Stimulation of Cultured Melanocytes in Medium Containing a Serum Substitute: Ultroser-G

Melanocyte cultures were established and maintained routinely in Ham's F-10 medium containing 12-O-tetradecanoyl-phorbol-13-acetate (TPA), isobutylmethylxanthine (IBMX), cholera toxin (CT) and fetal calf serum (FCS). Three serum substitutes (Ultroser-G, Nutridoma-Hu and Nutricyte-H) were tested in order to obtain a medium without FCS having a more constant composition. Melanocyte proliferation was examined in long-term culture experiments by in situ cell counts at different periods of time. Only with Ultroser-G (1-2%) was the proliferation of melanocytes maintained without both FCS and CT, whereas the addition of the other two serum substitutes resulted in stabilization of melanocyte densities in the cultures up to 28 days. In the medium containing 1% Ultroser-G and IBMX without TPA minimal or no increases in melanocyte density were found. Addition of basic fibroblast growth factor (bFGF, 1 ng/ml) to the medium without TPA resulted in a partial restimulation of growth in different experiments. In this system with 1% Ultroser-G and 1 ng/ml bFGF, IBMX could also be replaced by other factors (dbcAMP LTC4 and a purified form of α-melanocyte stimulating hormone). The culture medium with 1% Ultroser-G containing TPA and IBMX is now used for routine melanocyte culture. In this medium TPN/IBMX can easily be replaced by bFGF/dbcAMP with optimal growth stimulation. The combination bFGF/α-MSH and other more physiological stimulators offers an alternative to study responses of melanocytes in culture with respect to proliferation, metabolism, and phenotype.     

α-MSH and Melanogenesis in Normal Human Adult Melanocytes

Normal human skin melanocytes do not pigment consistently to α-melanocyte stimulating hormone (α-MSH) in culture. The aim of this study was to establish media conditions in which to obtain a reproducible melanogenic response to α-MSH in these cells. Twenty-five media of varying mitogen composition were examined. As previously noted by other workers, melanocyte morphology and proliferation are greatly affected by media composition. However, under the majority of media conditions that supported melanocyte survival and proliferation, cells did not respond to α-MSH with any consistent increase in dopa oxidase activity or melanin content. In only one medium condition, where basic fibroblast growth factor (bFGF) was the sole mitogen present, α-MSH induced both an increase in dopa oxidase activity (at 48%) and in melanin content (of 283%).     

Induction of Growth Factor Receptors on Cultured Human Melanocytes

Using a sensitive and selective culture system for human epidermal melanocytes, we have demonstrated that the morphologic changes induced by addition of phorbol 12-tetradecanoate 13-acetate (TPA) to proliferating newborn melanocytes are associated with induction of nerve growth factor (NGF) receptors, as measured by messenger RNA (mRNA) levels and protein accumulation and by cell surface immunofluorescent staining. Growth factor deprivation or addition of NGF similarly results in NGF receptor induction. NGF is believed to function in vivo and in vitro as a survival factor for many neural crest-derived cells and has been demonstrated to promote specific neural cell functions ranging from neurite outgrowth to enzyme induction, but to date no role for NGF has been identified with regard to normal human melanocytes. Our data demonstrate that, given appropriate stimulation, cultured human melanocytes may express the NGF receptor gene and therefore suggest that NGF may modulate human melanocyte behavior in vivo. This first demonstration of a growth factor receptor on human melanocytes provides an important opportunity to explore signal transduction relevant to their growth, differentiation, and malignant transformation.     

Corticotropin Expression by Human Melanocytes in the Skin

Highly dendritic melanocytes have been observed in rapidly proliferating seborrheic keratosis, epidermis overlying melanomas, and in melanomas. On staining for the presence of POMC with monoclonal antibody against human ACTH, the melanocytes show cytoplasmic positivity. Short term organ cultures of whole skin from the marginal zone of vitiligo patients show that 22.7% of controls and 45.5% on dark incubation in adriamycin and 87.5% exposed to a pulse of UV on adriamycin treatment show melanocytes positive for ACTH. The surrounding keratinocytes in the epidermis and in the seborrheic keratosis are negative, whereas in melanomas, isolated groups of melanocytes are positive for ACTH. These findings indicate that ACTH is expressed by the melanocytes in the G₂-phase, the activity being enhanced on UV exposure. Thus UV dependent pigmentation is associated with POMC production in human skin. From this work it is evident that the melanocyte network varies the MSH/ACTH levels in correlation with repigmentation and depigmentation in the marginal zone in vitiligo by expressing POMC locally and is related to the UV-sensitivity of the melanocytes.     

Analysis of the UV-Induced Melanogenesis and Growth Arrest of Human Melanocytes

Cultured human melanocytes derived from different skin types responded to frequent treatment with ultraviolet (UV) light with increased melanin synthesis, decreased proliferation, and morphologic signs of aging. These effects were augmented by increased frequency of irradiation with 15.5 mJ/cm² UV light. Stimulation of melanogenesis by UV light involved an increase in tyrosinase activity, without any change in the amounts of either tyrosinase or tyrosinase-related protein (TRP)-1, and a decrease in the amount of TRP-2, as determined by Western blot analysis. These results are different from the mechanisms by which other melanogenic agents, such as cholera toxin and isobutyl methylxanthine, stimulated melanogenesis, whereby the amounts of tyrosinase, TRP-1 and TRP-2 were increased. The decrease in the amount of TRP-2 might be significant in that it might alter the properties of the newly synthesized melanin. The UV irradiation protocol that was followed blocked melanocytes in G₂-M phase of the cell cycle without compromising cellular viability. Following three rounds of UV irradiation, melanocytes could recover from the growth arrest and resume proliferation. Treatment with 0.1 μM α-melanocyte stimulating hormone (α-MSH) postirradiation enhanced the melanogenic effect of UV light and stimulated the melanocytes to proliferate. The effects of α-MSH on the UV induced responses and their implications on photocarcinogenesis are being further investigated. Analyzing the mechanisms by which UV light exposure affects normal melanocytes might lead to a better understanding of how these cells undergo malignant transformation, and why individuals with different skin types differ in their susceptibility to skin cancers.     

Demonstration of Voltage-Dependent and TTX-Sensitive Na+-Channels in Human Melanocytes

The electrophysiological properties of cultured human melanocytes were investigated using the whole-cell configuration of the patch-clamp technique. Depolarizations to membrane potentials more positive than -30 mV resulted in the rapid development (<1 ms to peak) of an inward current. The maximum peak current was observed at +10 mV and reached an average amplitude of about 270 pA. During the depolarizations, the current inactivated with a time constant of about 2 ms. The current was abolished by the addition of 0.3 μM tetrodotoxin, a blocker of voltage-gated Na⁺-channels, and disappeared when Na⁺ was omitted from the extracellular medium. In addition, the melanocytes contain at least two types of outward K⁺-current. The first type, observed in every cell, was highly sensitive (K_i 1 mM) to the K⁺-channel blocker TEA, required depolarizations beyond zero to be activated and did not inactivate. The second type was less regularly observed (10% of the cells). This current activated at more negative voltages (–20 mV), was resistant to TEA (20 mM) but was blocked by 2 mM 4-aminopyridine and inactivated rapidly during depolarizations. We conclude that human melanocytes are equipped with voltage-dependent Na⁺-channels, a delayed rectifying K⁺-current and a K⁺-current similar to the A-current in neurones.     

Vero细胞微载体培养的化学成分明确无血清培养基的设计

目的：设计适用于Vero细胞微载体培养的化学成分明确无血清培养基。方法：以商品化的DMEM／F12合成培养基为基础培养基,应用Plackett—Burman实验设计和响应面分析法设计支持Vero细胞微载体培养的化学成分明确无血清培养基。结果：以细胞密度为评价指标,在单因素实验的基础上采用Plackett-Burman实验设计考察10种培养基添加成分对Vero细胞生长的影响,确定了3种对Vero细胞生长起明显促进作用的培养基添加成分,为胰岛素、血清素和腐胺。继而利用响应面法分析了这3种添加成分的最佳水平范围,设计了一种支持Vero细胞贴附培养的无血清培养基（VERO—SFM—A）。在Bellco搅拌式培养瓶中采用VERO-SFM．A和Cytodex1微载体培养Vero细胞,细胞密度由接种时的4×10^5cells／ml增加到培养6d后的22．3×10^cells／ml,细胞活力保持在96％以上。结论：VERO—SFM—A能够有效地支持Vero细胞在微载体表面固定化生长并达到较高的细胞密度,具有实际应用于Vero细胞微载体规模化培养的应用潜力。     

Responses of Cultured Melanocytes to Defined Growth Factors

To proliferate in vitro, normal melanocytes, unlike normal fibroblasts, require specific growth factors in addition to those supplied in serum. The substances that promote melanocyte proliferation, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and stimulators of cyclic adenosine monophosphate (cAMP), also promote pigmentation. Consequently, cell division and expression of at least some differentiated functions are not mutually exclusive for melanocytes. At present, the only known natural growth factor that can replace TPA in normal human melanocyte cultures is basic fibroblast growth factor (bFGF). Like TPA, bFGF is effective, most of the time, only in the presence of added cAMP. Some preparations of bFGF, however, may have a highly labile, intrinsinc cAMP stimulatory activity. It is thus possible that bFGF can assume two forms, dependent on and independent of cAMP stimulatory activity. Alternatively, a second factor may exist in pituitary glands that co-purifies with bFGF but deteriorates with storage. Abnormal melanocytes in culture, such as those derived from dysplastic nevi and primary melanomas, depend on the specific factors (bFGF and cAMP), whereas melanocytes from metastatic melanomas do not     

Luminol-Enhanced Chemiluminescent Response of Human Melanocytes and Melanoma Cells to Hydrogen Peroxide Stress

The response of human melanocytes and melanoma cells to hydrogen peroxide stress was measured. Cells were exposed to glucose/glucose oxidase or free H₂O₂ and reactive oxygen species measured by luminol-enhanced chemiluminescence. The response was distinctly different between the two types and the addition of superoxide dismutase to melanoma cells paradoxically enhanced the chemiluminescent signal. These findings coupled with other known differences between the way these two types of cells handle oxidative stress at a molecular level suggests that a therapeutic window may be avail-able for exploitation.     

一种新的胰岛素及生长因子促生长活性测定系统

ＧＲ２Ｈ６细胞是从小鼠乳腺癌细胞衍生来的成纤维细胞样细胞株。该细胞能在特定的无血清培养液中生长,我们发现在此无血清培养条件下,ＧＲ２Ｈ６细胞生长情况与胰岛素、表皮生长因子和类胰岛素生长因子－Ｉ的浓度有较好的相关性,根据细胞数目和ＤＮＡ总量的变化可定量测定胰岛素和上述生长因子对该细胞株的促生长作用。据此,本实验室建立了一种新的胰岛素与生长因子促生长活性测定系统。与已知的胰岛素和生长因子测活系统比较,     

Up‐regulation of Tyrosinase Gene by Nitric Oxide in Human Melanocytes

Ultraviolet light (UV) radiation causes skin‐tanning, which is thought to be mediated by stimulating the release of melanogenic factors from keratinocytes as well as other cells. Nitric oxide (NO) has been reported to be generated after UV radiation and to stimulate melanocytes as one of the melanogens. In a previous experiment by another group on melanogenesis induced by NO, increases in both tyrosinase activity and tyrosinase protein levels were observed after daily stimulation of NO for 4 days. In the present study, we investigated tyrosinase gene expression within the first 24 hr of NO‐induced melanogenesis. Tyrosinase mRNA expression was found to be induced 2 hr after a single treatment with S‐nitroso‐N‐acetyl‐ l ‐arginine. An increase of tyrosinase activity was also detected time‐dependently within the 24‐hr period, accompanied by an increase of tyrosinase protein levels. The induction of mRNA expression was suppressed by a cyclic guanosine 3′,5′‐monophosphate (cGMP)‐dependent protein kinase (cGMP/PKG) inhibitor. These results suggest that the enhancement of tyrosinase gene expression via the cGMP pathway may be a primary mechanism for NO‐induced melanogenesis.     

Growth of an Insect Trypanosomatid at 37 C in a Defined Medium*

SYNOPSIS. A defined medium for an insect trypanosomatid, “Leptomonas pessoai” (probably a member of the genus Herpetomonas) isolated from the reduviid Zelus leucogrammus, allows growth up to 37 C. No marked differences were evident for growth at 37 C. The requirements for amino acids, vitamins, purine, and hemin, and pH range were like those established for Crithidia fasciculata. Again like C. fasciculata, it remains alive at 4 C for at least 3 months in a çlycerolated defined medium.     

Growth of Phytomonas serpens in a Defined Medium; Nutritional Requirements

ABSTRACT. Three strains of Phytomonas serpens two from tomatoes, Lycopersicon esculentum one from the insect Phtia picta (Hemiptera, Coreidae), were cultivated in a chemically defined medium developed from a defined medium for cultivating insect flagellates. Besides organic growth factors required by other insect trypanosomatids this flagellate requires, serine and inositol. Glutamine stimulates growth, and, surprisingly, does not require heme.     

系统表达HB-EGF转基因小鼠的建立

目的建立系统表达肝素结合性表皮生长因子（HB-EGF）的转基因动物模型,利用转基因动物模型研究HB-EGF与组织纤维化的关系。方法RT-PCR法克隆小鼠HB-EGF基因,将其插入Chickenβ-actin启动子下游,构建Chickenβ-actin-HB-EGF表达载体,利用显微注射的技术把表达载体注射到受精卵的雄原核中,建立HB-EGF转基因小鼠。利用特异引物PCR的方法鉴定转基因的基因型,采用Western Blot方法鉴定HB-EGF基因在全身组织的表达。分别取HB-EGF转基因鼠与同窝阴性小鼠的肝、肾、肺及膀胱组织进行Massion染色。结果建立了系统表达HB-EGF转基因小鼠,Western Blot发现其HB-EGF在肝、肺、肾及膀胱的表达与同窝阴性对照小鼠相比明显增加。Massion染色结果表明转基因鼠肝、肺、肾及膀胱组织纤维化程度明显高于同窝阴性对照小鼠。结论成功建立了系统表达HB-EGF转基因小鼠,HB-EGF的过度表可显著加重组织纤维化程度。     

人角质细胞生长因子2基因的克隆及其在大肠杆菌中的表达

目的:构建人角质细胞生长因子2(hKGF2)基因的高效原核表达载体pET-30a( )-hKGF2,并在大肠杆菌BL21(DE3)中获得表达。方法:从培养的人胚胎肺成纤维细胞中提取总RNA,采用RT-PCR技术扩增出去除了信号肽部分的hKGF2基因,克隆到pMD18-T载体,经DNA序列分析后与pET-30a( )表达载体连接,在大肠杆菌BL21(DE3)诱导表达6×His-hKGF2,用SDS-PAGE及Western印迹鉴定表达蛋白。结果:构建了表达载体pET-30a( )-hKGF2,经IPTG诱导后,表达的重组蛋白理论相对分子质量约为23000,约占菌体总蛋白的20%。结论:6×His-hKGF2蛋白在大肠杆菌BL21(DE3)中为可溶性高效表达,为获得高纯度、高活性的产物,以及进一步的大规模生产和应用研究奠定了基础。