| Objectives:1、To establish an efficient technique of isolate and culture for epidermal stem cells and follicle stem cells in vitro, Culture primary and passage cells.2、Study on the biological characteristics and cell cycles of rabbits epidermal stem cells and follicle stem cells, Then compare differences of the two kinds of cells.3、To isolate and culture human follicle stem cells and epidermal stem cells, Observe cell morphology.4、 To compare biological characteristics between human follicle stem cells and human epidermal stem cells. Explore a more suitable seed cells of tissue engineering skin. The study provided basis for building artificial skin containing subsidiary organs in vitro and achieve a large area of skin damage from the structural repair to the reconstruction of function.Methods:1) Obtain follicle stem cells and epidermal stem cells from bilateral tentacles and abdomen respectively of the normal rabbits using two-step enzyme digestion method. Then culture the primary and passage cells, and observe cell morphology and activities using an inverted phase contrast microscope.2) Detect expression differences of molecular markers:(31integrin, keratin19of follicle stem cells and epidermal stem cells by Immunohistochemical staining. Observe proliferative capacity of follicle stem cells and epidermal stem cells using the MTT assay.3) Detect the cell cycle of passage5and passage15of follicle stem cells and epidermal stem cells using flow cytometry; Then compare biological activities of passage cells.4) Using fluorescence quantitative PCR detect keratin19(K19) and β1integrin mRNA expression differences of follicle stem cells and epidermal stem cells.5) Separate follicle stem cells of human hair follicles and culture cells applying a single cell and tissue explanted. Isolate and culture epidermal stem cells of human skin and observe morphology of primary and passage cells.6) Compare the differences between follicle stem cells and epidermal stem cells on adherent time in vitro, cell morphology, growth curves and proliferative capacities.Results:1) Applying two-step enzymatic successfully isolate rabbit follicle stem cells and epidermal stem cells. Follicle stem cells adhere to the culture dish after planted2day, adherent cells had aggregating tendency after planted3days, the clone increased to integrate after7days. The subcultured follicle stem cells adhere to the dish rapidly, cells can achieve complete integration after8days.Epidermal stem cells had clone-like growth trends after cultured10days Passage epidermal stem cells adhered to the culture dish slowly, and culture for2weeks cells to achieve fully integrate.2) Immunohistochemical staining showed that:rabbits follicle stem cells and epidermal stem cells express β1integrin and keratin19. The growth curves of Follicle stem cells and epidermal stem cells showed that:In addition to the second day, Proliferative activities of follicle stem cells is much higher than ESCs from the sixth day and at the0.05level, the difference between Follicle stem cells and epidermal stem cells is significantly different than the test difference (P<0.05). Proliferative activity of the10,14,18days follicle stem cells was gradually increased, the proliferative activity began to decaline until26days.Epidermal stem cells reached a proliferative peak and decline followed. Follicle stem cells showed sustaining high proliferation, the proliferative time is two times that of epidermal stem cells.3) The analysisi of cell cycle d showed that:S percentage and proliferative index (PI) of follicle stem cells and epidermal stem cells descended with extended passages. PI of follicle stem cells is higher than epidermal stem cells. The proliferative capacity of follicle stem cells was higher compared with epidermal stem cells.4) The result detected by fluoresence quantitative PCR demonstrated was that mRNA expression of β1integrin and keratin19of follicle stem cells was significantly higher than epidermal stem cells.5) A large number of K19,β1integrin positive expression of follicle stem cells can be obtained by two-step enzyme digestion method from the human scalp; Follicle stem cells adhere to the cultured dish within24hours using single cell culturing method. Passage cells proliferate rapidly,.80percent of cells merged about3days. Epidermal like cells climbed out of the tissue after culturing the tissue5days. Cells growed rapidly, fibroblast-like cells appeard around the epidermal like cells. Epidermal stem cells suspended after cultured3days, The passage cells proliferate slowly,80percent of cells merged after7days.6) Human follicle stem cells and epidermal stem cells were all express β1integrin and keratin19. Compared to the growth curve of epidermal stem cells, Follicle stem cells had higher proliferative activity and a long growth time to maintain a high proliferative activity.Conclusions:1) The method of "two step enzyme"digestion was a safe and efficient method to obtain follicle stem cells and epidermal stem cells. A large number of target cells were obtained in short period of time. It is convenient for the further study.2) Follicle stem cells and epidermal stem cells of human and rabbit express β1integrin and K19, however β1integrin and K19expression of follicle stem cells were higher than epidermal stem cells.3) Follicle stem cells and epidermal stem cells obtained have high proliferative capacity, follicle stem cell can amplify in a long time in vitro ant the cycle of cells were stable.4) Epidermal stem cells and follicle stem cells both can be used as seed cells for tissue engineering skin, but the follicle stem cells have higher proliferative activities and differentiating potentials.Follicle stem cells have more advantages to build full-thickness engineered skin. |
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