| Background: Spinal cord injury (SCI) is a frequently encountered disease of orthopedics. Respecting the severe result which the inconvertible lesion brought to individual and family, the therapy of functional restoration after SCI has being explored by medical scientists constantly, but few effect until nowadays. Because of nonreproducibility of the neural system, the method of transplantation with exogenous neural cell is a focus of SCI treatment. Neural precursor cell (NPC) consists of neural stem cell (NSC), and lineage-restricted precursor cell, and all of them have abilities to differentiate. So there will be a possibility to realize the recovery of the function of the injury neural system, if the NPCs which transplanted into the impaired spinal cord could differentiate to neurons and other construction of neural system.NSC is the most original cell of neural system, and possesses a high potentiality of differentiation. So it became the first target to investigate consequently. However some researches show NSCs transplanted into the injury spinal cord of adult rat only can differentiate into glial. The microenvironment of injury spinal cord does not support the differentiation to neuron of NSCs may be the reason. But another investigation indicates this microenvironment supports the survival and differentiation of neural lineage-restricted precursor cell and glial lineage-restricted precursor cell (NRP/GRP). So NRP/GRP become a new focus as the donator of cell transplantation.Suspension and adherent culture are most frequently used application in cultivations of NPCs. Under suspension culture, NPCs grew concentrately and showed typical morphology of neurospheres, proliferated robustly, and remained NSCs in major by purification gradually. While adherent culture means the adhesion the NPCs with culture flasks or plates which were after special handing. Proliferation of NPCs under adherent culture is inferior compared to suspension culture, but remaining lineage-restricted precursor cell in majoy after purification. Therefore , the adherent culture of NPCs was payed more attention to for the past few years. Until nowadays, the cell sourse utilized for transplantation by scholars abroad was aetio-generation of fetus rat spinal cord which was cultured for 3 days. And the time point is reference to our investigation.Objectives:1. To campare the proportion of survival, adherence, proliferation of NPCs from fetus rat spinal cord with different innoculation density, and of NSC, NRP, GRP before transplantation.2. Whether the NPCs in adherent culture could differentiate into mature neurocyte after induction.Methods: Spinal cord tissue of SD E13.5 day fetus rat was innoculated with differernt density, 1.25×104/cm2, 2.5×104/cm2, 5×104/cm2, 10×104/cm2, to the flasks and plates coated by laminin (20μg/ml) and polylysine (13.3μg/ml). After cultured for 3 days:1. Survival rate was detected by MTT, adherent rate was detected by counted number under high power lens, BrdU-labelled proliferating rate and celluar expression of Nestin, PSA-NCAM, and A2B5 was detected with FACS method.2. Culture solution was replaced with differential medium after 3 days, and kept for one week besides changing the medium per 3 days.Results:1. The means by MTT of the four differernt density from low to high were 0.2597,0.14535,0.076925,0.0474625;the rates of adherence were 96.673%,95.40%,94.173%,87.708%;the rates of BrdU-labelled cells and celluar expression of Nestin, PSA-NCAM, and A2B5 detected with FACS method were58.00%,37.45%,52.80%,41.62%;4.13%,1.04%,1.97%,2.27%;24.65%,40.75%,10.50%,8.65%;41.55%,39.52%,6.61%,12.88%。2. To induce the cells, innoculated with 2.5×104/cm2, by fetal bovine serum may become mature.The antibodies of NF-200, GFAP, Gal-C expreesed innegative.Conclusion: The rates of cell survival and adherence were degrade with the increase of innoculated density. The growth rate had no associativity with innoculated density in vitro. There is no variance in rate of neural stem cell, but the rate of lineage-restricted precursor cell in second group is the highest, exceeding 80%, and different with other groups (p<0.05). It suggests that the suitable innoculation density of NPC is 2.5×104/cm2, and the NPCs can differentiate into neuron, glial, oligodendrocyte. |
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