| Objective:To study the neuron-like differentiatation of BMSCs in vitro and to examine the Nestin expression. To study the tracking characteristics of BMSCs toward hypoxic-ischemic brain tissue.Methods:Twenty neonatal rats of 7 days old were randomly divided into control group and hypxic-ischemic group. Each group had 10 rats. Neonatal rats in the hypxic-ischemic group were induced HIBD by left carotid artery ligation and hypoxia exposure,in the control group the left carotid artery was seperated without ligation and hypoxia exposure. After 24h all the rats were killed.then the supernatant of brain tissue homogenate was made.Rat BMSCs were cultured by a whole bone marrow method. Bone marrow cells were flushed out from bone marrow cavity of femurs with DMEM and inoculated into plastic culture dishes. Then they were cultured under conditions of 37℃,5%CO2 and saturated humidity in a incubator. Monolayer adherent cells were passaged at ratio of 1:2 with 0.25% trypsin digestion solution when they reached 90% confluence. Afterwards the proliferating cells were always passaged at ratio of 1:2 when they were 80-90% confluent.P5 cells were digested and collected when they reached 90% confluence.1×106 cells were suspended in 100μl PBS and FITC conjugated anti-CD29, anti-CD31, anti-CD44, anti-CD45, anti-CD90 antibodies were added separately at their proper concentration. Staining reaction lasted for 30 min in dark. CD34 staining need 30min reaction of both anti-CD34 antibody and FITC conjugated second antibody one by one. At last the cells were suspended in 600μl 1% paraformaldehyde and examined by flow cytometry.In vivo, hypoxic-ischemic brain damage model of neonatal rat was established, followed by coculture for 24h of BMSCs with supernatant of brain tissue homogenate.The expressions of Nestin was detected after 24h by immunocyto-chemistry.24-well Transwell polycarbonate inserts with a pore size of 8μm coated with 60ul Matrigel were used in vitro. BMSCs (5×104 cells per well) were placed in the upper chamber of a Transwell system.Transwell coculture of BMSCs with supernatant of brain tissue supernatant for 24 hours. The number of BMSCs migrating to the lower chamber was mogenate was performed.In the HIBD group the commixture of supernatant of HIBD brain tissue homogenate (100μL) and DMEM(FBS15%) were placed in the lower chamber; in the normal group, supernatant of normal brain tissue homogenate and DMEM (FBS15%) were placed in the lower chamber; the DMEM+FBS group without supernatant,the contrast group only DMEM.In addition, BMSCs migration to the lower side of the insert was counted using light microscope.Results:A few cells became adherent 24h after inoculation. They continued to proliferate until many cells reached confluence 10-14 days after inoculation. P5 cells became homogenous and had fibroblast-like morphology. During long term culture senescent cells, ultra proliferating cells and multinucleated giant cells were occasionally found. Flow cytometry analysis showed that rat BMSCs expressed CD29(99.83%), CD44 (99.77%) and CD90(99.86%), but not CD31 (0.83%), CD34(1.78%) or CD45(2.90%).Bone mesenchymal stem cells body contracted into round or spindle shape.24h later,neuron-like cells could be seen. The immunocytochemical method showed the expression of Nestin. The expression of Nestin of BMSCs cultured in the medium with brain tissue extracts of HIBD rats were higher than those of BMSCs cultured with brain tissue extracts of normal rats(P<0.01).In the transwell migration system BMSCs displayed tropism toward normal brain tissue (20.18±4.86)%, hypoxic-ischemic brain tissue (39.42±2.81)%,and have higher migratory ratio toward brain tissue extracts of HIBD rats.Conclusion:BMSCs can be induced to differentiated into neuron-like cells and express characteristic marker for neurons. The BMSCs display strong tropism toward hypoxic-ischemic brain tissue.
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