卡泊三醇对鼠黑素瘤B16细胞维生素D受体表达的影响

目的:评价卡泊三醇(CPT)对鼠黑素瘤B16细胞维生素D受体(VDR)表达的影响.方法:用不同浓度CPT干预鼠黑素瘤B16细胞24h后,分别用荧光定量PCR法和Western印迹法检测B16细胞VDRmRNA和蛋白质表达情况.结果:与空白对照组相比,CPT浓度为10μg/mL时B16细胞中VDRmRNA和蛋白含量无明显变化(P＞0.05).在102μg/mL和103μg/mL时VDRmRNA和蛋白水平明显高于对照组,且103μg/mL组高于102μg/mL组,有统计学差异(P＜0.05).结论:CPT对鼠黑素瘤B16细胞VDR的mRNA转录和蛋白质表达有剂量依赖性的促进作用.     

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。     

角质形成细胞μ-阿片受体表达的研究

目的：研究不同特性的角质形成细胞μ-阿片受体的表达情况。方法：以体外培养的角质形成细胞株HaCaT细胞、人鳞状细胞癌(简称鳞癌)细胞株A431、神经母细胞瘤SK-N-SH细胞株为对象，采用逆转录(RT)-PCR方法研究细胞μ-阿片受体的表达。结果：在常规体外培养条件下，角质形成细胞株HaCaT细胞、人鳞癌细胞株A431的RT-PCR结果显示有μ-阿片受体mRNA的表达，后者的表达水平略高于前者。结论：μ-阿片受体在角质形成细胞的表达，为神经系统和皮肤通过神经肽直接发生作用提供依据。     

维生素D_3抑制角质形成细胞MCP-1的表达

目的　观察维生素D3 对角质形成细胞单核细胞化学吸引蛋白质 1(monocytechemoattractantprotein 1,MCP 1)的表达影响 ,探讨其在治疗银屑病中的作用机制。方法　培养角质形成细胞株HACAT ,并用不同浓度的维生素D3 处理 2 4h ,通过酶联免疫吸附 (ELISA)法检测上清液中MCP 1的表达 ;通过RT PCR法进一步检测细胞中MCP 1mRNA的水平。结果　经过浓度为 7.8× 10 -12 mol/L～ 6.2 5× 10 -11mol/L的维生素D3 处理过的角质形成细胞株HA CAT ,其分泌至上清中的MCP 1水平明显受到抑制 ,细胞中MCP 1mRNA的水平低于正常对照组。结论　维生素D3 在治疗银屑病过程中很有可能是通过抑制了角质形成细胞中MCP 1的表达 ,从而减弱了银屑病患者角质形成细胞趋化单核细胞及巨噬细胞的能力 ,阻止了银屑病皮损局部的炎症反应。     

卡泊三醇软膏联合卤米松乳膏治疗神经性皮炎疗效观察

目的评价卡泊三醇软膏联合卤米松乳膏治疗神经性皮炎的疗效和安全性。方法将87例神经性皮炎患者随机分为两组。治疗组:采用卡泊三醇软膏联合卤米松乳膏治疗;对照组:单用卤米松乳膏治疗,治疗1周和3周后进行疗效评定,随访6个月观察其复发率。结果治疗组的有效率为88.89%,对照组的有效率为69.05%,治疗组治疗1周及疗程结束后的总积分改善、有效率、复发率均优于对照组,差异有统计学意义。在不良反应方面,两组患者差异无统计学意义。结论卡泊三醇软膏联合卤米松乳膏治疗神经性皮炎的疗效较单独使用卤米松乳膏好,起效快,复发率低。     

卡泊三醇软膏和卤米松乳膏序贯疗法治疗寻常性银屑病疗效观察

笔者于2006年10月-2008年4月采用卤米松乳膏和卡泊三醇软膏序贯疗法治疗寻常性银屑病,取得较好疗效,现报告如下.     

卡泊三醇软膏和卤米松乳膏序贯疗法治疗斑块状银屑病临床观察

目的:观察卡泊三醇软膏和卤米松乳膏序贯疗法治疗斑块状银屑病的疗效及安全性.方法:76例银屑病患者随机分为卡泊三醇组(A组)36例和卡泊三醇软膏和卤米松乳膏序贯治疗组(B组)40例,A组患者每日涂药2次,B组患者按银屑病的序贯疗法用药.两组患者在疗后的第2周、4周、6周根据银屑病皮损而积和严重度指数(PASI)评分评判疗效,根据不良事件发生率分析其安全性.结果:随着疗程的增加两组患者的PASI评分均逐渐下降(P<0.01),B组下降更为明显(P<0.05);A组患者在治疗后2周、4周和6周的有效率分别为30.56%、55.56%和66.67%:B组患者的有效率分别为52.50%、67.50%和82.50%.B组的疗效明显优于A组(P<0.01);B组的不良事件发生率亦明显低于A组(P<0.01).结论:卡泊三醇软膏和卤米松乳膏序贯疗法治疗斑块状银屑病具有起效快、疗效好、不良反应小的优点.     

卡泊三醇与卤米松序贯疗法治疗稳定期斑块状银屑病临床观察

目的探讨卡泊三醇与卤米松序贯疗法治疗稳定期斑块状银屑病的疗效与安全性。方法将120例患者随机分为三组,治疗组早晨外用卡泊三醇1次/d,晚上外用卤米松1次/d,对照Ⅰ组仅用卡泊三醇2次/d,对照Ⅱ组仅用卤米松2次/d。均治疗4周后观察疗效。结果卡泊三醇与卤米松序贯治疗组有效率(81.58%)与卡泊三醇(62.86%)及卤米松组(60.00%)相比,差异有统计学意义(P均<0.05),且不良反应少。结论卡泊三醇与卤米松序贯疗法治疗稳定期斑块状银屑病疗效好,不良反应少。     

表皮生长因子受体及其配体与角质形成细胞

表皮生长因子受体是角质形成细胞自泌生长因子的主要受体 ,对其功能、功能调节、相关配体和活化后胞内信号转导的研究将有助于了解表皮的自身稳定机制和探讨有关疾病 (如银屑病 )的发病机制及治疗对策。     

表皮生长因子受体及其配体与角质形成细胞

表皮生长因子受体是角质形成细胞自泌生长因子的主要受体。对其功能、功能调节、相关配体和活化后胞内信号转导的研究将有助于了解表皮的自身稳定机制和探讨有关疾病（如银屑病）的发病机制及治疗对策。     

Calcipotriol (MC 903), a novel vitamin D3 analogue stimulates terminal differentiation and inhibits proliferation of cultured human keratinocytes

Summary The hormonally active form of vitamin D₃, 1,25-dihydroxy vitamin D₃ [1,25-(OH)₂-D₃; calcitriol], regulates the differentiation and proliferation of epidermal keratinocytes in vitro. MC 903 (calcipotriol) is a novel vitamin D₃ analogue which is at least 100 times less potent than 1,25-(OH)₂-D₃ in its effects on calcium homeostasis. The present study compared the effects of MC 903 and 1,25-(OH)₂-D₃ on terminal differentiation and proliferation of cultured normal human keratinocytes. Keratinocytes were grown in McCoy's 5A medium supplemented with penicillin (50 IU/ml), streptomycin (50 g/ml), l-serine (4×10^–4 M), and 10% human type AB serum. MC 903, 1,25-(OH)₂-D₃ or 1-OH-D₃ (10^–12 M–10^–8 M) was added with each feeding when cultures became preconfluent. After incubation for 24 h with D₃ vitamins, cultures were extracted for transglutaminase, and the enzyme activity was indexed against DNA content. The activity of transglutaminase, the enzyme reponsible for cross-linking the proteins of the cornified envelope, was maximally stimulated by 388% with MC 903 (10^–8 M), by 328% with 1,25-(OH)₂-D₃ (10^–8 M), and by 27% with 1-OH-D₃ (10^–8 M) compared with vehicle. After incubation for 2 weeks the number of keratinocytes with cornified envelopes had increased by 288% with MC 903 (10^–8 M), by 360% with 1,25-(OH)₂-D₃ (10^–8 M), and by 149% with 1-OH-D₃ (10^–8 M) compared with vehicle. Simultaneously the incorporation of (³H)thymidine into DNA was decreased by 64% with MC 903 (10^–8 M), by 71% with 1,25-(OH)²-D₃ (10^–8 M), and by 10% with 1-OH-D₃ (10^–8 M). There was a corresponding decrease in cell number. These results demonstrate that both MC 903 and 1,25-(OH)₂-D₃ are potent modulators of keratinocytes differentiation and proliferation in vitro. Because MC 903 is much less active than 1,25-(OH)₂-D₃ in causing hypercalcemia, this compound is a candidate for the treatment of skin diseases characterized by aberrant epidermal differentiation and proliferation.Parts of this study were presented at the ESDR meeting in Munich, May, 1988     

Intracellular free calcium and growth changes in single human keratinocytes in response to vitamin D and five 20-epi-analogues

Vitamin D, 1,25(OH)₂D₃, decreases proliferation and promotes differentiation of keratinocytes, and other keratinocyte differentiation stimuli have been associated with an early rise in intracellular free calcium, [Ca²⁺]_i. We therefore investigated the effect of 1,25(OH)₂D₃, its precursor D₃ and five 20-epi-analogues (EB1089, KH1060, KH1139, MC1288, MC1301) on growth and [Ca²⁺]_i levels of normal human keratinocytes. Cells were cultured in medium MCDB153 with an extracellular calcium concentration of 70 M or 1 mM. All the analogues were more potent than 1,25(OH)₂D₃ at inducing the morphological changes of differentiation, but D₃ was inactive. At concentrations down to 10^–8 M 1,25(OH)₂D₃, caused significant inhibition of growth, as assessed by counting cells and measurement of thymidine labelling. At 5 days 50% inhibition of growth occurred with 64 nM 1,25(OH)₂D₃ and 3330 nM D₃. All the analogues were more potent than 1,25(OH)₂D₃, and KH1060 inhibited growth at 10^–10 M. In single keratinocytes [Ca²⁺]_i was measured by micro-spectrofluorimetric techniques using the dye fura-2. No immediate rise in [Ca²⁺]_i was observed following addition of 1,25(OH)₂D₃ or the analogues up to 10^–6 M. However 10^–7 M 1,25(OH)₂D₃ or the analogues induced a gradual increase in [Ca²⁺]_i, significant at 4 h (P<0.001), which increased further over 2–3 days. D₃ had no effect on [Ca²⁺]_i. Increases in [Ca²⁺]_i following the differentiation stimuli of either 2 mM extracellular calcium or 1,25(OH)₂D₃ were similar at 48 h, increasing from 100 ± 3 nM (mean ± SEM) in control cells to 150 ± 3 nM with 2 mM calcium and 144 ± 6 nM with 10^–7 M 1,25(OH)₂D₃. The effect of extracellular calcium in raising [Ca²⁺]_i within minutes was more rapid than 1,25(OH)₂D₃, but in combination the two were not additive.     

Upregulation of vitamin D receptor levels by 1,25(OH)2 vitamin D3 in cultured human keratinocytes

Received: 15 May 1996     

Differentiation-regulated expression of Toll-like receptors 2 and 4 in HaCaT keratinocytes

Toll-like receptors (TLRs) play an important role in the recognition of pathogens in keratinocytes. In this study, we investigated whether the differentiation state of HaCaT keratinocytes correlates with the expression of TLR2 and TLR4 genes. The expression levels of TLR2 and TLR4 in a HaCaT differentiation model system were determined using quantitative real-time RT-PCR (Q-RT-PCR) and flow cytometry. The progression of keratinocyte differentiation was monitored by determining the level of involucrin gene expression using Q-RT-PCR. The expression levels of TLR2 and TLR4 increased with the stage of differentiation and there were strong correlations between the expression level of the involucrin gene and those of the TLR2 gene (r=0.809, P<0.0001) and the TLR4 gene (r=0.568, P<0.02). Increased cell surface expression of TLR2 and TLR4 was also found in differentiated HaCaT keratinocytes by flow cytometric analysis. Our findings suggest that upregulation of TLR expression during differentiation in keratinocytes could be a part of the differentiation process of keratinocytes and could have biological significance in protecting skin against microbes.     

Purinoceptor expression on keratinocytes reflects their function on the epidermis during chronic venous insufficiency

Purines are extracellular nucleotides that have long-term effects on keratinocyte proliferation, differentiation and death through P2Y₁, P2Y₂, P2X₅ and P2X₇ receptors. This study examined changes in expression of these P2 receptors on lower leg epidermal keratinocytes in control and chronic venous insufficiency (CVI) states. Lower limb skin biopsies from CVI (CEAP classification 4a and 4b) and control skin were immunostained for the above P2 receptor subtypes and epidermal area was calculated. Our results with CVI show an increase in P2Y₁ and P2Y₂ receptor expression in basal and spinosal layers of the epidermis and an increase of P2X₅ receptors mainly in the spinosal layer and extending further into the stratum granulosum. In contrast, P2X₇ receptors were reduced in the stratum corneum in CVI. In conclusion, a thinner epidermis was found in CVI, which might be the result of the changes in expression of P2Y and P2X receptors on keratinocytes: that is, increased proliferation via P2Y₁ and P2Y₂ receptors and reduced P2X₇ receptor-mediated cell death opposed by a dominant decrease in cell numbers as a result of increased P2X₅ receptor-mediated differentiation (which is in effect antiproliferative). Thus, increased keratinocyte P2X₅ receptor activity may, in part, be accountable for epidermal thinning in CVI.     

EGF Receptor expression and growth of psoriatic and normal human keratinocytes are modulated by 1.25 (OH)2-vitamin D3 ex vivo

Calcitriol or 1,25 (OH)₂-vitamin D₃ is used in the treatment of psoriasis as an inhibitor of cell proliferation. We studied the action of calcitriol ex vivo on the growth of psoriatic and normal human keratinocytes, and on the expression of the EGF receptor. Third passaged normal and psoriatic keratinocytes were seeded (10⁴/cm²) in 24-well dishes in serum-free medium (MCDB supplemented with amino acids, with either 0.1 or 1.1 mM of calcium) and 10⁻⁹ M calcitriol. When subconfluence was reached, cell counts and¹²⁵I-EGF binding studies were performed. Cell counts showed at least a 50% decrease in growth under all conditions studied (normal or psoriatic keratinocytes; 0.1 or 1.1 mM calcium) when calcitriol was added.¹²⁵I-EGF binding studies showed a decrease in total receptor numbers in the presence of calcitriol with acceleration of binding at low concentrations of¹²⁵I-EGF. Scatchard plot analysis showed only one type of high affinity receptor. Receptor sites were decreased (30% to 40% of controls) in the presence of calcitriol together with a decrease in the dissociation constant. In conclusion, at almost physiological concentrations ex vivo, calcitriol strongly decreased normal and psoriatic keratinocyte growth. This potent antiproliferative effect could in part be explained by the capacity of calcitriol to downregulate EGF receptor expression. Supported by grants of the Conseil Régional de la Région Aquitaine, and of the Université de Bordeaux II, to AT     

双氢睾酮对HaCaT细胞SREBP-1c表达的影响

目的 探讨双氢睾酮（DHT）在HaCaT细胞固醇调控元件结合蛋白-1c（SREBP-1c）表达中的作用。 方法 体外培养HaCaT细胞,分为4组,对照组不加任何刺激因素,DHT组分别加入3种不同浓度的DHT,LY294002 + DHT组在加入50 μmol/L PI3K抑制剂（LY294002）预处理40 min后加入100 nmol/L DHT,PD98059 + DHT组即在加入50 μmol/L MEK抑制剂（PD98059）预处理40 min后加入100 nmol/L DHT。用实时定量PCR和Western印迹法检测DHT对HaCaT细胞SREBP-1c mRNA和蛋白表达的影响。Western印迹法检测DHT作用于HaCaT细胞后蛋白激酶B（AKT）、胞外信号调控激酶（ERK）、p38丝裂原活化蛋白激酶（p38 MAPK）、c-Jun氨基末端激酶（JNK）的磷酸化情况。 结果 DHT可呈浓度依赖性上调HaCaT细胞SREBP-1c mRNA和蛋白的表达,并诱导AKT、ERK的磷酸化,但对p38、JNK的磷酸化无明显激活作用。LY294002预处理后HaCaT细胞SREBP-1c mRNA的表达较单纯DHT组明显降低（t = 9.406,P < 0.05）;SREBP-1蛋白水平降为0.7113 ± 0.0313,与单纯DHT组2.2577 ± 0.0601比较,差异有统计学意义（t = 39.498,P < 0.05）。而PD98059预处理后,SREBP-1c mRNA和蛋白的表达与单纯DHT组比较,差异均无统计学意义（P > 0.05）。结论 DHT可诱导HaCaT细胞SREBP-1c mRNA和蛋白的表达。