| Objective:1.To explore the optimal condition of isolation and cultivation of mesenchymal stem cells(MSCs) derived from human umbilical cord blood.2.To explore the optimal condition of MSCs derived from human umbilical cord blood directed differentiation into neural cells.Methods:1.Umbilical cord blood were collected from full term deliveries and preterm infant.The umbilical cord blood mononuclear cells(MNCs) were isolated by density gradient centrifugation with lymphocyte separation medium.Different condition influence on the growth of MSCs derived from human umbilical cord blood were analyzed.(1) the different culture medium:L-DMEM,DMEM/F12 and MesencultTM medium;(2) gestational age:full term deliveries and preterm infant;(3) planting density:5×105/cm2,1×106/cm2,5×106/cm2,1×107/cm2;(4) the time of the first medium changing:2d,3d,4d,5d,6d,7d.The biological characteristics of MSCs derived from human umbilical cord blood were observed by detecting the cell surface marker of CD29,CD34,CD44 and CD90 by immunofluorescence method. 2.The third passage of MSCs derived from human umbilical cord blood were detached with trypsin-EDTA.We induced MSCs when they were approximately 80% confluent by three methods:(1) the group of chemical induction:3mmol/Lβ-ME+ 20g/L DMSO+200mmol/L BHA;(2) the group of growth factor induction:20ng/ml EGF+20ng/ml bFGF;(3) the group of tanshinol+growth factor induction:3% Xiangdan injection+20ng/m lEGF+20ng/ml bFGF.Detected neural morphological characteristics(NeuN,β-TubulinⅢ) and glial fibrillary acid protein(GFAP) by the immuocytochemistry staining and(or) immunofluorescence method before and after induction.Result:1.When the other conditions were the same,umbilical cord blood from full term deliveries,MesencultTM medium was the best medium;5×106/cm2 was the best planting density;the best first medium changing was on the seventh day at primary culture.The achievement ratio of umbilical cord blood from preterm infant was higher than full term deliveries when the other conditions were the same.MSCs derived from human umbilical cord blood expressed the surface markers of MSCs such as CD29,CD44,CD90,et al,but not antigens of haemopoietic stem cell such as CD34.2.Neural cell marker of NeuN,β-TubulinⅢand GFAP of the group of chemical induction,growth factor induction and tanshinol+growth factor induction were negative before induction by the immuocyto -chemistry staining.After induction the rate of NeuN,β-TubulinⅢand GFAP positive cell respectively:the group of chemical induction were(71.6±4.6)%,(73.8±2.3)%and(12.6±2.0)%;the group of growth factor induction were(43.6±3.9)%,(54.6±4.1)%and(31.8±5.0)%;the group of tanshinol+growth factor induction were(78.6±4.5)%,(82.6±4.3)%and(15.6±3.6)%.Neural cell marker ofβ-TubulinⅢof three groups were negative before induction by the immunofluorescence,and after induction the rate ofβ-TubulinⅢpositive cell respectively:(72.6±2.7)%,(54.5±1.9)%and(79.8±3.0)%.Conclusion:1.When the other conditions were the same,umbilical cord blood from full term deliveries,MesencultTM medium was the best medium;5×106/cm2 was the best planting density;the best first medium changing was on the seventh day at primary culture,The achievement ratio of umbilical cord blood from preterm infant was higher than full term deliveries when the other conditions were the same.2.The combination of tanshinol and growth factor was able to effectively induced mesenchymal stem cells derived from human umbilical cord blood into neural cells.
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