Effects Of Y-27632 And HUC-MSCs Conditioned Medium On The Proliferation And Senescence Of Keratinocytes In Vitro

BackgroundAt present,one of the critical challenges in the treatment of severe burns is the lack of donor skin to repair the wound in time.In recent years,tissue engineering skin with keratinocytes as the seed cells provides a way to solve this problem.However,keratinocytes,the seed cells that make up tissue engineering skin,cannot proliferate quickly in vitro,limiting the development and clinical application of tissue engineering skin.Therefore,how to increase the proliferative capacity of keratinocytes in vitro has become a key point.It has been suggested that ROCK inhibitor can inhibit the apoptosis of human embryonic stem cells in the process of cell isolation.Subsequently,many scholars found that ROCK inhibitor Y-27632 can promote proliferation,delay senescence and inhibit differentiation of bone marrow mesenchymal stem cells,periodontal epithelial cells and corneal endothelial cells.And it can also enhance their paracrine ability to secrete a variety of cytokines.The results of our previous studies have shown that human umbilical cord mesenchymal stem cells(hUC-MSCs)can promote the proliferation of keratinocytes when they are co-cultured,and Y-27632 can promote the proliferation of hUC-MSCs and delay their senescence.Mesenchymal stem cell conditioned medium can also promote the proliferation of other cells and affect the differentiation potential of these cells.In vivo,Y-27632 and mesenchymal stem cell conditioned medium had a positive effect on wound healing.However,there is no report on the effect of Y-27632 and conditioned medium from hUC-MSCs in response to Y-27632 on the proliferation and senescence of keratinocytes isolated from the razor-thin graft of scalp.ObjectiveTo investigate the effects of Y-27632 on the proliferation,senescence and migration of human keratinocytes isolated from the razor-thin graft of scalp and cultured in serum-free medium,and the effects of the conditioned medium from hUC-MSCs pretreated with Y-27632 on the proliferation,senescence and migration of keratinocytes.And then to explore a more optimized method for proliferation of human keratinocytes in vitro.Methods1.Human keratinocytes isolated from the razor-thin graft of scalp,were cultured and passaged in DKSFM.Morphology and growth of keratinocytes were observed and cell biological identification was conducted.The cells were randomly divided into control group and Y-27632 group,and cultured respectively in DKSFM or DKSFM containing 5μM Y-27632.The cells in each group were observed under the microscope.The expression of cell markers CK14 and β1-integrin was detected by immunofluorescence.MTT assay was used to detect cell proliferation and the activity of cells.Cell senescence was detected by β-galactosidase staining.The migration ability of cells in each group was detected by scratch assays.2.Human umbilical cord mesenchymal stem cells were isolated from human umbilical cord.After hUC-MSCs reached 60-70%confluence,they were randomly divided into the hUC-MSCs-CM group and the hUC-MSCs-CMY group,which cultured in DMEM or DMEM with 5μM Y-27632.At 48h after culture,the culture supernatant was collected to prepare conditioned medium.The contents of EGF and VEGF in the conditioned medium were measured by ELISA.Keratinocytes were cultured with DMEM medium(control group)and two kinds of conditioned medium.The OD value was measured by MTT method to detect the proliferation ability and activity of keratinocytes.The cell migration ability of each group was detected by scratch assays.Cell senescence was detected by β-galactosidase staining.Results1.Human keratinocytes with proliferation activity and stemness were isolated from the razor-thin graft of scalp and expressed CK14 and β1-integrin.After hUC-MSCs reached 30-40%confluence,if culture medium was replaced every day,KC would senesce slowly in vitro,and when culture medium was replaced every 2-3 days,KC would senesce quickly after passage 3,which is not adverse to rapid amplification of KC.Y-27632 can promote KC adherence during KC isolation and culture.After treatment with Y-27632,the morphology of human keratinocytes did not change significantly.(1)Y-27632 can promote the proliferation of keratinocytes.The OD value in Y-27632 group was significantly higher than that in control group,which confirmed that the proliferation ability and activity of KC in Y-27632 group were higher than those in control group.(2)Y-27632 can alleviate the senescence of keratinocytes.The proportion of β-Galactosidase positive cells in the control group is(20.719±4.405)%,and that in Y-27632 group is(12.424±1.043)%only.(3)Y-27632 can accelerate the cell migration.Wound closure was faster in Y-27632 group than in the control group.2.Human umbilical cord mesenchymal stem cells were isolated from human umbilical cord.(1)The concentration of supernatant EGF and VEGF in hUC-MSCs-CMY group is higher than that in hUC-MSCs-CM group.(2)HUC-MSCs conditioned medium can promote the proliferation of keratinocytes.MTT results showed that the OD values in hUC-MSCs-CM group and hUC-MSCs-CMY group were higher than that in the control group without conditioned medium.Furthermore,the OD values in hUC-MSCs-CMY group were higher than that in hUC-MSCs-CM group.(3)HUC-MSCs conditioned medium can alleviate keratinocyte senescence,the proportion of β-Galactosidase positive cells in the control group is(21.936±5.260)%,while another two group were(16.684±1.194)%and(13.947±3.773)%respectively.(4)HUC-MSCs conditioned medium could accelerate the KC migration,scratch wound closure was faster in hUC-MSCs-CM group and hUC-MSCs-CMY group than the control group.And hUC-MSCs conditioned medium pretreated by Y-27632 has a stronger effect.Conclusion1.Human keratinocytes with proliferation activity and stemness were isolated from the razor-thin graft of scalp and expressed CK14 and β1-integrin.Y-27632 can promote KC adherence during KC isolation and culture.After treatment with Y-27632,the morphology of human keratinocytes did not change significantly.2.ROCK inhibitor Y-27632 can significantly promote the proliferation and migration of human keratinocytes,and alleviate its senescence in serum-free medium and without feeder cells.3.Y-27632 can stimulate hUC-MSCs to release EGF and VEGF.Conditioned medium from hUC-MSCs can also promote the proliferation and migration of human keratinocytes,and alleviate its senescence.Moreover,Y-27632 can significantly improve the effect of hUC-MSCs conditioned medium on proliferation and migration of keratinocytes.