Dicarboxylic acids inhibit the growth of keratinocytes in vitro

The antiproliferative effect of three straight-chained saturated dicarboxylic acids was examined with neonatal mouse keratinocyte cultures. Adipic acid (C6), azelaic acid (C9), and sebacic acid (C10) were added to the cultures in concentrations ranging from 1 to 50 mmol/l. Proliferation was assayed by liquid-scintillation counting of 3H-thymidine incorporation into DNA and by autoradiography. Fifty percent inhibition of 3H-thymidine incorporation was observed with 50 mmol/l adipic acid, 20 mmol/l azelaic acid, and 10 mmol/l sebacic acid, respectively. The antiproliferative effect was completely reversible after cessation of treatment. Moreover, treated cultures then showed a rebound effect with increased DNA synthesis. These results show that dicarboxylic acids exert reversible antiproliferative effects on keratinocytes.     

A toxicity index of skin and wound cleansers used on in vitro fibroblasts and keratinocytes

OBJECTIVE: To determine toxicity indexes of commercially available skin, wound, and skin/wound cleansers on in vitro fibroblasts and keratinocytes. DESIGN: Seventeen cleansers and 3 liquid bath soaps were evaluated for cytotoxic effect on human infant dermal fibroblasts and epidermal keratinocytes. Both skin cell types were exposed to serial 10-fold dilutions of each cleanser until treated cell viability was comparable to untreated controls. RESULTS: The experimental design allowed calculation of relative toxicity indexes ranging from 0 to 100,000. Shur-Clens, SAF-Clens, and saline were found to be the least toxic to fibroblasts (toxicity index 0); Dial Antibacterial Soap and Ivory Liqui-Gel were the most toxic (toxicity index 100,000). Biolex, Shur-Clens, and Techni-Care were the least toxic to keratinocytes (toxicity index 0); hydrogen peroxide, modified Dakin's solution, and povidone (10%) were found to be the most toxic (toxicity index 100,000). CONCLUSIONS: Successful cutaneous tissue repair depends on the viability of the principal cell types involved (fibroblasts and keratinocytes). Toxicity indexes provide helpful guidelines for subsequent in vivo evaluations and clinical applications. The study findings also suggest that judicious use of these supposedly innocuous agents should be considered in a clinical setting.     

Correlation of donor age and telomerase activity with in vitro cell growth and replicative potential for dermal fibroblasts and keratinocytes

Previous studies suggested telomerase activity as a determinant of cell replicative capacity by delaying cell senescence. This study aimed to evaluate the feasibility of adopting telomerase activity as a selection criterion for in vitro expanded skin cells before autologous transplantation. Fibroblasts and keratinoctyes were derived from the same consenting patients aged 9–69 years, and cultured separately in serum-supplemented and serum-free media, respectively. Telomerase activity of fresh and cultured cells were measured and correlated with cell growth rate, donor age and passage number. The results showed that telomerase activity and cell growth were independent of donor age for both cell types. Telomerase was expressed in freshly digested epidermis and dermis and continued expressing in vitro. Keratinocytes consistently showed 3–12 folds greater telomerase activity than fibroblast both in vivo and in vitro. Conversely, growth rate for fibroblast exceeded that of keratinocyte. Telomerase activity decreased markedly at Passage 6 for keratinocytes and ceased by Passage 3 for fibroblasts. The decrease or cessation of telomerase activity coincided with senescence for keratinocyte but not for fibroblast, implying a telomerase-regulated cell senescence for the former and hence a predictor of replicative capacity for this cell type. Relative telomerase activity for fibroblasts from the younger age group was significantly higher than that from the older age group; 69.7% higher for fresh isolates and 31.1% higher at P0 (p < 0.05). No detectable telomerase activity was to be found at later subcultures for both age groups. Similarly for keratinocytes, telomerase activity in the younger age group was significantly higher (p < 0.05) compared to that in the older age group; 507.7% at P0, 36.8% at P3 and the difference was no longer significant at P6. In conclusion, the study provided evidence that telomerase sustained the proliferation of keratinocytes but not fibroblasts. Telomerase activity is an important criterion for continued survival and replication of keratinocytes, hence its positive detection before transplantation is desirable. Inferring from our results, the use of keratinocytes from Passage 3 or lesser for construction of skin substitute or cell-based therapy is recommended owing to their sustained telomerase expression.     

人角质形成细胞的体外改良培养及生物学特征

目的：探讨体外人角质形成细胞的改良培养方法并观察其生物学特征。方法：取健康人包皮环切术包皮,采用EDTA预处理和冷消化法分离表皮,制成细胞悬液,通过倒置相差显微镜、电镜观察其形态学特征,绘制细胞生长曲线;免疫组化（ABC法）测定抗角蛋白单克隆抗体鉴定细胞。结果：体外培养的人皮肤角质形成细胞经过1～2天的潜伏期,即进入指数增生期,持续约5天,然后进入平台期。第7天左右细胞完全融合连成片状,形态呈扁平不规则多边形,免疫组化显示胞浆被染成棕黄色,为角蛋白阳性。透射电镜下见角质形成细胞胞浆内有大量束状张力纤维呈典型角质形成细胞特征。结论：采用改良体外培养方法获得的人角质形成细胞保持正常的生物学特征,从而建立一种简单易行的人角质形成细胞优化培养技术。     

咪喹莫特对人皮肤成纤维细胞活性和凋亡的影响

目的：观察咪喹莫特（imiquimod）溶液对人真皮成纤维细胞（FB）细胞毒性、细胞增殖及细胞凋亡率的影响，探讨其治疗瘢痕疙瘩的可能机制。方法：分离培养人真皮成纤维细胞，用四甲基偶氮唑蓝（MTF）法检测不同浓度咪喹莫特溶液对成纤维细胞毒性及细胞增殖的影响：用流式细胞技术检测咪喹莫特对成纤维细胞凋亡率的影响。结果：不同浓度咪喹莫特加入成纤维细胞培养体系孵育24h后，成纤维细胞形态均有不同程度受损，且细胞增殖活性下降，尤以20mg/L浓度时细胞活性下降明显；各浓度咪喹莫特处理组细胞凋亡率均高于对照组（P〈0．05），但在不同时间点检测的细胞凋亡率差异无统计学意义（P〉0．05）。结论：咪喹莫特可以降低成纤维细胞增殖活性并增加细胞凋亡率，这些可能是其促进皮肤瘢痕组织消退的相关机制。     

Cryotherapy modifies synthetic activity and differentiation of keloidal fibroblasts in vitro

In order to obtain a persuasive explanation for the beneficial clinical effect of cryotherapy on keloids, we developed a reproducible model to apply freezing temperatures on cell cultures, and investigated their influence on proliferation, viability, synthetic activity and differentiation of dermal fibroblasts in vitro. Cell cultures were established from 13 untreated keloids and 10 healthy skin specimens matched for age and skin localization to the donors. No significant influence of cell freezing on the proliferation rates of both keloidal and normal fibroblasts was documented, but mechanical cell destruction with a wide variation in lethality rates (29% average lethal effect on keloidal fibroblasts and 41% on normal ones) was observed. When comparing specimens of keloidal and normal tissue derived from the same four donors, the keloidal fibroblasts were similar regarding their synthetic activity but presented enhanced tenascin-C expression compared with the normal fibroblasts. After cryotherapy, delayed collagen III increase was detected in both cell types (P = 0.03). The collagen II/collagen I ratio increased from 1.6 to 2.8 in the keloidal and only from 1.9 to 2.2 in the normal fibroblasts after subcultivation. Normal fibroblasts exhibited a significantly lasting increase in fibronectin synthesis after freezing (P = 0.03). The intensity of staining against tenascin-C was decreased in five of nine keloidal fibroblast cultures after cryotherapy (P < 0.05) but increased in four of five normal fibroblast cultures (P = 0.016), so that the intensity of tenascin-C staining after freezing became identical in both cell types. Immunoblot studies in four patients and two controls confirmed a temporary decrease of tenascin-C in keloidal but not in normal fibroblasts immediately after freezing. Significantly decreased staining with two markers of myogenic differentiation, myosin in keloidal fibroblasts (P = 0.002) and desmin (P = 0.007) in normal fibroblasts, could also be detected after treatment. In summary, with the help of a model for controlled cell freezing in vitro, cryotherapy was found to modify collagen synthesis and differentiation of keloidal fibroblasts.     

金雀异黄素对体外培养的人皮肤成纤维细胞生物学活性和胶原合成的影响

目的 观察会雀异黄素对体外培养的正常人皮肤成纤维细胞生长和胶原合成的影响.方法 采用噻哗蓝(MTT)比色法检测不同浓度金雀异黄索对体外培养的人皮肤成纤维细胞存活力的影响,并绘制细胞的生长曲线;流式细胞仪检测细胞周期的变化;RT-PCR检测不同浓度金雀异黄素对成纤维细胞⒈型胶原mRNA表达的影响.结果 0.03125,0.0625,0.125,0.25,0.5,1 mg/L金雀异黄素作用24 h时,成纤维细胞增殖率分别为97.7%,113.8%,132.5%,116.4%,94.5%和83.3%.当金雀异黄素质量浓度大于0.5 mg/L,对成纤维细胞的增殖表现出抑制作用.浓度在0.0625～0.25 mg/L之间时,对成纤维细胞的增殖表现出促进作用.0.0625、0.125、0.25 mg/L金雀异黄素作用于成纤维细胞后,S期(41.15%±2.88%,61.89%±3.16%,48.18%±1.68%)和G2期(9.76%±3.99%,10.40%±0.54%,7.46%±2.47%)细胞明显高于对照组(S期为30.12%±0.60%,G2期为0.61%±0.16%),两组比较,差异均有统计学意义(P均＜0.05);而G1期细胞(49.08%±3.58%,30.04%±1.89%,44.36%±3.92%)明显低于对照组(69.27%±0.73%),两组比较,差异均有统计学意义(P均＜0.05);3个浓度组Ⅰ型胶原mRNA的表达(0.4814±0.0138,0.5767±0.0291,0.5675±0.0272)均高于对照组(0.4101±0.0236),两组比较,差异均有统计学意义(P均＜0.01).1 mg/L和0.5 mg/L的金雀异黄素作用成纤维细胞后,Ⅰ型胶原mRNA的表达(0.1662±0.0165,0.2017±0.0203)低于对照组,两组比较,差异均有统计学意义(P均＜0.01).结论 一定浓度的金雀异黄素可以促进正常人皮肤成纤维细胞的增殖和生长,并促进成纤维细胞Ⅰ型胶原mRNA的表达.     

Normal and psoriatic keratinocytes and fibroblasts compared in culture

The ability of psoriatic fibroblasts to stimulate growth of epidermal cells was studied by comparing the capacity of murine 3T3 cells, normal fibroblasts and psoriatic fibroblasts to act as feeder layers for cultured normal human keratinocytes. 3T3 cells consistently gave shorter times to confluency than normal or psoriatic cells which were about the same. The SDS polyacrylamide gel electrophoretic pattern of the fibrous protein was identical irrespective of the feeder layer use. Psoriatic epidermal cells could be grown from single cell suspensions by using 10(-9) M cholera toxin in the medium. The cultured psoriatic keratinocytes grew identically to normal cells and made the same fibrous protein. The psoriatic keratinocytes also grew better on 3T3 cells than normal or psoriatic fibroblasts.     

Heat shock proteins in cultured human keratinocytes and fibroblasts

Heat shock induces in cells the synthesis of specific proteins called heat-shock proteins. We have compared the induction of these proteins in human keratinocytes, skin fibroblasts, and a human epithelial tumor cell line following exposure to weak and strong inducing agents (heat, cadmium sulphate, and sodium arsenite). The induction of heat shock proteins was measured in cells by one-dimensional gel electrophoresis of [35S] methionine-labeled proteins and by immunofluorescence using a specific HSP72 monoclonal antibody. Both HSP90 and HSP116 were constitutively expressed in these cell types. Exposure of these cells to weak inducing agents such as heat or cadmium sulphate resulted in the synthesis of HSP72 and HSP90, whereas HSP28 and HSP116 synthesis was detected in keratinocytes and fibroblasts following exposure to the strong inducing agent sodium arsenite. In addition, sodium arsenite induced the synthesis of HSP46 in human keratinocytes. Immunofluorescence demonstrated a rapid and reversible accumulation of the 72-kD heat shock protein within the nucleolus of heat-stressed human keratinocytes and fibroblasts.     

芍药苷对黑素细胞生物活性的影响

目的:检测芍药苷对体外培养正常人表皮黑素细胞增殖活性、酪氨酸酶活性及黑素合成的影响。方法:建立正常人表皮黑素细胞培养体系,加入不同浓度(5~640μg/mL)芍药苷并作用一定时间后,采用MTT法、体外氧化DOPA反应法、NaOH法等分别测定黑素细胞增殖活性、酪氨酸酶活性及黑素含量的变化。结果:40~160μg/mL芍药苷呈剂量依赖性抑制黑素细胞增殖,与对照组比较差异有统计学意义(P0.05);5-20μg/mL芍药苷对酪氨酸酶活性呈剂量依赖性促进作用,与对照组比较差异有统计学意义(P0.05);10-20μg/mL芍药苷对黑素细胞黑素合成起促进作用,与对照组比较差异有统计学意义(P0.05)。结论:芍药苷在较高浓度范围内(40~160μg/mL)对体外培养正常人表皮黑素细胞增殖有剂量依赖性抑制作用;在较低浓度范围内(10-20μg/mL)对其黑素合成有促进作用。     

Effects of in vitro UVA irradiation and PUVA treatment on membrane fatty acids and activities of antioxidant enzymes in human keratinocytes

Human Keratinocytes (NCTC 2544) in culture were exposed to either plain ultraviolet A (UVA) irradiation or to 8-methoxypsoralen plus UVA (PUVA) treatment. Lipid peroxidation, activities of antioxidant enzymes, and percentage amounts of 14C-arachidonic acid in various cellular lipid subclasses and in the culture medium were measured. Both UVA irradiation and PUVA treatment induced significant changes in the distribution of arachidonic acid and increased the liberation of arachidonic acid from membrane phospholipids. At 24 h after either UVA irradiation or PUVA treatment the formation of thiobarbituric acid reactive material was significantly increased, whereas the amount of conjugated dienes was unaffected. The activities of the antioxidant enzymes, catalase and superoxide dismutase, were already significantly decreased at 0.5 h after UVA irradiation or PUVA treatment. The enzyme activities were partially restored during the following 24 h incubation. From the present study, we suggest that in keratinocytes both plain UVA irradiation and PUVA treatment induce changes in the distribution of membrane fatty acids and cause an impairment in the enzymic defense system against oxidative stress.     

Mast cell chymase and tryptase during tissue turnover: analysis on in vitro mitogenesis of fibroblasts and keratinocytes and alterations in cutaneous scars

In order to shed further light on the potential role of mast cells during tissue turnover, we have investigated the number of mast cells containing only tryptase and those storing both tryptase and chymase by enzyme histochemistry in normal versus healing skin. Furthermore, we have studied the in vitro effect of these enzymes on the mitogenesis of subconfluent quiescent fibroblast and HaCaT keratinocyte cultures, using flowcytometric DNA analysis. Chymase-containing mast cell numbers were markedly decreased in scars (P<0.001), whereas the overall number of tryptase-containing mast cells was not decreased, although these cells were smaller and stained more faintly in scars. Chymase (5 to 300 mU/ml) induced a marked, dose-dependent in vitro mitogenic response in 3T3 fibroblasts, whereas the effects of tryptase, at up to 60 nM, were only moderate, compared to the known fibroblast mitogens EGF, TGF-alpha, alpha-thrombin and trypsin at optimal concentrations. Coincubation of either protease with EGF or alpha-thrombin had additive effects. In contrast to fibroblasts, keratinocytes showed only minor mitogenic responses to tryptase and chymase, also in comparison to other known mitogenic stimuli, and responses to EGF and alpha-thrombin were inhibited on costimulation of cells with the proteases. These findings document for the first time a potential role of mast cell chymase in connective tissue repair, with tryptase being less active on fibroblasts, and with inhibitory effects of both mast cell proteases on keratinocytes.     

2%三氯醋酸治疗浅部真菌病的实验及临床研究

目的：寻找一种新的、安全有效、价格低廉、便于推广的抗浅部真菌病的药物。方法：采用2％三氯醋酸制剂进行体外抗真菌作用及最小抑菌浓度（MIC）和最小杀菌浓度（MFC）测定，并用此药治疗60例浅部真菌病患者。全部患者均做真菌镜检和真菌培养。结果：鉴定出菌株46株，分别为红色毛癣菌、须癣毛癣菌、絮状表皮癣菌。三氯醋酸对红色毛癣菌、须癣毛癣菌、絮状表皮癣菌的MIC范围分别为0.06-0.12mg/mL,0.30-0.60mg/mL,0.06-0.12mg/mL。2％三氯醋酸治疗浅部真菌病（手足癣、体股癣）60例，治愈率50％，有效率90％。结论：试验结果和临床观察显示三氯醋酸治疗浅部真菌病均有良好的疗效。