| ObjectiveTo observe the level of blood glucose,changes of retinal function and expression of brain-derived neurotrophic factor(BDNF) of diabetic retinopathy(DR) rats by caudal vein or intravitreal injection of human umbilical cord mesenchymal stem cells(hUCMSCs),and study the possible mechanism of hUCMSCs in DR treatment in order to lay a foundation for further clarifying therapeutic action of hUCMSCs for DR.MethodsThe model of diabetic rats was established by injecting with streptozocin (STZ) through caudal vein. The ratsof diabetic group was randomly divided into five groups:rats with DR only(B group),rats injected with1*10(?)6～7hUCMSCs0.5ml through caudal vein (C group),rats injected with placebo0.5ml through caudal vein(D group),rats with intravitreal injection of1*10(?)6-7hUCMSCs2μl (E group) and rats with intravitreal injection of placebo2μl (F group). Normal control(A group) and B group received no further treatment. Blood glucose of the rats was monitored every2-4weeks. Flash electroretinogram(F-ERG) was performed after intervention to evaluate the retinal function; morphological changes of rats retinas were observed by hematoxylin-eosin staining and optical microscope; Immunohistochemistry was performed to investigate BDNF-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 BDNF mRNA.Data collected was analysed by SPSS17.0software. T test of independent sample, One-way ANOVA, Kruskal-Wallis test and LSD-t test was used to compare the difference between groups. P<0.05showed statistical difference.Results1. Blood glucose:blood glucose of DM rats were much higher than normal control at the same time before transplanting(P=0.000). There was statistical difference of blood glucose among all groups after transplanting(P=0.000). Group C, which had great difference between2nd and4th week(P=0.012), had statistical difference of blood glucose with other groups(P<0.01).2. FITC-dextran fluorescent radiography and retinal flat mounts:Vascular morphology and distribution were fine in the retinal of normal control rats. It was fund that tortuous and expansive retinal blood vessel, microaneurysm, fluorescein leskage around vascellum and non perfusion areas in the retinal of DM rats.3.F-ERG:There was statistical difference among all groups in a-and b-wave implicit times and amplitude of b-wave and OPs(P<0.05). There was no significant difference among all groups in amplitude of a-wave (P>0.05)。4. HE staining:The each retinal layer and cellular morphology of group A were regular.In group B,D,F, 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,E, 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, BDNF mainly located in RGCs layer and positivity in other retinal layers as well. In B,D,F group. BDNF had a weak positivity in each retinal layers. In group C,E, it was turn to more expression of BDNF. especially in group E.It was significant difference of the expression of BDNF among each group in every time point(P=0.000). The expression of BDNF fewer and fewer along with the time and there was no statistical significance among B,D,F group(P>0.05). In group C,E, the expression of BDNF was gradually increased and it was statistical significance with other groups in6th and8th week(P<0.01). There was significant difference of the expression of BDNF along with the time among all DR group (P<0.01).6. RT-PCR:There was no statistical significance of the relative expression of BDNF among all DR group in2nd week(P=0.301). There was significant difference in4th,6th and8th week(P=0.000). There was statistical significance between group C,E and group B,D,F (P<0.01).Compared with group E, group C had significant difference in8th week(P=0.009). There was statistical significance of the relative expression of BDNF along with the time among all DR group(P<0.01).Conclusion1.hUCMSCs can significantly reduce blood sugar through caudal intravenous injection.2.hUCMSCs can improve the retinal function of DR and increase the expression of BDNF through caudal intravenous or intravitreal injection.The intravitreal injection gets better effect.3. RGCs.perithelial cells and other retinal layers will damage in the early stage of DR.4.The expression of BDNF and the nervous function of retina will descend in the early stage of DR. |
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