| BackgroundAs the largest organ of the body and the first natural barrier, the skin iscomposed of the epidermis, the dermis, the subcutaneous tissue and its subsidiaries.Epidermal stem cells are the main function cells to maintain the metabolism of skin,and also the differentiation cradle of the skin and its appendages.Scar is the inevitable outcome in the healing process of the skin wound. Whenthere is wound in the skin, the Epidermal stem cells can be actively participate in thewound healing and the re-epithelialization, however the mechanism may be as follows:①When skin is trauma, with the repair signals startup, the epidermal stemcells start to proliferate and differentiate, then migration to the epidermis and heal thewound actively.②When the hair folliclestem cells around the wound edge receivedthe instructions to heal the wound, then the cells were activated and participated inthe healing.In recent years, with the research about the unique biological characteristics ofepidermal stem cell, people began to think about using the epidermal stem cells toprovide a new strategy for clinical treatment from the biological point. However, theepidermal stem cells in research are mainly from normal skin, and there are no reportsabout the epidermal stem cells from mature hypertrophic scars. In the plastic surgery,there were a lot of mature hypertrophic scar skin that was discarded, as a result, manyreusable precious resources were wasted. Therefore, the biological characteristics ofthe epidermal stem cells from mature hypertrophic scars were comparatively studiedwith the epidermal stem cells from normal skin, and as the result, the researchfoundation about the applications of the epidermal stem cells from maturehypertrophic scars in the clinic and experimental study was proposed.Object1．To comparatively study the biological characteristics of the epidermal stemcells from mature hypertrophic scars and normal skin, and to define the biologicalcharacteristics of the epidermal stem cells from mature hypertrophic scars.2．By to define the biological characteristics of the epidermal stem cells frommature hypertrophic scars, to find a new way for the prevention and treatment ofhypertrophic scars treatment;3．To provide a pre-research base for scientific research and clinical applicationof HS-ESCs.Method1．Organizational structure of the mature hypertrophic scars and normal skin wascompared by HE staining. 2．The expression of Keratin19, Integrin β1and Nucleoprotein p63of theepidermal stem cells from the mature hypertrophic scars and normal skin werecompared by immunohistochemistry staining, then in order to kown whether therewere epidermal stem cells in the mature hypertrophic scars.3．The epidermal stem cells were isolated from the mature hypertrophic scarsand normal skin by two-step enzymatic digestion, then the cells were rapidly sorted,purified, adhere to the dish by collagen-Ⅳ,and the cell morphology was observed.4．The growth characteristics of the primary cultured epidermal stem cells fromthe mature hypertrophic scars and normal skin were compared by CCK-8, and inorder to determine the growth characteristics of the primary cultured epidermal stemcells from the mature hypertrophic scars.5．The expression of keratin Keratin19, Integrin β1and Nucleoprotein p63inprimary cultured epidermal stem cells from mature hypertrophic scars and normalskin were tested by immunocytochemistry and in order to determine whetherepidermal stem cells from mature hypertrophic scars had the same biologicalcharacteristics with that from normal skin.6．The expression of keratin Keratin19, CD29and CD49f of the primarycultured epidermal stem cells were texted by flow cytometry and in order to examinethe percentages of positive cells from mature hypertrophic scars and normal skin.7．The expression of Keratin19, Integrin β1and Nucleoprotein p63in primarycultured epidermal stem cells from mature hypertrophic scars and normal skin weretested by Western-Blot technique to determine whether the HS-ESCs had the samebiological characteristics with NS-ESCs.8．The expression of the gene of Oct4and Nanog of the primary culturedepidermal stem cells were texted by real-time PCR technology to determine whetherthe HS-ESCs had the same biological characteristics with NS-ESCs.Results1．The cells in the epidermis of the mature hypertrophic scars and normal skinarranged neatly and structured by HE staining. And the expression of keratin19and integrin β1in the mature hypertrophic scars and normal skin were shown positivelyby immunohistochemistry, and there were no significant difference between themature hypertrophic scars and normal skin(P﹥0.05), so there were ESCs in themature hypertrophic scars.2．The morphology of primary cultured ESCs were looked like teardrop-shapedor oval, and arranged like cobblestone-like. The proliferation of primary culturedESCs was detected with CCK-8. The cell proliferation was slow in the1-2days, likeda logarithmic growth in the3-5days and flatten in the6-7days, and there was nostatistically significant difference between the two proliferative capacity of ESCs(P>0.05).3．The expression of keratin19, integrin β1and nucleoprotein p63were testedby immunocytochemistry, and the results showed no statistically significantdifference between the both ESCs (P>0.05). Flow cytometry was used to examine themarkers of integrin CD29, integrin CD49f and Keratin19of the both ESCs, and theresults of the percentage of positive cells showed that there were significantdifferences between the both ESCs (P<0.05).The protein expression of Keratin19,integrin β1and nucleoprotein p63were tested by Western Blot, and the result showedthere were no statistically significant difference (P>0.05). The genes of Oct4andNanog were tested by quantitative reverse transcription-polymerase chain reaction(qRT-PCR) and there is no statistically significant difference (P>0.05).Conclusion1．There were ESCs exist in mature hypertrophic scars.2．The ESCs were separated by two-step enzymatic digestion from maturehypertrophic scars and then enriched and adhered using collagen-Ⅳ. And the ESCsfrom mature hypertrophic scars had the same biological characteristics with that fromnormal skin, but the quantity of ESCs in the mature hypertrophic scars were less thanthan in normal skin.3． The ESCs of mature hypertrophic scars have the same biologicalcharacteristics with the ESCs of normal skin, and the ESCs of mature hypertrophic scars can replace the ESCs of normal skin are studied in the scientific research, forexample, as the cell seeds of artificial skin being used in the tissue engineeringresearch; And also being used in the clinical applying research, for example, as thethe source of skin-forming and skin-updating, ESCs of mature hypertrophic scars canbe used to repair the refractory wound or large skin defect, that not only avoiding thewaste of valuable resources containing a lot of ESCs, but also reduce the injury to thepatient. |
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