| Objective：Culture of rabbit bone marrow mesenchymal stem cells In vitro and differentiationinto neuron-like cells, Implant neuron-like cells in its superior cervical sympatheticganglion injury model in order to observe the change of the pupil, check the expression ofcervical sympathetic ganglion NGF, VEGF stem cells and investigate the possible ofrecovery mechanism of the injured nerve.Method：Select4month old New Zealand young rabbit, get the femur bone marrow and obtainthe BMSCs after in vitro culture,and induced to differentiate into neuron-like cells.Prepare the sympathetic ganglion damage model of rabbit carotid by using microforceps crush rabbit superior cervical sympathetic ganglion.Select24healthy adult male New Zealand rabbits, randomly divided into four groups,group A named normal control group, normal rabbit, gave no treatment; group B namedanimal model control group, cervical sympathetic ganglion damage which has not beengiven with other intervention; group C named culture medium control group, whichsuperior cervical sympathetic ganglion after the cell culture fluid intervention (notincluding cell); group D named BMSCs transplantation group, which cervicalsympathetic ganglion damage to neuron-like cells intervention. After the model has beensuccessful prepared for7days, group C will be injected with microinjection pump via earvein injection of medium1ml, group D wil be injected with microinjection pump via earvein injection concentration of1×109neuron-like cells culture fluid1ml, and respectivelyrecord the changes of the pupil after transplantation in the1d,3d,7d,14d,21d; aftertransplantation of21d, the rabbit will be killed, and get the superior cervical sympatheticganglion, staining the HE and detecting the nerve growth factor NGF and vascularendothelial growth factor VEGF expression. Result：1. Cultured the rabbit BMSCs successfully; observed by optical microscope, it wasshown that the primary rabbit with less BMSCs adherent, most of which were round oroval, and a small part of a short fusiform; the adherent cells haa been gradually increasedafter purification, amplification, experessed as colony growth, shown aslong fusiform, andpartly as swirl shape; with the passage number increased, till6~7generation, cell growthgradually slow, cells show as flat; and the immunocytochemical staining for detection ofBMSCs surface specific marker training: CD34(-), CD54(+).The differentiated cellidentification of most cell neuron-specific protein and nestin positive expression.2. The comparison of the diameter of the pupil:In1d,3d,7d,14d,21d, normal pupildiameter (mm) were:7.21±0.77,7.72±0.99,7.33±0.35,7.45±0.56,7.41±0.65, modelcontrol group, the pupil diameter (mm) were:5.25±0.30,5±0.66,5.27±0.45,5.39±0.24,5.30±0.49, medium control group pupil diameter (mm) were:5.07±0.41,5.32±0.36,5.16±0.43,5.14±0.49,5.41±0.84, BMSCs transplantation group pupil diameter(mm) were:5.35±0.34,5.52±0.25,5.40±0.61,6.38±0.60,6.87±0.40. After neckinjury of sympathetic ganglion, the pupil of model control group、multure medium controlgroup and BMSCs transplantation grouphas been significantly reduced comparing withnormal control group, whiile, the pupil of normal control group with no changes, and withthe passing of the time, the pupil of model control group and culture medium control grouphas been restored in some degree, but not significantly, the pupil of BMSCs transplantationgroup has been gradually restored after the transplantation of BMSCs,became larger in14d,and the recovery is more obvious in21d, then the difference becomes smaller with normalcontrol group. At the same time, different groups: in the time point of1d、3d、7d and14d,the diameter of the pupil of model control group、culture medium control group andBMSCs transplantation group are significantly less than normal control group(P<0.05); and at the time poin of21d, model control group and culture medium controlgroup were less than group normal control group and BMSCs transplantationgroup(P<0.05), BMSCs transplantation group and normal control group showed no significant difference (t=-0.469, P=0.184); comparison within the same group at differenttime points: no significant difference was found between for normal control group, modelcontrol group and culture medium control group at different time points (F=0.483,P=0.748; F=0.618, P=0.0.654; F=0.407, P=0.802). Comparison of BMSCs transplantationgroup in different time points, significant difference has been shown (F=13.150, P=0.000),the pupil diameter in the time points14d,21d was significantly higher than that of1d,3d,7d (P<0.05), and for the time points of14d and21d, the diameter of the pupil has notshown big different (t=-0.486, P=0.080).3.Ganglion HE staining indicated that: for the normal control group under lightmicroscope with more neural cells and the cell morphology is normal, the size is consistent,and the distribution is uniform, whose interstitial almost with no vascular distribution; forthe model control group: neurons are sparse, cell size is not the same, the distribution is notuniform, the majority of cells decreased, karyopyknosis, interstitial cell hyperplasia wasobvious,whose interstitial with little angiogenesis; for the cell morphology mediumintervention control group: the interstitial and vascular proliferation is roughly equivalentwith the model of natural recovering control group. BMSCs transplantation group: cellshape, size and distribution is uniform, only a small number of cell nuclear pyknosis,stromal hyperplasia was not obvious, but in stroma, lots of blood vessels.4. Immunohistochemical staining of NGF (unit: each): the number of NGF positivecell expression showed an upward trend, it shown differences compared the betweengroups (F=31.615, P=0.000), BMSCs transplantation group was significantly higher thanthat in normal control group, model control group and culture medium control group(t=54.267, P=0.000; t=50.382, P=0.000; t=44.859, P=0.000); there was no significantdifference when comparing the normal control group, model control group and culturemedium control group in pairs (P>0.05).5. Immunohistochemical staining of VEGF (unit: each):the number of VEGF positivecell expression showed an upward trend, compared the differences between groups(F=18.634, P=0.000), BMSCs transplantation group was significantly higher than that in normal control group, model control group and culture medium control group (t=22.147,P=0.000; t=17.987, P=0.000; t=16.693, P=0.000); there was no significant differencewhen comparing the normal control group, model control group and culture mediumcontrol group in pairs(P>0.05).Conclusion:1: The rabbit bone marrow mesenchymal stem cells can help the recovery of the rabbitsuperior cervical sympathetic ganglion damage;2: One of the mechanism of rabbit bone marrow mesenchymal stem cells to repair thedamaged ganglion may be through the secretion of NGF, VEGF and other neurotrophicfactors. |
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