Effects of olfactory ensheathing cells on hydrogen peroxide-induced apoptosis in cultured dorsal root ganglion neurons / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Olfactory ensheathing cells (OECs) can promote many kinds of neuron growth and axonal extension. The aim of the study was to investigate the effects of co-culturing with OECs on neuron apoptosis in vitro.</p><p><b>METHODS</b>Apoptosis was induced by treatment of cultured dorsal root ganglion neurons with 1 mmol/L hydrogen peroxide (H(2)O(2)). Cells were randomly arranged into the following treatment groups. In group 1, OECs at different density (10(4)/ml to 8 x 10(5)/ml) were added immediately after H(2)O(2) treatment and cells were co-cultured for 24 hours. In group 2, OECs were added at different time points (0, 4, 8, 12 and 24 hours) after H(2)O(2) treatment. Apoptotic cell death was determined by Hoechst 33258 staining and flow cytometry (FCM). Cell viability was determined by using methyl thiazoleterazolium (MTT) assays.</p><p><b>RESULTS</b>The results showed in the Hoechest 33258 staining, FCM and MTT that OECs have both the density-dependent protection and time-dependent protection on neuron apoptosis. The apoptosis decreased and the dorsal root ganglion neuron viability increased, when the density of OECs was increased in co-culture groups. But further increasing OEC density above 2 x 10(5)/ml (i.e. 8 x 10(5)/ml) failed to exert additional protection. As the interval between adding H(2)O(2) and adding OECs was increased, the amounts of apoptosis cells were also increased. When OECs were added 24 hours after H(2)O(2), no significant protection was observed.</p><p><b>CONCLUSION</b>These results indicated that OECs could protect dorsal root ganglion neurons from apoptosis induced by H(2)O(2) in a density- and time-dependent manner.</p>

Isolation and differentiation of embryonic stem cells from BALB/c mouse / 神经科学通报·英文版

Objective To investigate the efficient method which can culture and induce embryonic stem cells to neurocyte in vitro. Methods Isolate the blastula of 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass (ICM) which were isolated by mechanical method on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem cells were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunochemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 (stage specific embryonic antigen 1). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated cells presented the characters of ESCs. Then the isolated cells were able to differentiate into neurocytes in vitro. Conclusion Mouse embryonic stem cells isolation, culture and differentiation system has been established.

Morphology research of the rat sciatic nerve bridged by collage-heparin sulfate scaffold / 中华外科杂志

<p><b>OBJECTIVE</b>To observe the treating effect of collage-heparin sulfate after the 10 mm rat sciatic nerve defect was bridged by it.</p><p><b>METHODS</b>A new kind of nervous tissue engineering scaffold was produced by freeze-drying technique from collagen-heparin sulfate. Thirty-two SD rats were randomly divided into A, B, C and D groups. Sciatic nerve defect in group A was bridged by collagen-heparin sulfate. In group B, sciatic nerve was bridged by auto-nerve transplantation. Group C was the blank control group. Animals in group D were normal. And 10 mm sciatic nerve defect was bridged in the experiment. Thirty-six weeks after the operation, the experimental animals were detected by HRP labeled retrograde trace, HE staining, toluidine staining, silvering staining, S100, GAP-43 and NF immunohistological staining, MBP immunofluorescence staining and transmission electron microscope to observe the nerve regeneration inducing effect of this new scaffold.</p><p><b>RESULTS</b>Nine months after operation, the collage-heparin sulfate scaffold was replaced by newly regenerated nerve. The number of HRP labeled spinal cord anterior horn cells and the area of sensation nerve fiber at the posterior horn were similar with that was repaired by auto-nerve. GAP-43, NF and S100 labeled regenerated nerve fiber had passed the total scaffold and entered the distal terminal. The regenerated nerve fibers were paralleled, lineage arranged, coincide with the prearranged regenerating "channel" in the collagen-heparin sulfate scaffold. MBP immunofluorescence staining also proved that the newly regenerated nerve fiber could be ensheathed. In the experimental group, the area of myelinated nerve fiber and the thickness of the myelin sheath had no obvious difference with that of the group repaired by auto-nerve, except that the density of the regenerated myelinated sheath fiber was lower than that of the control group.</p><p><b>CONCLUSION</b>Nervous tissue engineering scaffold produced by collagen-heparin sulfate can guide the regeneration of nerve fibers. The nerve function recovers fine. This kind of material has great application potential.</p>