Experimental Study On The Effect Of Y-27632 On The Proliferation And Senescence Of Human Umbilical Cord Mesenchymal Stem Cells Under Serum-free Condition

BackgroundThe key to the treatment of severe burn patients is effective wound coverage and wound healing.However,patients with extensive burns often face the problem of insufficient autologous skin,which is expected to be solved by constructing tissue engineered skin.In recent years,mesenchymal stem cells（MSCs）based cell therapy and the construction of tissue-engineered skin are expected to become effective methods to solve this problem.However,cell therapy and the construction of tissue-engineered skin need a large number of mesenchymal stem cells.Therefore,the rapid expansion of mesenchymal stem cells is of great significance for cell therapy and the construction of tissue-engineered skin.Mesenchymal stem cells are derived from a variety of human tissues including bone marrow,adipose tissue,and umbilical cord.Among them,human umbilical cord mesenchymal stem cells（hUC-MSCs）derived from human umbilical cord tissue Wharton’s jelly have higher proliferation capacity and lower immunogen compared with bone marrow or adipose-derived MSCs,can be used as one of the best seed cells for tissue engineering skin.HUC-MSCs cultured in serum-free medium are believed to maintain stable MSCs’morphology,surface markers expression,and high proliferation potential,and avoid the risk of exogenous pathogenic factors and immune rejection caused by the addition of animal serum,which help us obtain safer and more effective cell products.ROCK inhibitors have high safety and have been widely used in scientific research and clinical medicine.Y-27632 has been proven to promote the expansion rate of a variety of cells including human embryonic stem cells（hESCs）,bone marrow mesenchymal stem cells（BM-MSCs）and epithelial cells and delay the process of cell senescence in vitro.It is an effective method to increase stem cell proliferation.Up to now,there is no related study about the effect of ROCK inhibitor Y-27632 on the proliferation and senescence of hUC-MSCs in China and abroad.ObjectiveTo study the effect of Y-27632 on the proliferation and senescence of hUC-MSCs cultured in serum-free medium,and to preliminarily explore its mechanism,and find a method that can rapidly expand hUC-MSCs in vitro.MethodsHUC-MSCs were isolated from umbilical cord tissues and then sub-cultured in serum-free medium.The cells were biologically identified（observing the morphology and growth of cells,detecting the surface markers CD90,CD 105,CD34 and HLA-DR of hUC-MSCs by flow cytometry,and detecting their osteogenesis,adipogenesis and chondrogenic differentiation potential）.The P3 hUC-MSCs were randomly divided into two groups:experimental group and control group.10 μM Y-27632 was added to the culture medium of the experimental group while the same amount of DMSO was added to the control one（changing the solution and adding Y-27632/DMSO solution once every 48 hours）.The total number of cells in each passage was calculated,the cell proliferation curve was drawn,the cell proliferation ability was detected by MTT method,the cell cycle of hUC-MSCs was detected,the cell proliferation index was evaluated,and the concentrations of epidermal growth factor（EGF）and vascular endothelial growth factor（VEGF）in cell supernatant were detected by ELISA.In the end,the cells were stained by β-galactosidase and the p16 and p21 proteins were detected by Western Blot to evaluate the cell senescence.Results1.In this study,hUC-MSCs were isolated from Wharton’s jelly of umbilical cord.After being cultured in serum-free medium,hUC-MSCs had good proliferation ability in vitro and triline differentiation potential,and could stably express cell markers,which met the MSC standard proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society of Cell Therapy.2.In different cell passage periods,the total number of cells in the experimental group was significantly higher than that in the control group at the same period（P<0.05）,In each generation of samples,the fold increase value of experimental group was higher than that of control group（P<0.05）.In P3,P8 and P12 cells,the proliferation-promoting effect of Y-27632 was more significant with the increase of cell passage times（P<0.05）.3.Y-27632 accelerated the cell cycle process of hUC-MSCs,and increased the proportion of cells in S phase and G2/M phase（P<0.01）.4.Y-27632 promoted the secretion of EGF and VEGF by hUC-MSCs（P<0.05）.5.Y-27632 decreased the proportion of β-galactosidase positive cells in hUC-MSCs（P<0.05）,and suppressed the expression of p16 and p21.ConclusionY-27632 promoted the proliferation and inhibited the senescence of hUC-MSCs under serum-free condition.