IL-1α and ATP mRNA expression after ultraviolet irradiation in human keratinocyte original-SCC 12F cells

Background Generally speaking, undifferentiated keratinocytes may synthesize a larger amount of IL-α and its production is decreased as cells complete differentiation gradually. Ultraviolet B (UVB)light can signigicantly stimulate the production and release of some cytokines. In this study we investigated the influence of UVB irradiation on IL-1α and adenosine triphosphoric (ATP) mRNA expressions in the human keratinocyte (KC) of original squamous cell carcinoma line (SCC 12F cells).Methods The cultured SCC 12F cells were irradiated with 30 mJ/cm^2 of UVB. Northern blot was employed to analyze the expression of IL-1α and ATP mRNA.Results There was a constitutive expression of IL-1α mRNA in SCC 12F cells. The expression increased in culturing time in regular KC medium and reached the highest expression at 120 hours.The expression level of IL-1α was up-regulated with two peaks at 6 hours and 72 hours, respectively after UVB irradiation. In comparison with IL-1α mRNA expression, ATP mRNA was down-regulated,with similar biphasic peaks, compared with the sham irradiated group.Conclusions SCC12F cells may express IL-1α mRNA constitutively. After UVB irradiation, the mRNA expression of IL-1α and ATP will show opposite effect because of inflammation/immunity and energy consumption mechanisms.     

Human epidermal melanocyte and keratinocyte melanotropin receptors: visualization by melanotropic peptide conjugated macrospheres (polyamide beads)

Abstract The objectives of this research were to determine whether melanotropin receptors are characteristic membrane markers of human epidermal melanocytes. Methodologies were developed to visualize these receptors by light microscopy. Multiple copies (up to a thousand) of [Nle⁴,D-Phe⁷]α-MSH, a superpotent analog of α-melanocyte stimulating hormone (α-MSH), were conjugated to a macromolecular carrier, large polyamide beads (macrospheres). Incubation in (the presence of the I conjugated macrospheres resulted in binding of human epidermal melanocytes to the macrospheres. Specificity of the binding of melanocytes of the melanotro-pin-conjugated macrospheres was demonstrated by several studies: (i) Binding of melanocytes to the conjugate was specific since it could be blocked by prior incubation of the cells in the presence of (the unconjugated hormone analog: (ii) The macrospheres after removal of the bound ligand did not bind to the melanocytes: (iii) Another peptide hormone ligand (e.g., a substance-P analog) attached to the macrospheres failed to bind to the melanocytes: (iv) BI6/F10 mouse melanoma cells known lo express melanotropin receptors bound to the macrospheres; (v) Cells of nomnelanocyte origin (e.g., mammary cancer cells, lung cancer cells, fibroblasts) did not bind lo the macrospheres. One exception was that human epidermal keratinocytes also expressed melanotropin receptors as determined by all the criteria established for epidermal melanocyles. Thus, cell specific melanotropin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.     

Construction of large area organotypical cultures of oral mucosa and skin

BACKGROUND: Skin and oral mucosal keratinocytes grown in vitro usually lose their normal patterns of differentiation, unless grown as organotypical cultures that are constructed by allowing collagen gels containing fibroblasts to contract before they are plated with keratinocytes and raised to the air/medium interface. However, the contraction process tends to produce small irregular cultures. METHODS: To generate uniformly differentiating large cultures, we have investigated several aspects of the factors involved in the culture construction. By adjusting the number of fibroblasts used and by plating the matrices with keratinocytes prior to contraction, cultures of up to 72 cm2 were constructed. RESULTS: The cultures retained almost the full surface areas of the original matrices and showed uniform patterns of epithelial plating and differentiation. Immunostaining for cytokeratins and integrins indicated restoration of in vitro phenotypes similar to those of the epithelial tissues of origin. CONCLUSIONS: These methods successfully generate cultures required for certain types of investigations and tissues that are suitable for clinical use as grafts.     

The perinuclear factor, a rheumatoid arthritis-specific autoantigen, is not present in keratohyalin granules of cultured buccal mucosa cells.

Rheumatoid arthritis patients have antibodies in their serum directed against the perinuclear factor, a protein component present in keratohyalin granules in the cytoplasm of human buccal mucosa cells. The anti-perinuclear factor (APF) can only be detected by an indirect immunofluorescence test performed on fresh buccal mucosa cells from 'selected donors'. To obtain a more reliable antigen source and to gain more insight into the origin and nature of the perinuclear factor we attempted to culture perinuclear factor-containing buccal mucosa cells. Here we describe the successful culturing of such cells, which, however, did not contain keratohyalin granules nor the perinuclear factor. By adding the phorbol ester 12-o-tetradecanoylphorbol-13-acetate (TPA) we were able to induce keratohyalin granules in both cultured primary buccal mucosa cells and a squamous carcinoma cell line of the cheek (SqCC/Y1). These induced keratohyalin granules do contain the protein profilaggrin, which in vivo, in fresh buccal mucosa cells, co-localizes with the perinuclear factor. However, we were not able to demonstrate the presence of the perinuclear factor, not even after induction of terminal differentiation of the cultured cells nor after Epstein-Barr virus infection. Our results suggest that the perinuclear factor, in contrast to profilaggrin, is not an integral component of buccal mucosa cells.     

The expression of the gap junctional protein Cx43 is restricted to proliferating and non differentiated normal and transformed keratinocytes

Abstract The passage of specific growth modulating signals through gap junctions may regulate the proliferation and differentiation of human keratinocytes. To investigate this, we correlated the proliferation of normal human keratinocytes and a transformed squamous cell carcinoma cell line, SCC4. with the expression of the gap junctional proteins Cx43. 31 and 31.1, known to be expressed by keratinocytes. Proliferation was con-lined to preconfiuent and confluent cultures of normal keratinocytes, falling to undelectable levels once poslconfluency was achieved. Cx43. at both the message and protein levels, paralleled these changes, being elevated predominantly in prcconiluent and confluent cultures, and downregulated in postconfluency. Similar results were found for Cx31 and 31.1 at the message level. In contrast, the proliferation of SCC4 cells cultured in media supplemented with 5.0° FCS was maintained at n substantial level from preconfluency through 2 weeks postconfluency. Cx43, 31. and 31.1 RNA and Cx43 protein expression mirrored the levels of proliferation within SCC4 cultures. Cx26 and 32 were not found in normal keratinocytes or SCC4 cells at any stage of differentiation. These data, illustrating a tight correlation between proliferation and Cx43, 31 and 31.1 expression, suggest that these connexins may represent proliferation-specific gap junctions within keratinocytes. and may therefore transmit signals that control keratinocyle division.     

抗角蛋白自身抗体对白细胞介素1β诱发角朊细胞增殖的影响

目的：研究抗角蛋白自身抗体（ＡＫ ａｕｔｏ Ａｂ）对白细胞介素１β（ＩＬ－１β）诱发角朊细胞增殖的影响。方法：采用角朊细胞无血清培养方法，以＾３Ｈ－ＴｄＲ标示角朊细胞增殖程度，检测ＩＬ－１β刺激角朊细胞及ＡＫａｕｔｏ Ａｂ处理角朊细胞的增殖代谢。结果：１６００Ｕ／ｍｌ及以上浓度ＩＬ－１β对角朊细胞增殖作用有明显促进作用（Ｐ〈０．０１），正常血清浓度ＡＫ ａｕｔｏ Ａｂ对ＩＬ－１β引起的角朊细胞增殖     

无血清条件下紫外线诱导表皮细胞凋亡的实验研究

目的：观察紫外线照射诱导新生大白鼠表面细胞凋亡。方法：用不同剂量的紫外线照射培养条件下和活体状态下表皮细胞，采用Tunel法检测表皮细胞凋亡，流式细胞仪观察表皮细胞P53、Bcl-2、CD95表达。结果：传代培养12－24h ,et 1500J/m^2紫外线照射，照射后继续培养24－48h表皮细胞凋亡率达高峰。紫外线照射后的表皮细胞P53表达升高。相同剂量紫外线照射，活体状态下表皮细胞凋亡率低于培养表皮细胞。结论：表皮细胞凋亡与P53表达升高相关，活体上的表皮细胞能较强抵搞紫外线诱导的凋亡作用。