| ObjectiveTo observe retinal function and expression of nerve growth factor (NGF) of diabetic retinopathy(DR) rats by intravitreal injection of different concentrations human umbilical cord mesenchymal stem cells(hUCMSCs),and study the possible mechanism of hUCMSCs in DR treatment in order to further proof that the best concentration of hUCMSCs DR treatment.Methods48male Sprague-Dawley(SD) rats were randomly devided into normal group(Group A) and diabetes mellitus groups,10rats and30rats respectibely.Diabetic rat models induced by streptozocin(STZ) caudal vein injection.The diabetic rat models was randomly divided into four groups:rats with DR only(B group), rats with intravitreal injection of1*10Λ6～7hUCMSCs2μl (C group), rats with intravitreal injection of1*10Λ5-6hUCMSCs2μl (D group), and rats with intravitreal injection of placebo2μl (E group). Normal control(A group) and B group received no further treatment. Blood glucose of the rats was monitored every2-4weeks. The model of DR was tested by ethidium-bromide(EB) staining the retinal flat mounts. morphological changes of rats’ retinas were observed by hematoxylin-eosin staining and optical microscope; Immunohistochemistry was performed to investigate NGF-positive staining in rats’ retinas. Image-Pro Plus6.0was performed to analyze integrated optic density(IOD) of staining region on rats’ retinal slices in each group.Real-time fluorescence quantitative PCR(RT-PCR) was applied to detect relative expressions of NGF mRNA.Data collected was analysed by SPSS17.0software. T test of independent sample, One-way ANOVA, LSD-t test was used to compare the difference between groups. P<0.05showed statistical difference.Results1. Blood glucose:A week after tail vein injection of STZ,the average blood glucose value is (5.43±0.81)mmol/L in the normal control group,and blood glucose value of DM rats is (27.59±3.56) mmol/L. Measured at different time points DM rats blood glucose level is higher, but the basic keep in a stable level; The difference had statistical significance compared with normal control group rats (P<0.01).2. Weight:DM rats at the beginning of the body quality and normal control group of rats are similar, both are no statistically significant difference (t=0.805, P=0.433). After the success of the building normal control group rats increased with the increase of weeks body quality is growing rapidly, DM rat body quality slow pace than normal control group, the longer the weeks of age, body mass is the greater difference, in different time points measured the body quality between the two groups have statistical significance (P<0.01).3.EB staining and the retinal flat mounts:Vascular morphology and distribution were fine in the retinal of normal control rats. Vascular morphology and distribution were changed in the retina of DR rats and lose the normal distribution patterm.Retinal vascular was disorder,Fluorescein leakage and microaneurysms was showed in the fluorescence image of retina in DR rats.4. HE staining:The each retinal layer and cellular morphology of group A were regular. Nerve fiber layer (NFL) is relatively sparse horizontally arranged; Ganglion cell layer (GCL) order, large nuclei, assumes the circular or elliptic, dyeing was light; Inner plexiform layer (IPL) thicker, mesh structure is more obvious; The inner nuclear layer(INL) cell nuclei is bigger, dyeing a bit deep; outer plexiform layer(OPL) than from within the slant thin layer; Outer nuclear layer (ONL) is composed of multi-layer cell arrangement, dyed deep closely packed; Photoreceptor cell layer(PRL) section morphology neat inside and outside.In group B and E,each retinal layers were disordered and the amount of cells were reduce. The inner limiting membrane was not completed and became local bodiness in part. It was also found that expansive retinal vascular and proliferative retinal vascular endothelial cells. In group C,D, each retinal layers were less regular, the amount of each layers’ cells were less and expansive retinal vascular can not be found. There was occasionally found partly abscence of inner limiting membrane.5. Immunohistochemistry staining:In group A, Group A:NGF plays express positive, positive cells in the boundary is clear, mainly in the retinal ganglion cells (retinal ganglion cells, RGCs) layer, IPL, an OPL, external cortex on membrane, pigment, INL and ONL only small amounts of expression. B and E group:2w model NGF expression positive, increased expression in each layer, positive staining is deeper.4w model NGF positive expression began to abate, decreased expression of each layer.8w model NGF positive expression was significantly decreased, the layers of positive of dyeing are shallow. C, D group:2w model NGF expression increased, with the passage of time, the model NGF positive expression gradually strengthen, reaches its peak when the8w, C group is more apparent, dyed deeply.6. RT-PCR:There was no statistical significance of the relative expression of NGF among all DR group in2nd week(P=0.920). There was significant difference in4th,6th and8th week(F=13.737,25.060,123.277, P=0.000). There was no statistical significance in4th,6th and8th week between group B and group E (P=0.703,P=0.801,P=0.819),compared with group C, group D had significant difference in in4th,6th and8th week (P<0.05). There was no statistical significance in4th,6th week between group C and group D, There was significant difference in8th week((P<0.05).Conclusions1. The DM rats induced by STZ is to simulate a reliable animal model of diabetic retinopathy;2. DR early which will cause damage to the cells of the layers of retina;3. DR early cause the expression of NGF plays was increased, with the passage of time model NGF expression is on the decline, the retina nerve function impairment;4. hUCMSCs intravitreal injection can improve the DR retinal function, increase the expression of NGF plays, improve the function of the retina;5. High concentrations of hUCMSCs on DR protective effect is superior to the low concentration of hUCMSCs effect. |
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