A Study Of The Expression Of HSP90 In Rat Epidermal Stem Cells After Heat Injury

Objective Isolate, purify and culture the SD rat epidermal stem cells(Ep SCs), use the two or three generation cells to establish heat injury models. Detect the expression of HSP90 in rat Ep SCs which were heat injured at different temperatures, and explore the role of HSP90 in thermal injured Ep SCs.Methods The epidermis was mechanically separated from the dermis. Ep SCs were obtained by using a “two-step trypsinization and Ⅳcollagen differentially adhere cells for 10minutes” method. The rapidly adherent epidermal cells were cultured in keratinocyte serum-free medium(K-SFM). Observe the morphology of cells, calculate the colony forming efficiency by clonogenic assays, and draw the cell growth curve, so as to assess the colony formation and proliferation of the cultured cells.In order to determine the phenotype of cells and assess the purity of cells, we use immunofluorescence to detect the expression of surface markers, such as β1-integrin, K19 and K10.The heat-injured experimental cells are from P2-P3 Ep SCs, which will be put in the water bath and incubated for 10 minutes. The temperature of control group is 37℃, the temperatures of experimental group are 41℃, 43℃, 45℃, 48℃, 51℃, 53 and 55℃ ℃. After thermal injury, we use Hoechst33342 and propidium iodide(PI) co-staining to measure the apoptosis rate and viability of Ep SCs. Use RT-PCR and Western Blot methods to detect the gene transcription and protein expression of HSP90 in heat injuried Ep SCs.Results The cultured Cells are small and irregular polygonal, which have high nuclear-cytoplasmic ratio, and the cell membranes have strong refraction. The cells growing as clones, and have typical cobblestone-like structure.The Colony formation and growth curves show that the cultured cells have strong clonogenic capacity and proliferative capacity in vitro. The immunofluorescence shows that our cultured cells have high purity which were expressed β1-integrin and K19, but not expressed K10.Hoechst33342 and PI co-staining shows that, between 41℃~51℃, apoptosis rate and mortality rate are increased with temperature by compared with 37℃control group cells. At 51℃, the cells reached a peak of apoptosis, but at 55℃, the mortality rate of cells was highest. RT-PCR and Western Blot assay consistently show, HSP90 expression level is relatively low in 37 ℃control group, but in the experimental groups are significantly changed.Between 41℃~48℃, the HSP90 expression levels increased with temperatures, at 45 and 48℃ ℃ thermal stimulation conditions, the roughly at the same level.But over 48℃, the expression levels gradually decreased.Conclusion The cultured cells were consistent with the biological characteristics and phenotypes of Ep SCs.So we successfully established a method to get Ep SCs.Within a certain temperature range, the levels of gene transcription and protein expression of HSP90 in Ep SCs were increased after heat stress.Meanwhile, HSP90 may be involved in heat stress response and had a protective effect on these cells. Scald cells 10 min under 48℃, which is a suitable condition for the construction of heat-injured model. And this model can be used to study the protective effect mechanism of HSP90.