环孢素A及雷公藤内酯醇对CD4^＋T细胞,角质形成细胞和Hela细 …

目的 比较环孢素Ａ（ＣｙＡ）和雷公藤提取物Ｔ０对ＣＤ４＾＋Ｔ细胞，角质形成细胞和Ｈｅｌａ细胞增殖的影响。方法 采用人外周血单一核细胞（ＰＢＭＣ），人表皮角质形成细胞和Ｈｅｌａ细胞培养，＾Ｈ－ＴｄＲ掺入法测定药物对细胞ＤＮＡ合成的影响，结果Ｔ０和ＣｙＡ都能抑制ＣｏｎＡ诱导的人外周血Ｔ淋巴细胞增殖。Ｔ０在体外可抑制生长于低无血清角质形成细胞培养基中的正常人角持形成细胞和Ｈｅｌａ细胞株的生长，而ＣｙＡ     

盐酸博宁霉素对人表皮角质形成细胞、宫颈鳞癌细胞和表皮癌细胞增殖的影响

目的:探讨盐酸博宁霉素对人角质形成细胞HaCaT、人宫颈上皮癌细胞HeLa及人表皮癌细胞A431增殖的影响.方法:采用MTT法检测盐酸博宁霉素处理后培养的HaCaT细胞、HeLa细胞和A431细胞的增殖率.结果:盐酸博宁霉素能剂量依赖性抑制HaCaT细胞和HeLa细胞的增殖,IC50值分别为11.57 μg/mL和16.47 μg/mL, 但对A431细胞增殖的抑制作用较弱.结论:盐酸博宁霉素的细胞毒性是抗尖锐湿疣药物的治疗学基础.     

姜黄油对角质形成细胞体外增殖分化影响的研究

目的：探讨姜黄油对角质形成细胞体外增殖分化的影响。方法：以人角质形成细胞株COLO-16细胞为模型，噻唑盐比色法(MTT)检测细胞活性和生长情况；免疫组化SABC法检测姜黄油对增殖细胞核抗原(PCNA)表达的影响；电镜观察姜黄油对细胞超微结构的影响。结果：姜黄油抑制角质形成细胞增殖，随药物浓度增加，其抑制增殖能力增加，PCNA表达逐渐减弱，且对角质形成细胞超微结构有直接损伤作用。结论：姜黄油具有抑制体外角质形成细胞增殖分化的作用，有望成为一种治疗增殖性皮肤病的外用药物。     

p53抑制剂PFT-α对阿霉素诱导的人皮肤角质形成细胞凋亡的影响

目的探讨p53抑制剂PFT-α对阿霉素诱导的人皮肤角质形成细胞凋亡的影响。方法用MTT法通过比色分析测定吸光度(OD)值,检测0,4,8,12mg/LPFT-α和阿霉素给药对角质形成细胞增殖的影响,用流式细胞仪检测细胞凋亡。结果阿霉素抑制体外培养的人皮肤角质形成细胞增殖,先给予8,12mg/LPFT-α处理细胞,然后给予阿霉素,与对照组比较,PFT-α升高OD值(P<0.05),降低细胞凋亡率(P<0.05)。结论先行给予适当剂量PFT-α处理人皮肤角质形成细胞,能一定程度减轻阿霉素对人皮肤角质形成细胞的损伤,表现出对阿霉素致凋亡的保护作用。     

齐墩果酸对阿霉素诱导的培养人皮肤角质形成细胞凋亡的影响

目的探讨齐墩果酸对阿霉素诱导的培养人皮肤角质形成细胞凋亡的影响。方法用MTT法通过比色分析测定吸光度(A)值,检测不同剂量齐墩果酸和阿霉素给药对角质形成细胞增殖的影响,用流式细胞仪检测细胞凋亡。结果阿霉素抑制体外培养的人皮肤角质形成细胞增殖,先给予齐墩果酸处理细胞,然后给予阿霉素,齐墩果酸升高A值(P<0.05),降低细胞凋亡率(P<0.05)。结论先行给予适当剂量齐墩果酸处理细胞,能一定程度减轻阿霉素对细胞的损伤,表现出对阿霉素致凋亡的保护作用。     

芦荟对银屑病患者外周血单核细胞和角质形成细胞增殖和凋亡的研究

目的探讨芦荟对银屑病患者外周血单一核细胞(PBMC)和角质形成细胞增殖和凋亡影响.方法选择寻常性银屑病患者PBMC,共20例,正常人16例,及角质形成细胞株Colo-16细胞.采用MTT方法检测细胞增殖,ELISA方法检测细胞凋亡.结果芦荟对正常人和银屑病患者PBMC增殖均有抑制作用(P＜0.01),无促进细胞凋亡作用,对角质形成细胞株Colo-16细胞有明显的抑制增殖和促进凋亡作用(P＜0.01).结论芦荟抑制银屑病患者PBMC的增殖和诱导角质形成细胞凋亡可能是芦荟治疗银屑病的重要药理作用之一.     

MTX对正常人表皮角质形成细胞体外培养增殖的作用及影响

目的　探讨甲氨蝶呤 (MTX)对表皮角质形成细胞的作用及影响 ,进而了解MTX治疗银屑病的机理。方法　将人体离体角质形成细胞培养、分离传代并测定其自然生长曲线作对照 ;将不同浓度MTX与角质形成细胞混合培养 ,测定在MTX作用下的角质形成细胞生长曲线 ,比较MTX对细胞生长的影响。结果　MTX对角质形成细胞的体外生长具有明显的抑制作用 ,不仅明显抑制角质形成细胞的正常生长 ,而且可以促使细胞的提前凋亡。结论　MTX通过抑制角质形成细胞的DNA及RNA合成调节细胞的生长代谢及自然凋亡过程 ,尤其对银屑病患者生长过度活跃的表皮角质形成细胞产生调节作用 ,使细胞的过度生长受到抑制 ,保持角质形成细胞的自然生长平衡     

柳蘑多糖对角质形成细胞作用实验研究

目的观察柳蘑多糖对角质形成细胞株(Ha Cat细胞)生物学性状的影响,并探讨其相关作用机制。方法MTT法检测柳蘑多糖对Ha Cat细胞增殖活性的影响;Annexin V-PI染色法测定柳蘑多糖刺激Ha Cat细胞后的凋亡情况;PI法测定应用柳蘑多糖刺激后Ha Cat细胞周期的改变;重组人γ-干扰素(rh IFN-γ)诱导Ha Cat细胞活化,然后加入柳蘑多糖,用ELISA法检测IL-8和ICAM-1细胞因子分泌情况。结果 MTT法证实柳蘑多糖对Ha Ca T细胞的增殖有抑制作用,且最佳抑制浓度为0.1μg/m L,最佳作用时间为48 h;用Annexin V-PI染色法证实了柳蘑多糖可以促进Ha Ca T细胞凋亡,尤其是晚期凋亡;用PI法证实了柳蘑多糖可以影响Ha Ca T细胞的细胞周期,对细胞DNA合成期有影响;用ELISA法证明了rh IFN-γ活化后Ha Ca T细胞分泌的白介素-8(IL-8)及细胞间黏附分子-1(ICAM-1)明显增多,而柳蘑多糖可以使活化后Ha Ca T细胞分泌的IL-8及ICAM-1显著减少。结论柳蘑多糖可以抑制体外培养的角质形成细胞的增殖、促进角质形成细胞凋亡、改变角质形成细胞的细胞周期,减少细胞因子(IL-8,ICAM-1)分泌,提示柳蘑多糖有抑制角质形成细胞增殖、减轻炎症反应的潜力。     

小剂量环孢素A治疗严重的银屑病引起外周血嗜酸粒细胞减少

环孢素 A(CyA)是非骨髓毒性的免疫抑制剂,能有效地抑制 T 淋巴细胞产生的细胞素,包括 IL-2.IL-2的减少抑制了 T 淋巴细胞产生IL-5,IL-5是强的嗜酸粒细胞生成的特异性刺激剂。在小剂量系统性应用 CyA(3～6mg/kg/d)治疗银屑病和其他炎症性皮肤病时,很少发现有外周血嗜酸粒细胞减少。但曾有报道口服 CyA 后,导致外周血嗜     

肿瘤坏死因子与角质形成细胞

肿瘤坏死因子(ＴＮＦ)是一类由巨噬细胞和激活的淋巴细胞等分泌的细胞因子,具有广泛的生物学活性。近年来,随着对ＴＮＦ理论和临床研究的深入,发现ＴＮＦ与角质形成细胞有密切的关系;角质形成细胞可以产生ＴＮＦ,ＴＮＦ又可影响角质形成细胞的形态、功能。现综述如下。一、角质形成细胞ＴＮＦ的产生及影响因素人类正常皮肤角质形成细胞、肿瘤及一些自身免疫性疾病的角质形成细胞均能通过自分泌或旁分泌的方式产生ＴＮＦα[1],抑制正常人和银屑病皮损角质形成细胞的生长。培养的人角质形成细胞亦可表达ＴＮＦ受体(ＴＮＦ-Ｒ),对ＴＮＦ引起的…     

Effects of cyclosporin and ultraviolet radiation on growth and ornithine decarboxylase activity in cultured human epidermal keratinocytes

Both cyclosporin (CyA) and ultraviolet radiation are effective in the treatment of psoriasis, but their precise mechanisms of action are uncertain. We investigated their effects on ornithine decarboxylase (ODC) activity, ODC gene expression, and cellular proliferation stimulated by epidermal growth factor (EGF), in cultured normal human epidermal keratinocytes. CyA (5 μg/ml) inhibited ODC activity, ODC mRNA level, and cell growth induced by 50ng/ml EGF. Ultraviolet B (10 mJ/cm²) irradiation suppressed the induction of ODC, ODC mRNA, and cell proliferation stimulated by EGF, but ultraviolet A (0-15 J/cm²) irradiation inhibited neither EGF-stimulated ODC activity nor cell proliferation. These findings indicate that reduction of ODC activity in CyA- or ultraviolet B-treated human keratinocytes may contribute to the antiproliferative mechanism of these agents. These results also suggest that the regulation of ODC activity by ultraviolet B and A irradiation may be mediated by different signal transduction pathways.     

Cyclosporin A induces the unfolded protein response in keratinocytes

Psoriasis vulgaris is a chronic inflammatory disorder of the skin, in which activation of keratinocytes and crosstalk between keratinocytes and T cells or dendritic cells are considered to be involved in the pathogenesis of psoriasis vulgaris. Cyclosporin (Cy) A, an immunomodulator, has been used for the treatment of psoriasis vulgaris, but the mechanism of its action on keratinocytes has not been well elucidated as its function on T cells is well known. Previous study indicated that the expression of the unfolded protein response (UPR) markers, GRP78/Bip and HRD1 were poorly expressed in psoriasis vulgaris. To investigate if the UPR in keratinocytes is involved in the pathogenesis of psoriasis vulgaris we assessed immunocytochemistry of normal human skin and psoriatic lesions, quantitative PCR of keratinocyte cell line (HaCaT) treated with TGFβ. Moreover, to elucidate how CyA effects on the UPR in keratinocytes, we set out quantitative PCR and western blotting, HaCaT and squamous cell carcinoma cell lines (HSC-1) treated with CyA and CyA analog, cyclosporin D. Furthermore, the siRNA-mediated knockdown effect of cyclophilin (Cyp) A, Cyp B and Cyp C on HaCaT cells were also examined. As a result, the UPR was downregulated in keratinocytes from psoriatic lesions, characterized by immunocytochemical staining of GRP78/Bip, CHOP/GADD153, HRD1 and C/EBPβ. TGFβ induced UPR markers in HaCaT cells. CyA treatment and siRNA-mediated knockdown of Cyp B induced the UPR in HaCaT cells or HSC-1 cells. Altogether, we demonstrate that in psoriasis vulgaris CyA or reduction in Cyp B by RNA interference might induce the UPR in keratinocytes.     

Response of the clinically uninvolved skin of psoriatic patients to repeated tape stripping during cyclosporin A treatment

Summary It is well established that cyclosporin A (CyA), a widely used immunosuppressant in human organ transplantation, is an effective drug in the treatment of psoriasis. Although it has been postulated that the effect of CyA in psoriasis is mediated through antilymphocyte activity, there is also evidence suggesting that CyA exerts a direct cytostatic effect on epidermal keratinocytes, but results of studies relating to the latter have been contradictory.
Using immunohistochemical methods we investigated the influence of systemic CyA on proliferation and differentiation in the tape-stripped uninvolved skin of psoriatic patients, a model which provides the opportunity of studying epidermal regeneration in the absence of a significant accumulation of T lymphocytes. We addressed the question of whether CyA (3–5 mg/kg/day) modulates epidermal proliferation and differentiation following standardized injury in uninvolved skin of psoriatic patients. Ten patients with severe psoriasis participated in this study. The dosages of CyA were sufficient to induce a marked and statistically significant improvement (PASI, week 0, 20.5·4.4; PASI, week 16, 4.3 ± 0.6). Before CyA treatment, and during week 16 of treatment, Sellotape stripping was carried out on a 2-cm² area of the uninvolved skin of psoriatic patients. After 48 h punch biopsies were taken. Immunohistochemical assessment of recruitment of cycling cells (Ki-67), filaggrin, involucrin, T lymphocytes and tenascin, was carried out. We did not find any significant alteration during the treatment period in the tape-stripped uninvolved skin of psoriatic patients. We conclude that epidermal hyperproliferation and abnormal keratinization are not modulated directly by CyA at therapeutic doses in vivo. Furthermore, our study provides indirect evidence that the antipsoriatic effect of CyA is mediated by the immune system.     

Time-dependent inhibition of growth of human keratinocytes and fibroblasts by cyclosporin A: effect on keratinocytes at therapeutic blood levels

The effect of cyclosporin A (CyA) on the growth of cultured normal human keratinocytes and fibroblasts was studied. Tritiated thymidine incorporation per DNA content was used for short-term studies (1-2 days) and cell counting for longer term studies (up to 14 days). CyA caused a dose- and time-dependent inhibition of growth of both cell types. Keratinocytes (serum free, 70 microM Ca2+) were more sensitive to CyA than fibroblasts (10% serum). For keratinocytes, at 2 days growth was 61% and 6% of controls with 3 and 10 micrograms/ml CyA, respectively, at 4 days growth was 51% with 1 microgram/ml with complete inhibition at 10 micrograms/ml, and at 11 days growth was 58 and 39% with 0.1 and 1 micrograms/ml. For fibroblasts, at 4 days growth was 72 and 58% of controls with 10 and 30 micrograms/ml CyA, respectively, at 10 days growth was 76, 43 and 10% with 3, 10 and 30 micrograms/ml, and at 14 days growth was 41 and 11% with 3 and 10 micrograms/ml with complete inhibition with 30 micrograms/ml. For keratinocytes the effect of 24 h of CyA on growth was reversible. Growth of keratinocytes was inhibited by 10% delipidized serum, and 1% serum had a small stimulatory effect, but the action of CyA was unaltered. We conclude that increasing the duration of exposure to CyA increases the concentration-dependent growth inhibition. At therapeutic blood levels of CyA the growth of cultured keratinocytes but not fibroblasts is inhibited. The benefit of CyA in reducing the hyperproliferation of psoriasis may include a direct effect on keratinocytes.     

Effects of cyelosporin A on resident and passenger immune cells of normal human skin and UV-induced erythema reactions

Cyclosporin A (CyA) has proved effective in various dermatological diseases, but its mechanisms of action within the skin are still ill-defined. In order to characterize more clearly the cellular targets of CyA we examined its effects on skin immune cells in normal and UVB- and PUVA-irradiated skin by means of a three-step immunoperoxidase reaction and immunofluorescence double-labelling technique. The CyA-induced depletion of epidermal Langerhans cells equals that seen with UVB or PUVA alone. CyA alone has no effect on the number and distribution of dermal cells. CyA modulates the UV irradiation-induced changes by: (i) inhibiting the UVB- and PUVA-induced ICAM-1 expression by keratinocytes and (ii) suppressing the PUVA-induced upregulation of CD11a expression by macrophages (72 +/- 12% of Ki-M8+ macrophages express CD11a with PUVA, compared with 20 +/- 5% with CyA + PUVA, P < 0.001). Neither treatment affected ICAM-1 expression by endothelial cells. In addition, CyA increases PUVA minimal phototoxicity dose from 10 +/- 2.6 J/cm2 (PUVA alone) to 15.3 +/- 3.1 J/cm2 (CyA + PUVA), (P < 0.001). In conclusion, the effects of CyA on the skin include a down-modulation of the PUVA-erythema reaction, associated with a direct or indirect modulation of adhesion molecule expression.     

血小板活化因子刺激后中性粒细胞对角朊细胞增殖的影响

研究了１０例正常人中性粒细胞（ＰＭＮ）血小板活化因子（ＰＡＦ）刺激后调节培养基对人角阮细胞增殖的影响及ＰＡＦ拮抗剂ＢＮ ５２０２１对其抑制作用。结果表明单纯ＰＭＮ培养基对角朊细胞有促增殖作用（Ｐ<０．０５），而经ＰＡＦ刺激后，ＰＭＮ调节培养基促角朊细胞增殖作用明显增强（Ｐ＜０．０１）。ＢＮ５２０２１能明显抑制ＰＡＦ对ＰＭＮ刺激作用，间接抑制其促角阮细胞增殖作用，提示ＰＡＦ在银屑病皮损表皮过度增殖中起一定作用。     

Differential effects of retinoids on DNA synthesis in calcium-regulated murine epidermal keratinocyte cultures

To study the possibility that the state of proliferation of epidermal keratinocytes can influence the action of retinoids, the rate of proliferation of murine epidermal keratinocytes was manipulated by growing the cells in media containing high or low concentrations of Ca++. In contrast to what other investigators have reported, keratinocytes cultured in medium containing 1.4 mM Ca++ proliferate faster, instead of slower, than cells cultured in medium with 0.09 mM Ca++. Other experiments showed that Ca++ was stimulatory to keratinocytes in medium containing a low level of growth factors, and inhibitory in medium containing a high level of growth factors, suggesting that the discrepancy could be due to a difference in the sera used. The high Ca++ cells prominently expressed the 48kD/56kD pair of keratin, showing that they were in a hyperproliferative state. Exposure of the faster growing high Ca++ cells to all-trans retinoic acid, 13-cis retinoic acid, etretinate, etretin, and arotinoid ethyl ester caused dose-dependent inhibition of DNA synthesis. In contrast, exposure of the slower growing low Ca++ cells to these retinoids resulted in dose-dependent stimulation of DNA synthesis. In addition, all-trans retinoic acid caused dose-related increases in cell number in the low Ca++ cultures. These findings correlate with the reported differential effects of retinoids on normal and hyperproliferative epidermis, and suggest that Ca++ and low growth factor-regulated keratinocyte cultures are useful for studying the mechanism of hyperproliferation and retinoid actions.     

Cyclosporin A inhibits keratinocyte cytokine gene expression

Summary The immunosuppressive peptide cyclosporin A (CyA) is an extremely effective therapy for severe recalcitrant psoriasis, although its mechanism of action is unknown. In this study, we examined the effect of CyA on keratinocyte growth and cytokine expression, and showed that CyA inhibits the growth of murine and human keratinocytes (KC) and KC cell lines. In addition, CyA inhibits the expression of cytokine genes in a dose-dependent fashion. After 2 days' incubation with 20 μM CyA, interleukin-lα (IL- α), interleukin-1β (IL-l β), and interleukin 8 (IL-8) mRNA were decreased by 4-fold, 3·3-fold and 3·3-fold, respectively, in COLO-16, a keratinocyte cell line. IL-1 biological activity recovered from COLO-16 culture supernatants decreased to one-fifth of that of controls. In the murine KC cell line PAM 212, 10 μ CyA treatment for 2 days downregulated IL-1α, tumour necrosis factor-α (TNF-α) and IL-1 receptor by 60%, but had no effect on the message for interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), ornithine decarboxylase and β-actin. Cells cultured for 5 days in the presence of CyA required much lower concentrations (2 μM) to achieve the same degree of inhibition of IL-1α. Similar tissue concentrations of CyA have been reported in psoriatics undergoing CyA therapy. The inhibition of KC growth caused by CyA treatment could be partially overcome by the addition of 0·l–1·0 ng/ml of recombinant IL-1α simultaneously with CyA, suggesting that the growth inhibitory effect of CyA on KC cultures is due in part to loss of autocrine cytokine stimulation. Collectively, these data suggest that CyA may exert some of its therapeutic effects in psoriasis by inhibiting the release of KC cytokines, so terminating epidermal hyperproliferation. As some of these cytokines (IL-1, IL-8 and TNF-α) are chemotactic for leucocytes, or induce endothelial adhesion molecules, infiltration and inflammation may also be reduced by their inhibition.     

rhFGF-7对体外培养人皮肤角质形成细胞增殖的影响

目的建立一种人皮肤角质形成细胞分离和培养的方法,研究重组人成纤维细胞生长因子7(rhFGF-7)对人皮肤角质形成细胞增殖的影响。方法取环切术后包皮,分别用胰酶和dispaseⅡ酶消化法分离表皮,用无血清培养基进行无滋养层培养,采用免疫细胞化学方法对培养的细胞进行鉴定,并用细胞计数法测定细胞的生长曲线,MTT法检测rhFGF-7对人皮肤角质形成细胞增殖的影响。结果与胰酶消化分离表皮相比,dispaseⅡ酶消化获得的表皮,其细胞贴壁率高,增殖速度快,且成纤维细胞污染少;荧光显微镜下细胞胞浆呈黄绿色,为角蛋白阳性染色;培养的角质形成细胞可持续增殖至第8代,10ng/mL的rhFGF-7促角质形成细胞增殖作用最显著。结论所建立的人皮肤角质形成细胞分离和培养方法,可获得高纯度的人皮肤角质形成细胞,rhFGF-7可促进体外培养人皮肤角质形成细胞的增殖。     

Granulocyte macrophage--colony-stimulating factor (GM-CSF) decreases CD1a expression by human Langerhans cells and increases proliferation in the mixed epidermal cell-lymphocyte reaction (MELR).

Langerhans cells (LC) undergo a variety of phenotypic and functional changes in vitro. To determine the effects of granulocyte macrophage--colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1-alpha (IL-1) on LC phenotype in vitro, epidermal cell suspensions were enriched for LC by density-gradient centrifugation and cultured in the presence of 10 ng/ml of these cytokines. The percentage of cells expressing the surface protein CD1a was determined by flow cytometry. This percentage typically dropped after 48 h culture in both control and cytokine-treated medium to less than half that of the starting value. By the fifth day, the percentage of cells expressing CD1a in TNF-alpha and IL-1--treated cultures was still near half of the starting value, slightly above that of control cultures. Treatment with GM-CSF caused large and consistent decreases in the percentage of epidermal cells expressing CD1a. Cell viability in each of the three cytokine-treated cultures was identical to the control cultures, with essentially all cells having died by the sixth day after isolation. To determine the functional effects of these cytokines, the cytokine-containing medium was replaced after 72 h with medium containing purified allogeneic T cells and proliferation measured. Preliminary experiments showed no increased proliferation induced by IL-1 or TNF-alpha--treated epidermal cells. GM-CSF-treated epidermal cells induced 2-3 times more T-cell proliferation than epidermal cells cultured without additional cytokines. We conclude that GM-CSF, a cytokine known to be produced by keratinocytes in vitro, decreases CD1a expression by human LC and increases their ability to stimulate proliferation by allogeneic T cells.