了皮肤类似物模型，并且利用该模型观察了成纤维细胞分泌的可溶性因子对黑素细胞增殖和黑素合成的影响。方法：①从新鲜环切的成人包皮(Ⅳ型皮肤)分离表皮角质形成细胞、黑素细胞和真皮成纤维细胞，分别培养至第二代；②用去表皮的真皮作底物构建     

Histamine effect on melanocyte proliferation and vitiliginous keratinocyte survival

Repigmention of vitiligo requires melanocyte proliferation and migration. Keratinocytes have been shown to play a role in this process. Data from this laboratory showed that bee venom (BV) stimulated melanocyte proliferation and migration as well as melanogenesis. As histamine release is associated with BV, its effect on melanocyte proliferation and migration was examined. Cultured normal human melanocytes treated with histamine were studied with and without receptor-specific antagonists or agonists. The effect of histamine on vitiliginous keratinocytes, in cultured cells treated with a PI3K inhibitor in the presence of TNF-α, was also examined. Histamine exerted a more significant effect on melanocyte proliferation than on melanogenesis. This occurred through the H2 receptor with complex signalling to ERK, CREB, and Akt activation, which stimulated melanocyte migration. Histamine and the H2 receptor agonist also increased survival of vitiliginous, but not normal, keratinocytes, with NF-κB activation. Because expression levels of the H2 receptor was significantly decreased in depigmented compared to normally pigmented epidermis, in patients with vitiligo, histamine may increase the survival of vitiliginous keratinocytes. Overall, histamine stimulated the proliferation and migration of melanocytes and the vitiliginous keratinocyte survival, providing the basis for novel therapeutic approaches to vitiligo repigmentation.     

Effects of psoralen plus ultraviolet A irradiation on cultured epidermal cells in vitro and patients with vitiligo in vivo

BACKGROUND: Both psoralen plus ultraviolet (UV) A (PUVA) and narrowband UVB (NB-UVB) irradiation are effective treatments for vitiligo vulgaris. However, the mechanisms of PUVA and NB-UVB in repigmentation are not thoroughly clarified. Our previous results showed that NB-UVB irradiation directly promotes melanocyte (MC) migration and stimulates MC proliferation via keratinocytes (KCs). OBJECTIVES: In the present study, we used NB-UVB as a reference for comparison to investigate the immediate effects of PUVA on MC proliferation and migration. METHODS: Cultured MCs and KCs were treated with PUVA or irradiated with NB-UVB. The direct impact of PUVA treatment on MCs was assessed in terms of its effect on MC proliferation and migration. The indirect effect of PUVA treatment and NB-UVB irradiation on MC proliferation via KCs was also investigated. The activities of matrix metalloproteinase (MMP)-2 and MMP-9, known for their influence on cell migration, were evaluated in the PUVA-treated MC and KC supernatants. The concentrations of MC mitogens/growth factors in the PUVA-treated KC supernatants were also determined. In addition, the serum levels of MC mitogens/growth factors in healthy controls, in patients with active vitiligo and in patients with repigmenting vitiligo after PUVA treatment were determined to elucidate the mechanisms of how PUVA induces vitiligo repigmentation in vivo. RESULTS: Our results demonstrated that PUVA treatment did not significantly stimulate the release of MC mitogens/growth factors from KCs. The migration of MCs was also not enhanced after PUVA treatment. The expression of MMP-2 activity in supernatants derived from PUVA-treated MCs was significantly increased as compared with the control group. However, neither MMP-2 nor MMP-9 activity in KC supernatants was stimulated by PUVA treatment. In contrast to NB-UVB, immediate effects of PUVA on MC proliferation and migration were not observed in this study. Sera from patients with repigmenting vitiligo after PUVA treatment contained higher levels of basic fibroblast growth factor, stem cell factor and hepatocyte growth factor as compared with healthy controls and patients with active vitiligo. CONCLUSIONS: Our results indicate that in addition to immune suppression, PUVA treatment creates a favourable milieu for promoting the growth of MCs in patients with vitiligo instead of directly stimulating the regrowth of MCs. Based on our results, we propose that in the active stage of vitiligo, PUVA treatment is the therapy of choice to slow down the destruction of MCs and to create a favourable environment for MCs to survive. In the stable stage of vitiligo, NB-UVB irradiation should be used to stimulate the proliferation and migration of MCs directly.     

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.     

FK506 promotes melanocyte and melanoblast growth and creates a favourable milieu for cell migration via keratinocytes: possible mechanisms of how tacrolimus ointment induces repigmentation in patients with vitiligo

BACKGROUND: Vitiligo is an acquired pigmentary disorder characterized by depigmentation of skin and hair. As the pathogenesis of this disease is still obscure, the treatment of vitiligo has generally been unsatisfactory and often disappointing. Topical tacrolimus (FK506) ointment has recently been added to the armamentarium against this pigmentary disorder. Despite its clinical efficacy, the underlying mechanisms of how topical tacrolimus induces repigmentation in vitiligo have rarely been investigated. As tacrolimus ointment is applied directly to the skin, its impact on keratinocytes (KCs) requires thorough investigation. OBJECTIVES: To investigate the effects of FK506 on melanocyte (MC) and melanoblast (MB) growth via KCs. METHODS: Cultured MCs and MBs were treated with supernatant of KC cultures conditioned with various concentrations of FK506. The impact of supernatant on MCs and MBs was assessed in terms of its effect on MC/MB proliferation, melanin formation and cell migration. The activities of matrix metalloproteinase (MMP)-2 and MMP-9, known for their influence on cell migration, were evaluated. The concentrations of MC/MB growth factors in the KC supernatant were also determined. RESULTS: Results demonstrated that proliferation of both MCs and MBs was significantly enhanced by FK506-treated KC supernatant. In addition, the concentration of stem cell factor in KC supernatant increased dose-dependently with FK506 treatment. The supernatant from FK506-treated KC culture showed a significant increase in MMP-9 activity. CONCLUSIONS: Our study provides in vitro evidence demonstrating that direct interaction between FK506 and KCs creates a favourable milieu for MC growth and migration. Furthermore, our findings provide a possible mechanism explaining how tacrolimus ointment induces repigmentation in patients with vitiligo.     

Autologous melanocyte–keratinocyte suspension in the treatment of vitiligo

Background In stable vitiligo, several techniques of autologous transplantation of melanocytes are used. Autologous melanocyte transplantation of non‐cultured melanocytes is one of those techniques with variable reported outcomes. Objective The objective of this study was to evaluate the response to autologous melanocyte–keratinocytes suspension transplantation in cases of stable vitiligo. Methods A total of 25 cases of vitiligo were treated by autologous melanocyte–keratinocytes suspension transplantation. After 6–17 months, patients’ response was evaluated according to the extent of pigmentation (excellent 90–100%, good 50–89%, fair 20–49% and poor response <20%). Results Of the 25 patients treated, 22 continued the follow‐up period. Five (23%) patients showed excellent response, 7 (32%) good, 6 (27%) fair and 4(18%) showed poor response. Conclusion Unlike transplantation of cultured melanocytes, which requires experience in culture technique, autologous melanocyte–keratinocytes suspension transplantation is an easy economic technique, which may be used in resistant areas of stable vitiligo.     

白癜风相关黑素细胞膜抗原VIT150、VIT90、VIT75纯化及分析

目的：纯化和分析白癜风相关的黑素细胞膜抗原，为蛋白质微量测序及筛选、克隆黑素细胞膜抗原打下基础。方法：培养高纯度的正常人黑素细胞，活细胞ELISA法和蛋白免疫印迹法(Western blot)检测并筛选抗黑素细胞的高滴度IgG抗体血清，生物素标记可溶性黑素细胞膜抗原，加入筛选血清与蛋白A-琼脂糖凝胶(protein A-sepharose)进行免疫共沉淀．沉淀后的抗原抗体复合物行平极电泳及电转印，碱性磷酸酶标记的亲合素进行化学发光法检测及鉴定。结果：活细胞ELISA法和Western blot检测结果中共筛选了10例白癜风患者高滴度IgG血清，免疫沉淀、免疫印迹、化学发光法检测后发现10例患者均有阳性条带，其抗原相对分子质量为150000、90000、75000、60000。结论：白癜风患者血清中存在抗黑素细胞膜抗原的自身抗体，抗原相对分子质量主要为150000、90000、75000，通过免疫共沉淀初步纯化了150000、90000和75000抗原。     

Transplant of cultured autologous pure melanocytes after laser-abrasion for the treatment of segmental vitiligo

Segmental vitiligo is a special type of vitiligo with unilateral distribution of lesions and has a stable course. Clinically, many patients with segmental vitiligo have unsatisfactory responses to topical corticosteroid or UV phototherapy. We have developed a technique for the isolation of melanocytes from a small specimen of normally pigmented skin obtained via a suction blister. The melanocytes can be proliferated in culture and then replanted onto laser-abrased vitiliginous areas. We used this procedure to treat 25 patients with segmental vitiligo that were refractory to medical therapy. The repigmented portion of the total treated area amounted to 95-100% in 21 patients and 65 to 94% in 4 patients. The response rate to treatment was 100% in this study. No scarring or other side-effects developed. The results of this study demonstrate that this method is a valuable tool for the treatment of patients with segmental vitiligo.     

白癜风发病过程研究进展

白癜风是皮肤科常见的色素脱失性疾病,其发病机制目前尚不明确,主要有遗传、氧化应激、黑素细胞丢失、免疫等学说,其发病可能经历了始动阶段、效应阶段及致病阶段,本文对其进行了综述。