砷剂对角质形成细胞生长和IL-8、TNF-α分泌的影响

目的研究不同浓度砷剂对角质形成细胞生长和IL-8、TNF-α分泌的影响。方法以体外培养的角质形成细胞株为对象,不同浓度砷剂处理细胞后,用MTT比色分析法反映细胞增殖的变化。ELISA方法测定细胞上清中IL-8分泌,用L929细胞检测上清TNF-α的生物活性。结果0.5～5μmol/L三氧化二砷对角质形成细胞株HaCaT细胞有明显抑制作用,并呈剂量依赖关系。在0.001μmol/L至0.015μmol/L药物浓度范围内,同对照组相比细胞增殖加快;未药物处理的对照组HaCaT细胞可分泌一定水平的IL-8和TNF-α,低浓度砷剂(0.001～0.015μmol/L)可刺激其分泌增加。结论低浓度砷剂可促进角质形成细胞株HaCaT细胞的生长,但高浓度能抑制增殖和影响细胞活性。并在一定浓度范围刺激IL-8和TNF-α的合成分泌,可能在砷相关皮肤病的发病机制中具有重要意义。     

Expression of midkine in normal human skin,dermatitis and neoplasms: Association with differentiation of keratinocytes

Midkine is a 13‐kDa heparin‐binding growth factor. It promotes growth, survival, migration and gene expression of various target cells and play roles in many diseases. In normal adult tissues, midkine expression is highly restricted; however, midkine expression levels are high in various malignant tumors. The major biological roles of midkine can be categorized into three areas, namely, the nervous system, cancer and inflammation. Thus far, midkine has not been studied extensively in diseased human skin. We performed immunohistochemistry tests by using anti‐midkine antibodies to study the expression of midkine in normal skin and skin samples of 26 different cutaneous diseases. In addition, we investigated the expression pattern of the midkine gene in cultured keratinocytes. In normal skin, midkine expression was observed in the secretory coils of the eccrine sweat glands, outer root sheath and inner root sheath. Among the cutaneous tumors, the majority of keratinocyte‐derived neoplasms were positive for midkine. Tumors that were not derived from keratinocytes were negative for midkine. In cultured keratinocytes, the midkine gene was expressed earlier than the genes required for keratinization, for example, cytokeratin 10 and transglutaminase 1. Because midkine is expressed in the keratinized areas of normal skin, neoplasms and inflammation, it may play a role as a modulator of keratinization in the skin.     

人角质形成细胞体外扩增与生物学特征变化

目的 研究人角质形成细胞体外扩增过程中生物学特征的变化,观察其角蛋白19(K19)mRNA表达及克隆形成率与扩增受限的关系。方法 取2-3岁健康儿童包皮20例,通过2.4 U/mL dispase和0.05％胰酶(tripsin)分离,获得角质形成细胞体外培养。细胞计数仪计数描绘生长曲线。光镜观察细胞体外扩增过程中形态学改变,同时计数克隆形成率。RT-PCR检测角质形成细胞K19和Involucrin的mRNA表达情况。结果 儿童包皮角质形成细胞体外平均最大扩增能力为(700±37)倍。细胞平均获得率为(1.64±0.297)×106/cm2包皮。角质形成细胞原代(P0)、第2代(P2)、第4代(P4)、第5代(P5)克隆形成率分别为:(45±4)％,(37±4)％,(28±3)％,(9±2)％,第6代(P6)克隆形成率消失。RT-PCR检测发现P0、P2、P4、P5有K19 mRNA表达,P6未见表达;Involucrin各代均可见表达。结论 人角质形成细胞体外扩增能力随传代逐渐降低,扩增受限与角质形成细胞中K19 mRNA表达及克隆形成率消失相关。     

人角质形成细胞培养的实验研究

目的：探讨实验室条件下人体角质形成细胞的培养技术，为角质形成细胞的多方面研究提供可靠的细胞来源。方法：采用改良的无血清培养技术进行人体角质形成细胞的培养，用显微镜及电子显微镜观察角质形成细胞的形态特征。结果：培养的角质形成细胞在多次传代后仍保持了正常的形态。结论：用改良的培养角质形成细胞的技术可为角质形成细胞进一步的实验和临床提供可靠的、丰富的细胞来源。     

Optimization of murine keratinocyte culture for the production of graftable epidermal sheets.

The aim of the present study was to optimize murine epidermal cell cultures in order to obtain graftable sheets. Newborn (1-3 days old) Balb/c mice skin were used to optimize culture media and plating cell concentration, then epidermal sheet production, and grafting. Epidermal cells were plated at various concentrations in different culture media containing low (0.1 mM) or high (greater than 1 mM) Ca2+ levels. After a 3 day culture at the 10(4) cells/cm2 plating cell concentration, the percentage of differentiated cells was more than 80% in the high Ca2+ culture medium and less than 50% with bulky cells in the low Ca2+ culture medium. Under these conditions confluence was not obtained. At the 10(5) cells/cm2 seeding inoculum, the percentage of confluence increased to 95-100% during the first 72 h of culture in both high and low Ca2+ culture media. Three-day-old culture showed stratified multilayer epidermal sheets in the high calcium medium, and monolayer epidermal sheets were present in the low calcium medium after seeding keratinocytes in fibronectin precoated flasks. Seven days after plating, post confluent cultures were composed of a high percentage of differentiated cells (90%) with an increase in shedding cells in the medium. Considering the above morphological observations, sheets obtained with 10(5) cells/cm2 in MCDB-153 (A), DME-HAM (B) or GMEM (C) media after 3 days in culture were grafted. Twenty days after grafting, histological analysis of biopsies showed an epidermal structure and organization comparable to normal murine epidermis without hair follicles. Epidermal transplants showed a complete basement membrane, hemidesmosomes, and tonofilament bundles. Sheets obtained after seven day culture in all media showed lower coverage of the wound bed. These studies point out the importance of the plating cell and Ca2+ concentrations, and the culture time for murine keratinocyte confluence and differentiation to obtain graftable epidermal sheets.     

角质形成细胞源促成纤维细胞增殖的细胞因子研究

目的:研究正常角质形成细胞分泌的促进成纤维细胞增殖的细胞因子。方法:采用免疫组化法测定角质形成细胞所合成分泌的TGF-β1,IL-1α,分别单独及联合应用TGF-β1抗体I、L-1α抗体中和角质形成细胞条件培养基,以Cellcountingkit-8测定中和前后其对成纤维细胞增殖的影响。结果:正常角质形成细胞可合成分泌大量TGF-β1,IL-1α,抗体中和前后角质形成细胞条件培养基对成纤维细胞的增殖作用有明显差异。结论:正常角质形成细胞分泌TGF-β1,IL-1α及一些未知的活性因子,可促进成纤维细胞增殖。     

Adenylate cyclase-cyclic AMP system in pure epidermis isolated by use of dispase

Epidermal adenylate cyclase systems following dispase treatment were investigated. Dispase is a bacterial neutral protease obtained from Bacillus polymyxa. Following the treatment with dispase, the epidermal sheet is easily peeled off the dermis. Dispase-treated pure epidermal sheets were shown to contain three major (beta-adrenergic-, adenosine-, and histamine-) receptor adenylate cyclase systems. Without phosphodiesterase inhibitors, the intracellular cyclic AMP (cAMP) level reached the maximal level at 3 min. This effect was markedly enhanced by the addition of cAMP phosphodiesterase inhibitor. Among these epidermal adenylate cyclase systems, the most marked cAMP accumulation was observed by histamine, followed by adenosine, and then by epinephrine. The separation of epidermis and dermis following dispase treatment revealed that epidermis contained most of the beta-adrenergic response (87%), whereas the dermis retained a significant proportion of adenosine (26%) and histamine (40%) responses when 0.3 mm thickness skin was studied. Specific antagonists of epinephrine, adenosine, and histamine inhibited the effects of these agents completely. The simultaneous addition of two stimulators into the incubation medium resulted in an additive effect. Beta-augmentations by hydrocortisone, colchicine, and retinoid all remained in the dispase-treated pure epidermal sheets, but beta-augmentations by these drugs were spoiled by trypsin treatment. These results indicate that dispase-treated pure epidermis contains three major (beta-adrenergic-, adenosine-, and histamine-) specific and independent receptor adenylate cyclase systems. Dispase is a very useful tool for investigating the metabolism and regulatory system of keratinocytes without any significant damage to epidermal membrane receptor systems.