| Objective To investigate the effect of intravitreal injection of neural stem cells(NSCs) induced from human umbilical cord mesenchymal stem cells(h UCMSCs) by the number of reinal ganglion cells(RGCs) and expression of brain-derived neurotrophic factor(BDNF).Methods The neural differentiation medium was adopted to induce h UCMSCs to NSCs. Neural markers, nestin and neurogenic differentiation factor1(Neuro D1), were detected by immunocytochemistry. Fifty-two adult male Sprague-dawley(SD) rats were randomly divided into normal group(group A) and diabetes mellitus group which were injected streptozocin(STZ) by intraperitoneal injection to make diabetic rat models. One month after the diabetic rat models were confirmed successfully, diabetic rats were randomly divided into diabetic group(group B), h UCMSCs group(group C) and h UCMSCs-induced NSCs group(group D). And thirteen diabetic rats were included in each group. Immunohi-stochemistry was applied to observe BDNF and thymosin-1(Thy-1) staining in rat’s retina when post-intervention every 2 weeks. Then mean integrated optical density(IOD) of the staining region on rat’s retina slices were analyzed by Image-Pro Plus 6.0. The number of RGCs which shows positive in labeled Thy-1 was record. SPSS 20.0 software was used to analysis all of the data. P<0.05 were considered as statistical difference.Results 1. After three days induced by neural differentiation medium,60~70% h UCMSCs started to aggregate to form clusters of cells forming cell clusters, and became either bigger or richer than over time.. 2. Immunocytochemistry showed that the h UCMSCs- induced NSCs highly expressed nestin and Neuro D1, which reached to 87.3% and 73.6% respectively. 3. Thirty-eight diabetic rats were confirmed successfully. There were two rats from the twenty-six rats injected cells by intravitreal injection occurred vitreous hemorrhage. Finally, twelve rats were included in each group.4. Results’ of Immunohistochemistry:(1) BDNF:The staining region on rat’s retina slices from group A showing that BDNF located in the ganglion celllayer(GCL) and the other retinal layers.The staining intensity on rat’s retina slices from group B became week and the expression of BDNF gradually decrease with time,and those from group C and group D were positively, especially in group D. Statistical significant differences were found among the different follow-up times in the four groups(F=20.325,13.540, 16.896, P=0.000).Except for the second week since post-intervention between group B and group C, statistical significant differences of the expression of BDNF were found among the four groups(F=37.144,27.419, 83.659,89.654, P=0.000).(2)Thy-1:The staining region on rat’s retina slices from group A showed that Thy-1 mainly located in the GCL and NFL. The staining intensity on rat’s retina slices from group B became week and the expression of Thy-1 gradually decrease with time, and those from group C and group D were positively, especially in group D. Besides to the second week since post- intervention between group C and group D, statistical significant differences of the expression of Thy-1 were found among the four groups(F=16.087, 15.239, 10.950, P=0.000). In addition to the sixth week and the eighth week in group B and group D since from post-intervention, and significant differences were found among the different follow-up times in the four groups(F=26.463, 51.360, 61.820, 101.597, P=0.000).(3) The number of Thy-1 labeled RGCs: there were about to thirty-three RGCs in group A. RGCs’ s number in group B gradually decrease, while in group C and in group D which were reversed(F=7.273, 17.606, 16.290, P=0.000). Except for the fourth week and the sixth week in group C since post-intervention, statistical significant differences were found among the four groups(F=100.333, 117.404, 70.212, 90.012, P=0.000). 5. Significant positive relationship between the expression of BDNF and the number of RGCs were found by the Pearson correlation analysis(r=0.964, P=0.000).6. Conclusion 1. In vitro, h UCMSCs can successfully induced into NSCs, which highly expression nestin and Neuro D1. 2. Diabetic rat models induced by STZ had lost of RGCs when diabetes durationabout six weeks. Intravitreal injection of h UCMSCs- derived NSCs in vitro to diabetic rat models may reduce the loss of RGCs 3. Transferring from intravitreal injection of h UCMSCs- derived NSCs in vitro to diabetic rat models can promote the retina to express BDNF. 4. Transferring from intravitreal injection of h UCMSCs- derived NSCs in vitro to diabetic rat models protect retina may related to the potential of promoting BDNF expression. |
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