Effects Of Pathogen-associated Molecular Patterns On Differentiation Of Epidermal Cells And Sebocytes In Vitro

Part I: Effects of pathogen-associated molecular patterns on proliferation and differentiation of epidermal cells Background: Skin forms the first barrier outside the human body.As a part of skin microecology,skin microorganisms are critical to the structure and physiology of skin.Microbes can promote the self-regulation of our skin,but the specific mechanism is unclear.In this section,we used human keratinocytes Ha Ca T and isolated 3D-Sebo Skin model to study the effects of Pathogen-associated molecular patterns(PAMPs)on the proliferation and differentiation of human epidermal cells in vitro,and to explore the effects of skin microorganisms on skin physiological and pathological functions.Methods: Western-blot was used to detect the expression of IVL and LOR on Ha Ca T,after stimulated by lipopolysaccharide(LPS)5 mg/ml,lipoteichoic acid(LTA)5 mg/ml and peptidoglycan(PGN)10 mg/ml;then we used human skin co-cultured with immortalized SZ95 human sebocytes which called 3D-Sebo Skin model,treated with different concentrations of PAMPs(2?20?200 ?g/ml LPS,LTA and PGN).After 7days culture,the epidermal skin area was calculated by using the Photo Shop software.The proliferation and differentiation of epidermal cells were observed by immunohistochemical labeling with cell proliferation nuclear antigen Ki67 and cytokeratin 10(CK10).The Image J software and the Image-Pro Plus software were respectively used to calculate the stained area and the integrated optical density(IOD)of each image.Results: Under the stimulation with LPS,LTA and PGN,a dose-depended increase of epidermal thickness was detected(P <0.01),which achieved maximum effects at 200 ?g/ml for LPS and LTA(P <0.01)and 20 ?g/ml for PGN(P <0.001).In addition,the expressions of Ki67 in the basal epidermal layer and of CK10 at the suprabasal epidermis were also enhanced.Conclusion: PAMPs promote the proliferation and differentiation of epidermal cell in a dose-dependent manner in vitro,which indicated that the skin microorganisms may be play an important role in the formation of skin epidermis and barrier.Part II:Effects of pathogen-associated molecular patterns on lipogenesis of sebocytes in 3D-Sebo Skin model Background: In the first part,we explored the role of PAMPs on the epidermal cells.In the second part,we further explored the effects of PAMPs on the differentiation and lipogenesis of human sebocytes in vitro to evaluate the effects of microorganisms on the morphology and function of sebocytes.Methods: 3D-Sebo Skin model was treated with different concentrations of PAMPs(2,20,200 ?g/ml LPS,LTA,PGN).After 7 days of culture,we used HE-stained sections to observe the changes in the structure of sebaceous glands;frozen sections were oil-red stained to observe the lipid secretion of sebocytes under the stimulation of different concentrations of PAMPs.Results: Sebum secretion of sebocytes in each experimental group increased under the stimulation of LPS,LTA and PGN at 2 ?g/ml(P<0.05);when the concentration was increased to 20 ?g/ml,the volume of sebaceous glands and lipid secretion decreased(P <0.05);When the stimulation concentration was increased to 200 ?g/ml,the sebaceous glands atrophied and lipid secretion was further reduced(P <0.01).At the same time,abnormal keratinization of sebaceous glands could be observed in HE-stained sections.Conclusion: PAMPs can regulate the differentiation and lipogenesis of sebocytes,which may be one of the mechanisms that the sebaceous glands realize self-regulating.