| Background and purpose:The skin is the largest organ in the body. It consists of the epidermis, derived from the ectoderm, and the dermis of mesodermal origin. The skin serves a number of vital physiological functions including thermoregulation, maintenance of fluid balance, and protection against a variety of environmental assaults. In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. The regeneration of the epidermis is sustained by epidermal stem cell, which also participates in the repair of the skin after injuries. However, the extensive damage caused by massive burns still constitutes a major surgical challenge for surgeons. Ideally, this would be best accomplished with full thickness skin graft or flap. However, donor site availability limits full thickness skin use to only select cases and applications. Stem cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues with cultured cells. The clinical use of cultured skin substitutes for wound has reduced the morbidity of donor site and avoid the restriction of skin resources. Stem cell is "seed cell" of tissue engineered skin. However, due to the lack of stem cells from autologous epidermal, large amplification of cells cultured in vitro is a key problem. This hindered the clinical application of tissue-engineered skin.Cell growth factor is a class of biologically active substances that can regulate cell growth and development. Cell growth factors regulate cell proliferation and differentiation by autocrine and paracrine. In this study, to change the ESC growth of micro-environment affect the proliferation and the maintenance of the phenotype of ESC through bFGF and IGF-1and TGFa added to the medium.Methods:MaterialsThe skins were obtained from16to22-year-old circumcision patients, department of Urology, Nanjing Drum Tower Hospital. Patients have no urinary tract infections and other diseases. Patients are informed consent.Methods of Part1:1. The isolation and culture of the primary stem cell:to obtain the primary epidermal stem cell with two step enzyme digestion method and type IV collagen coated chosen methods.2. Identification of the cultured cell:to take good growth of cells for cell slide. When cells reached an appropriate density, in accordance with the Boster ABC immunohistochemical staining kit instructions identify the cell by immunohistochemical staining, then DAB color.3. The IGF-1, bFGF, and TGFa were divided into group A, group B and group C according to the respective concentration; corresponding adding cytokines Ong/ml, lOng/ml and20ng/ml. Five days later, determination of wavelength of490nm absorbance A value of MTT assay was used.Methods of Part2:1. Epidermal stem cells divided into group A (K-SFM), group B (K-SFM+10ng/ml, bFGF), group C (K-SFM+10ng/ml of IGF-1) and group D (K-SFM+1Ong/ml of TGFa) were cultured.2. The cell growth curve determination:the epidermal stem cells were seeded in 96-well plates with2×103density, added the appropriate medium, and measured by MTT assay under the absorbance wavelength of490nm that calculate the average, continuous count7days.3. Determination of cell colony formation rate:200/well cells were seeded to24-well plates, divided into A-E group, added the corresponding medium, and cultured for one week. Then record the number of clones.4. Determination of cell proliferation:to observe the culture of the fifth day of cell proliferation (each of different cytokines, each determination of the6holes) by CCK-8detection (refer to CCK-8kit instructions)5. Cell cycle analysis:cell suspension was prepared by0.25%trypsin and0.02%EDTA to digest the cells, the cell suspension was washed two times with PBS, fixed overnight in70%ethanol,4℃. Then using flow cytometry to detect the cell cycle.Results:Results of Part1:1. The primary epidermal stem cell can be obtained with two step enzyme digestion method and type IV collagen coated chosen methods.2. Immunohistochemical observations:β1integri, CK19, immunocytochemical staining showed positive expression.3. Three cytokines act on cells to detect the A value increased, but not the cytokine concentration the bigger the better. There were not significant difference between10ng/ml and20ng/ml(P>0.05).Results of Part2:1. Four groups of cell growth curve showed inverted "S" shape into the multiplication of cells in the first five days. The ESC of the group that did not add cytokines reached the peak at nine days. The ESC of the group than add cytokines entered into the plateau at11days.2. Group B, group C and group D cell clones rates were significantly higher than group A (P<0.05). Group D cell clones rates were significantly higher than Group B and group C(P<0.05)(A:10.0±0.71%, B:14.0±0.89%, C:13.7±0.61%, D:16.4±0.92%).3. Group B, group C and group D absorbance A values were significantly higher than group A (P<0.05), while group D was higher than group B and group C (P <0.05).There were not significant difference between group B and group C (P>0.05)(A:0.502±0.006, B:0.603±0.013, C:0.584±0.015, D:0.677±0.027).4. The proportion of G0/G1cells of group B and group C were significantly higher than in group A and group D (P<0.05), while no significant difference between group B and group C. There were not significant difference between group A and group D (P>0.05)(A:55.6±2.1, B:62.8±2.4, C:63.3±2.3, D:56.0±1.0).Conclusions:1. Two enzyme digestion method can obtain the epidermal stem cells.2. bFGF and IGF-1and TGFa can promote the proliferation of epidermal stem cells.Three cytokines in the culture medium of the optimum concentration are lOng/ml.3. bFGF and IGF-1can maintain the epidermal stem cell phenotype. |
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