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[Objective] The rat bone marrow mesenchymal stem cells (BMSCs), olfactory ensheathing cells (OECs) in vitro isolation, purification and cultivation methods, and transected rat spinal cord injury model to investigate the BMSCs, OECs transplantation alone and BMSCs OECs transplantation for spinal cord injury in rats Ⅷ, Ⅸ lamellar survival of neurons and glial scar expression of GFAP and APC changes and nerve fiber regeneration for cell transplantation in the treatment of spinal cord injury provide theoretical and laboratory evidence. [Method] were isolated and cultured rat BMSCs and OECs for cell staining; clean healthy male SD rats were 25, 5 animals in each group were randomly divided into sham group (SG group), surgical control group (CG group ), BMSC transplantation alone group, OEC transplantation alone group and BMSCs OECs group. Under conditions in vitro bone marrow-derived mesenchymal stem cells (Dil mark) and OECs (Hoechst33342 mark) Stereotactic transplanted into the chronic phase (14 days after complete transection) rat spinal cord injury scar, cephalic and caudal . After spinal cord injury in the first 6m taken after the end of perfusion-fixed and head and tail side of the spinal cord scar Paraffin sections OK hematoxylin staining and immunohistochemical staining, were counted in each group spinal Ⅷ, Ⅸ plate layer neuron number and size; count in each group Scar GFAP and APC-positive cells and analyze their immunostaining OD value, while observing the host PKCγ ,5-HT and CGRP-positive fiber regeneration. SPSS11.5 software package for statistical analysis. [Results] 1, cell culture results: Cultured cells were treated with CD44 and p75 immunostaining positive result, identification of BMSCs and OECs; 2, the host cell transplant survival of neurons: surgical control group Ⅷ, Ⅸ board layer neuron number and area were less than the sham group (p lt; 0.05); ① compare each experimental group VIII lamellar number of neurons showed: BMSC group and the surgery group had no significant difference (p gt; 0.05), OEC surgery group than the control group (p lt; 0.05), BMSC OEC transplantation group than BMSC group or OEC group (p lt; 0.05); ② Ⅷ board in each group showed layer neurons area: BMSC group and the operation control There was no significant change (p lt; 0.05), OEC group than in the control group surgery (p lt; 0.05), BMSC OEC combination group than BMSC group, OEC group (p lt; 0.05); ③ Ⅸ board in each group layer neurons The results showed that the number of: BMSC group or OEC surgical control group with no significant difference (p gt; 0.05), BMSC OEC combined group more with BMSC group (p lt; 0.05), BMSC OEC OEC combined group with no significant difference (p gt; 0.05); ④ Ⅸ board in each group showed layer neurons area: BMSC group and the control group, no significant difference in surgery (p gt; 0.05), OEC surgery group than the control group (p lt; 0.05), BMSC OEC joint group than in the BMSC group or OEC group (p lt; 0.05). 3, the host cell transplant scar tissue astrocytes and oligodendrocytes effects: 3.1, the host cell transplant scar formation in astrocytes effects: spinal cord injury, scar tissue in the host GFAP number of positive cells or immune staining OD values ??were significantly higher than the sham group (p lt; 0.05). ① compare the cell transplantation group GFAP positive cells showed: BMSC group or the OEC group were less than the surgical control group (p lt; 0.05), in both groups scar header corticospinal tract (corticol spinal tract, CST) site and surgery group showed no difference (p gt; 0.05), two parts of the scar head and tail segment posterior horn of the gray matter area were less than surgery group (p lt; 0.05); BMSC OEC combination group than BMSC group or OEC group ( p lt; 0.05), in which parts of the combined group header CST than BMSC group (p lt; 0.05), with no significant difference between OEC group (p gt; 0.05), the combined group header or gray area than BMSC group OEC group (p lt; 0.05), part of the posterior horn of the combination group than BMSC Endian or OEC group (p lt; 0.05). ② compare the cell transplantation group OD value of GFAP immunostaining showed: BMSC group or the OEC group were weaker than the surgical control group (p lt; 0.05), which BMSC group header CST site scar and scar tail horn parts are weaker than surgery group (p lt; 0.05), scar Tau gray area with no significant difference in CG (p gt; 0.05), OEC group header CST scar site and CG group had no significant difference (p gt; 0.05), scar Tau gray area and scar posterior horn parts are weaker than the tail section surgery group (p lt; 0.05), BMSC OEC OEC combined group or weaker than BMSC group (p lt; 0.05), in which parts of the scar and scar header gray tail rear corner parts are weaker than BMSC or OEC group (p lt; 0.05), scar Tau CST site and BMSC group or the OEC group showed no significant difference (p gt; 0.05). 3.2, the host cell transplant scar oligodendrocytes effects: spinal cord injury, scar tissue in the host cell number or APC-positive immunostaining OD values ??were significantly less than the sham group (p lt; 0.05). ① APC cell transplantation group compared the number of positive cells showed: BMSC group or the OEC group were more surgery group (p lt; 0.05), which BMSC group head section scar surgery group than CST site (p lt; 0.05), OEC group header CST scar site and surgery group had no significant difference (p gt; 0.05) BMSC or OEC group head section scar scar tail gray and posterior horn parts are more than surgery group (p lt; 0.05), BMSC OEC combined group more than BMSC group (p lt; 0.05), in which scar Tau CST site and BMSC group had no significant difference (p gt; 0.05), gray head section scar and scar tail horn parts parts are more than BMSC group (p lt ; 0.05); BMSC OEC OEC combined group with no significant difference (p gt; 0.05), but in the head section scar CST and gray parts of the tail section scar posterior horn parts are more than OEC group (p lt; 0.05). ② compare the cell transplantation group OD values ??APC immunostaining showed: BMSC group or the OEC group were more surgery group (p lt; 0.05), where the two sets of scar header CST site and surgery group showed no significant difference (p gt; 0.05), two gray head section scar and scar tail horn parts are more than surgery group (p lt; 0.05), BMSC OEC combined group is stronger than BMSC group (p lt; 0.05), in which scar Tau CST and the gray area is stronger than BMSC group (p lt; 0.05), part of the posterior horn of the tail section scar was no significant difference (p gt; 0.05), BMSC OEC OEC group is stronger than the combined group, in which scar header CST and gray parts, scar Endian posterior horn parts are stronger than the OEC group (p lt; 0.05). 4: cell transplantation on the spinal nerve regeneration group: ① PKCγ positive fiber regeneration: Full transection injury PKCγ positive fibers in each group were found to have axonal regeneration, scar tissue found no PKCγ staining. ② 5-HT-positive fiber regeneration: the spinal cord 5-HT-positive fibers are incorporated into the scar tissue, which operated group into lengths 532.60 145.74μm (distance from the scar head); BMSC group to enter the length of 1698.00 ± 880.81μm; OEC group to enter the length ( ?) 659.80 ± 271.54μm, BMSC OEC transplantation group into lengths 1492.00 337.52μm, comparison group and BMSC OEC found that BMSC transplantation group, 5-HT-positive fiber regeneration longer than surgery group (p lt; 0.05), combined transplantation group graft better than the OEC group (p lt; 0.05), with no significant difference between BMSC group (p gt; 0.05). ③ CGRP-positive upstream fiber regeneration: surgical group did not enter the scars, scars from the tail section 962.40 ± 441.14μm; BMSC group also did not enter the scar from the tail section 313.60 ± 109.79μm; OEC head into the scar into a length of 198.00 ± 85.26μm ; BMSC and OEC transplantation group CGRP uplink fiber ingrowth scar into a length of 531.80 162.62μm, each cell transplantation group CGRP uplink fiber regeneration length greater than CG group (p lt; 0.05), BMSC and OEC transplantation group CGRP-positive fiber regeneration length greater than or OEC alone BMSC transplantation group (p lt; 0.05). [Conclusion]: 1, the host completely transected spinal cord injury resulting Ⅷ, Ⅸ lamellar neuronal death, reducing the number of surviving neurons atrophy and decreased size. 2, BMSC transplantation group alone failed to promote the host spinal cord Ⅷ, Ⅸ lamellar neuronal survival, but also failed to inhibit host neuronal degeneration atrophy; OEC transplants alone can increase the host spinal lamina Ⅷ neuronal survival, not promote IX lamellar neuronal survival, OEC transplantation alone can inhibit the host spinal cord Ⅷ, Ⅸ plate layer neuron atrophy; BMSC OEC transplantation group than in the BMSC group to better promote the host VIII, IX lamellar neuronal survival, combined transplantation group than in the OEC group to better promote neuronal survival lamina VIII, IX lamina in promoting neuronal survival respects with the OEC was no significant difference in the inhibition of host neuronal atrophy aspects BMSC transplantation group than alone or OEC transplantation better. 3: Full transected spinal cord tissue after spinal cord injury in glial scar formation, GFAP positive response enhancement, spinal cord axons appear demyelination of nerve fibers, APC positive reaction weakened. BMSC or OEC cell transplantation can improve scar microenvironment, inhibit glial scar formation and promote oligodendrocyte survival, BMSC and OEC transplantation role better. 4: spinal cord injury, PKCγ positive fibers and CGRP-positive upward into the scar tissue can not be regenerated fibers ,5-HT-positive fibers can be recycled into the scar tissue a certain length; BMSC transplantation alone or in combination with OEC PKCγ can promote the regeneration of nerve fibers; BMSC transplantation or BMSC OEC transplantation could promote regeneration of 5-HT positive fibers, OEC transplantation does not promote regeneration of 5-HT-positive fibers; BMSC and OEC alone or in combination CGRP-positive cell transplantation could promote regeneration of nerve fibers in the uplink, which OEC transplantation or BMSC OEC transplantation promotes upward CGRP positive fibers into scar tissue regeneration-promoting cell transplantation combined effect is better.
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