Research Of In Vitro Cultivation Of Human Bone Marrow Mesenchymal Stem Cells And Differentiation To Sensory Neurons

Chapter Ⅰ:Isolation, Expansion and Identification of hBMSCs Objective:To establish a reliable procedure to gain numerous hBMSCs and identify the cells, lay foundation for subsequent experiments.Methods:The adherence method and density gradient centrifugation method were used to isolate and expand hBMSCs. The colony forming time, number of colonies and growth curve of P3hBMSCs were compared. Flow cytometry was used to assay CD33,CD44and CD45expression. The osteogenic and adipogenic culture medium were used to induce hBMSCs differentiatated to osteoblasts and adipocytes, Alizarin red staining and Oil red O staining were used to stain calcium nodules and lipid droplets.Results:Both adherence method and density gradient centrifugation method can isolate and expand hBMSCs. The colony forming time of adherence method is shorter compared with gradient centrifugation methold, The number of colonies of adherence method is more compared with gradient centrifugation methold. P3hBMSCs proliferation ability of adherence method is more vigorous than density gradient centrifugation. Flow cytometry showed the P3hBMSCs CD34positive rate is2.18%, CD44positive rate is98.14%, CD45positive rate is0.41%. The calcium nodules and lipid droplets can be stained by Alizarin red and Oil red O stainning after osteogenic and adipogenic induction.Conclusions:Both adherence method and density gradient centrifugation method can be used to isolate and expand hBMSCs. The adherence method have advantage over density gradient centrifugation method. Objective: To investigate the feasibility of hBMSCs differentiated to sensory neurons.Methods:RT-PCR was used to detect the expression of RAR α, RAR β, RAR γ, PTCH1, Smoothened, Gli1, Gli2. Gli3Frizzled,β-catenin in undifferentiated hBMSCs.Induce hBMSCs in RA group, Shh group, Wntl group, RA+Shh group, RA+Wntl group, Shh+Wntl group and RA+Shh+Wntl group. Observe morphological changes after induction, measure β-tubulin3, MAP-2, Tau, GATA-3, Calretinin, Brn3a RNA expression level in induction group and undifferentiated hBMSCs by real-time quantitative PCR, detect GATA-3, Calretinin, Brn3a protein expression by immunofluorescence.Results:RAR a, RARβ, RARγ, PTCH1, Smoothened, Gli1, Gli2、Gli3、 Frizzled, β-catenin are expressed in undifferentiated hBMSCs at different level. There are neuron-like cells in all induction group, Immunofluorescence assay showed GATA3positive cells in RA+Shh group and RA+Shh+Wntl group, Calretinin positive cells in RA+Shh group and RA+Shh+Wntl group, Brn3a positive cells in Wntl group, RA+Wnt1group, Shh+Wntl group and RA+Shh+Wntl group. Real-time quantitative PCR showed β-tubulin3, MAP-2, Tau were significantly upregulated in all induction group, no difference between induction group. GATA3was upregulated24.4fold in RA+Shh group,68.1fold in RA+Shh+Wntl group after induction. Calretinin was upregulated339.2fold in RA+Shh group,572.9fold in RA+Shh+Wntl group after induction. Brn3a was upregulated143.0fold in Wntl group,94.2fold in RA+Wntl group,132.3fold in Shh+Wntl group,424.3fold in RA+Shh+Wntl group.Conclusions:The RA, Shh and Wnt related genes are expressed in undifferentiated hBMSCs. GATA3and Calretinin can not be upregulated when induced with RA or Shh separately, but can be upregulated when induced with RA and Shh in combination. Brn3a can be upregulated by Wnt1. The in duction effect of RA+Shh+Wntl group was the best among all induction group.