| Objective: To culture and obtain human umbilical cord mesenchymal stem cells(h UMSCs), detect its surface markers and analyze the conditions and functions of edavarone inducing h UMSCs to differentiate into neuron-like cells in different concentrations as well its cell-morphological changes. This study is aimed at providing primary theoretical support for the further application of h UMSCs to the treatment of nervous system diseases and experimental support for in vivo study in the future.Methods: The human umbilical cord was obtained from full-term caesarean section patients and rinsed thoroughly by D-Hank’s after being removed of the umbilical artery and umbilical vein. Then the umbilical cord mesenchymal tissue was cut into size of 1mm3 and digested by 0.2% collagenaseⅡ before put into the culture bottle containing 2ng/ml EGF, 20% FBS, 25 m M L-Glu, 100U/ml penicillin and 100μg/ml streptomycin mixture. Next observe the morphology and growth tendency of the primary cells. Replacing half of the culture medium after 24 hours, and replaced all of them every 3 days. When the cellular confusion achieved 80%-90%, added trypsin(trypsin 0.25%–EDTA 0.2g / L) to digest the adherent cells into single cell, and passaged.Flow cytometry was used to monitor the expression of surface antigen profile, including CD105, CD90, CD73, CD19, CD34, CD45, CD11 b as well as the histocomp-atibility antigens HLA-DR(MHC-II). Ig G1-PE and Ig G1-FITC were used as isotype control.The primary h UMSCs were passaged by a ratio of 1:1 and marked as P1. We observed their morphological changes under inverted phase contrast microscope and continued to passage by a ratio of 1:2 or 1:3 once adherent cells reached approximately 90% confluence. At the mean time, drew the cell growth curve. The 4th generation of h UMSCs which were in logarithmic growth phase were inoculated to 6 well cell culture cluster and 25 cm2 culture bottles and nurtured by full-quality of culture medium. When the cellular fusion rate reached 70~80%, we randomly combined one 6 well cell culture cluster and three culture bottles as a group and there were Group A, Group B, Group C and Group D, the first three of which were experimental groups while the last one was the control group. The first three groups were successively infused with Edaravone inductive liquid at the concentration of 75 mg/L, 150 mg/L and 300 mg/L. All the inductive liquid was confected by L-DMEM culture medium and in each group, every hole of 6 well cell culture cluster contained 3ml inductive liquid and every culture bottle contained 5ml. Group D was control group which contained L-DMEM culture medium only. Then we put all the groups in CO2 incubator with the conditions of culture same as before for 24 more hours and observed the cell morphological changes with inverted phase contrast microscope. It turned out that each group respectively discarded the inductive liquid after 2, 4, 6, 12 and 24 hours. We applied the methods of immunocytochemical staining, Western blot and RT-PCR to detect the expression of NSE, Nestin, GFAP and MAP-2 of the cells in each group and calculate the cell positive percentage. The result was expressed as X ±s.Results: After 12 hours’ inoculation, 60% of the most primary cells were adherent and grew into diamond, dot and long-spindle shapes. As time passed by, the amount of cells gradually increased and the cells all grew into more uniform long-spindle shape with high refraction. When the cellular confusion rate reached 90%, the cells showed radiated or swirling growth under low-magnification microscope. When the cells completely confused, subcultured them at the ratio of 1:2, and they could keep exuberant proliferation capacity till P15 th generation.Flow cytometry analysis of cultured P3th、P5th、P10th generration h UMSCs demonstrated the phenotype, CD73,CD90,CD105 but not CD11 b,CD1,CD34, CD45 and HLA-DR.After one hour’s induction, sporadic cells in Group A shrank into long-ellipse or round shape with increased refractivity and one or two lathy protuberances,these cells can be regard as Neuron-like Cell.After four hours, 20% of the cells had changed with more and longer protuberances and furcations. After six hours the changed cells accounted for 40% and in 12 th hours 50% cells had changed with the protuberances intermingling with each other into web form. After 24 hours, the amount of neutron-like cells reached its highest scale and only few cells hadn’t changed. We could observe a small amount of cell floating in the inductive liquid. As for Group B, obvious change could be detected after one hour with 40% cells turning into neutron-like cells. In the 4th hour, 70% of the cells had changed and 12 hours later the amount of the changed cells reached its peak meanwhile a few cells began to fall off. In Group C, a great amount of cells had changed only after one hour and the number peaked in the fourth hour. The rate remained high in the next twenty hours with few cast-off cells. Group D was the control group which had no obvious reaction in the 24 hours. In the 12 th hour, the experimental groups showed that the expression of the NSE is 54.03±1.76%、80.87±2.03%、90.97±4.06% and the expression of the Nestin is 59.64±2.38%、81.70±3.08% 、 92.98±3.04%,as well as the expression of the GFAP is 53.53±2.93%、78.26±4.17%、88.27±2.36%while the MAP-2 was negative. Group C(the concentration is 300mg/L) showed the highest and steadiest positive expression rate. Focusing on the cells of Group C, we observed the Mrna expression of specific markers for neurons as well as GAPDG respectively in 0th hour, 2nd hour, 4th hour, 6th hour, 12 th hour and 24 th hour and calculated the relative transcript level. The m RNA expression of GFAP is 0、0、0.238±0.014、0.298±0.010、0.504±0.696、0.597±0.054 in proper order;The m RNA expression of NSE is 0、0.275±0.020、0.499±0.021、0.877±0.022、0.859±0.018、0.438±0.016 in proper order; The m RNA expression of Nestin is 0.244±0.009 、 0.654±0.025 、 0.794±0.041 、 0.915±0.049 、 0.898±0.044 、0.408±0.012 in proper order. There is no expression of MAP-2 before and after induction and all the groups showed high expression of GAPDH in every time period. Then we observed the protein expression of specific markers for neurons as well as GAPDG respectively in each point of time and calculated the relative transcript level. The protein expression of GFAP is 0、0、0.203±0.021、0.221±0.023、0.353±0.022、0.746±0.037 in proper order;The protein expression of NSE is 0、0.323±0.038、0.403±0.026、0.557±0.021、0.553±0.025、0.214±0.030 in proper order; The protein expression of Nestin is 0.385±0.014 、 0.507±0.017 、 0.842±0.008 、 0.861±0.007 、 0.886±0.018 、0.466±0.010 in proper order. There is no expression of MAP-2 before and after induction and all the groups showed high expression of GAPDH in every time period.Conclusions: h UCMSCs can be isolated and purified from umbilical cord and be cultured and passaged steadily in vitro with the ability of keeping exuberant proliferation capacity till P15 th generation. All generations cells co-expressed CD105, CD90 and CD73, but not CD11 b, CD34, CD19, CD45, and histocompatibility antigens HLA-DR(MHC-II). The edavarone can induce h UMSCs to differentiate into neuron-like cells effectively at a certain concentration and the best concentration was 300mg/ L. After induction, the Neuron-specific enolase(NSE)and neurofilament protein(NF-H)of the neuron-like cells were positive but the glial fibrillary acidic protein 2(GFAP-2) was negative. |
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