| Sulfur mustard (HD), "the king of war gases," is a potent vesicant agent that has a long history of use as a chemical warfare agent and remains a significant military and civilian threat even today. Researchers worldwide have been showing great interest in the study of sulfur mustard.Skin is the major target for HD. Exposure to HD in either the liquid or vapour form produces dose-dependent injuries, which range in severity from erythema and mild erythema to severe blister and necrosis. The basal keratinocytes are one of the most sensitive cells population in the skin for the toxic effects of HD. There are still no specific antidotes available to prevent or minimize injury induced by exposure to HD. Presently, most therapeutic measures such as anti-inflammatory drugs are only supportive. The reasons that antidotes to HD have not yet fielded are mainly that its molecular mechanism is not fully understood, apart from multiple stratification and multiple targets of HD in the cell. HD can cause the severe damage to the epidermic basal cells, which are vital for the repair and replacement or reconstruction of epidermal tissue. Whether proliferative dynamics of the epidermic basal cells especially keratinocyte stem cells will be changed in mouse skin injury induced by sulfur mustard have not been well understood.Our research aims at an experimental study of the possible characteristics of the proliferative dynamics which sulfur mustard induced and a probe of the possible approach to identify the keratinocyte stem cells, in the hope of providing a theoretical basis for further therapeutic studies such as amelioration of their niche or microenviroment, stimulation of their proliferation and differentiation and transplantation of stem cells.The results are as follows: 1. The effects of HD on the proliferative dynamics of mouse epidermic basal cells.Freshly isolated mouse epidermal cells with proteolytic enzymes (trypsin) were identified mainly as positive-β1 integrin cells, namely epidermic basal cells.Changes of the mouse cell proliferative cycle related to a single topical application of 5μL of 32, 160, 320 mg/mL HD were evaluated at different time points post-exposure by flow cytometer. The cells of S and G2/M phase were promoted at 48h after the exposure of 32mg/mL HD. The dominant form of cell death was dose and time dependent, with apoptosis predominating at the doses of 160, 320 mg/mL HD.The cells were stained with PI & HO and identified by confocal laser scanning microscopy (CLSM). The distinctive morphological changes had been observed in the nucleus, such as the condensation of chromatin and the formation of nuclear fragmentation at 72h after the 320mg/mL HD exposure, hi order to further verify apoptosis induced by HD, skin ultrastructure at different time and concentration of HD2004 Abstractwere investigated by transmission electron microscope (TEM). HD induced the classical morphological features of apoptosis in nucleus at 24h after the 32mg/mL HD exposure, which accompanied by condensation of chromatin and breakage of nuclear into small fragment. A small number of apoptotic cells were observed in a study of exposure to 160mg/mL HD at 24h. However, at 72h after the dose of 160 and 320mg/mL HD exposure, necrosis was the predominant form of cell death. The changes of proliferative dynamics of mouse epidermic basal cell exposed to HD were significant. Sulfur mustard caused basal cells death, namely, induction of terminal differentiation and apoptosis.2. Identification of keratinocyte stem cells (KSCs) and effects of HD on KSCs.Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells, which are charactered by the slowly cycling cells that retain BrdU label, termed label-retaining cells (LRCs). We separated KSCs from TA cells through the use of in vivo cell kinetic analysis. Young growing mice were given with l0mg/mL BrdU on the 10th and 11th days after birth and then were allowed to grow for 30 days with no further labeling. Cell samples...
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