The aim of this work was to validate an image analysis method, based on cell nuclei form factor determination, for counting fibroblasts within human dermis. We first used reconstructed dermal equivalents in which fibroblasts can also be counted directly after lysis of the collagen matrix. We found a good correlation between the results of direct counting and those of image analysis from day 10 to day 28 of culture. When applied to young normal donors' skin biopsies fixed in Bouin's solution and embedded in paraffin, the image analysis method yielded mid-dermis fibroblast counts of between 2100 and 4100 per mm3 of fresh tissue. A nuclear form factor (FF) comprised between 0.35 and 0.84 was found to be a biologic marker of fibroblasts. This was confirmed after fibroblast discrimination from other cell types, which had rounder nuclei (FF >/= 0.85) and were identified either by their location (e.g. endothelial cells) or by labeling with specific antibodies (e.g. lymphocytes and monocytes/macrophages). Similar results were obtained with seven healthy donors' skin biopsies that had been frozen in nitrogen liquid and cryostat-sectioned, showing that this counting method is independent of the histologic procedure. Finally, analysis of samples of hypertrophic scars from two patients revealed that fibroblast density in some parts of the dermis was more than twice the value found in other parts presenting a fibroblast density almost normal, showing that this cell counting method can also be used to assess fibroblast heterogeneity within a given tissue. 相似文献
: To examine the incidence of radiation-induced late rectal complications by analyzing the data of measured rectal doses in patients with cancer of the uterine cervix treated with high-dose-rate intracavitary brachytherapy.
: We measured doses to the rectum in 105 patients with cancer of the cervix during high-dose-rate intracavitary brachytherapy with a semiconductor dosimeter that can measure five points in the rectum simultaneously. On the basis of these measurements, equivalent doses, to which the biologically equivalent doses were converted as if given as fractionated irradiation at 2 Gy/fraction, were calculated as components of the cumulative dose at five rectal points in intracavitary brachytherapy combined with the external whole pelvic dose.
: The calculated values of equivalent doses for late effects at the rectum ranged from 15 to 100 Gy (median 60 Gy for patients who did not develop complications and 76 Gy for patients who subsequently developed Grade II or III complications). When converted to a graph of absolute rectal complication probability, the data could be fitted to a sigmoid curve. The data showed a very definite dose-response relationship, with a threshold for complications at approximately 50 Gy and the curve starting to rise more steeply at approximately 60 Gy. The steepest part of the curve had a slope equivalent to approximately 4% incidence/1 Gy increase in equivalent doses.
: The radiation tolerance dose, 5% and 50% complication probability, was about 64 and 79 Gy, respectively. Our data almost agree with the prescribed dose for the rectum for the radiation tolerance doses on the basis of the recorded human and animal data. The probability of rectal complications increased drastically after the maximal rectal dose was >60 Gy. 相似文献
For the investigation of the skin irritancy potential of chemicals in an in vitro model it is necessary to have sensitive endpoints that predict the effects of those compounds on native human skin. Recently, we have identified that 27-kDa heat shock protein (HSP27) can serve as a sensitive marker of skin irritation, as exposure of human skin to sodium lauryl sulfate (SLS) both in vitro and in vivo induced relocalization of HSP27 from the cytoplasm to the cell nucleus. The aim of the present study was to determine whether nuclear localization of HSP27 could be used as a parameter for evaluation of potential skin irritants in screening assays in vitro. For this purpose, human skin equivalent consisting of epidermis reconstructed on de-epidermized dermis was exposed to SLS or UV light. Stress-induced nuclear relocalization of HSP27 was observed in excised skin exposed to SLS or UV light and in reconstructed epidermis only when the latter was generated in the absence of vitamin C. The omission of vitamin C results in an impaired barrier function. In the presence of vitamin C, however, the barrier function was comparable with excised skin, suggesting that vitamin C may control the response to stress in the reconstructed epidermis. Besides the presence of vitamin C, the response of skin equivalents may strongly depend on other conditions under which they are generated, because the stress-induced HSP27 relocalization was not detected in the commercially available epidermal kit EpiDerm. The results of the present study show that HSP27 nuclear staining can serve as a sensitive marker for skin irritation or cellular stress in excised skin as well as in certain well-characterized human skin equivalents in vitro. 相似文献