Primary Study On Construction Of Novel Engineered Human Skin In Which HaCaT Was Used As Seed Cell

Background: One of the most important burn cure methods is how to heal wound. Quick and effect cover on wounds is vital for the patients suffering from burn.Much investigation have focussed on covering for wound surface in these days.It include construction and application of engineered human skin in which seed cells and many kind of acelluar dermal matrix(ADM) were used.As a promising select,keratinocyte stem cells(KSC) were used in new engineered human skin.As far as KSC was concerned,the investigations include its separation , purification , molecule identification and multiplication control, etc.However,some questions were suspended,which restrict the development of engineered human skin.The cell line HaCaT had been reported the spontaneous transformation in vitro of human keratinocytes from histologically normal adult body skin.That the higher genetic stability and immortalization are its characters.But there were not reported about stem cell phenotypic analysis of human immortal keratinocyte line HaCaT by flow cytometry.If HaCaT cells have similar biological characters with keratinocytes,we can use it instead of KSCs.So we selected served several stem cell phenotypes of human immortal skin keratinocyte line (HaCaT) ,cell cycle and atoptosis as a supply to study HaCaT by flow cytometry. The xenogeneic ADM was made from the reticular layer of rat(SD) dermis and prepared by physical and chemical methods.On these bases,we can try to construct engineered human skin in which HaCaT was used as seed cells.Objectives1. To observe several stem cell phenotypes of human immortal skin keratinocyte line (HaCaT) and provide experimental evidences for applying these cells to engineered human skin and gene modification.2. The xenogeneic ADM was made from the reticular layer of rat (SD) dermis.3.Try to construct engineered human skin in which HaCaT was used as seed cell. Methods1. The HaCaT cells were cultured in defined keratinocyte-serum free medium (DK-SFM) and harvested in log phase growth period. The relevant stem cell like phenotypes were labeled by immunofluorescence., ie. direct dual labeling for CD49f and CD71, direct singular labeling for CD29, indirect labeling for CD133 and K19. The percentages of these phenotypes were analyzed for flow cytometry.2. ADM was prepared by physical and chemical methods;And its result was evaluated by HE staining.3. HaCaT reached a conflunent monolayer after seeding,which was detected by MTT method; Contrast of effect before and after removel of cells or monolayer or even mutilayer on ADM by HE staining.4. To observe the cell grow state after the spread of HaCaT for one and five days through scanning electron microscope.Results1. The phenotypic characteristics of human immortal skin keratinocytes (HaCaT) indicate that they have higher percentages of keratinocyte stem cell and much stronger capacities of adhesion and proliferation than normal human keratinocytes. These suggest that HaCaT cells would be used as a substitute of keratinocyte stem cells for fabricating engineered skin and manipulating epidermis relevant genes.2. It is ideal ADM after removel of cells with its tissue looseness;There were many natural microbores on which cells can easily grow.3. The time of monolayer formation is 5 days if HaCaT cells were seeded as 2x10~5/cm~2 ,which was observed on cell growth curve by means of MTT.4.In the same methed,HaCaT spread fully on ADM in one and five days,which were detected through scanning electron microscope.The growth of HaCaT was observed after they were seeded for one,five and ten days by HE staining.Conclusion1. The phenotypic characteristics of human immortal skin keratinocytes (HaCaT) indicate that they have higher percentages of keratinocyte stem cell and much stronger capacities of adhesion and proliferation than normal human keratinocytes. These suggest that HaCaT cells would be used as a substitute of keratinocyte stem cells for fabricating engineered skin and manipulating epidermis relevant genes.2. It is ideal ADM (SD rat) that made by improved methed for this investigation.3. It is sure that we can construct new engineered human skin in which HaCaT was used as seed cell on ADM from SD rat.