Transplantation for retinal diseases

我们报告一例视网膜下虹膜色素上皮细胞（IPE）移植手术。当这些细胞通过基因工程表达神经营养因子时，移植将光感受器细胞从光毒性中拯救出来。我们使用腺相关病毒（AAV2）将神经营养因子，如脑源性神经营养因子（BDNF）传递到移植细胞。如果我们使用超过1x107个衣壳/ml的AAV-BDNF进行转染，在AAV-BDNF- ipe移植中观察到具有统计学意义的光感受器保护。然而，救援效果不依赖于剂量。然后，我们检测了BDNF受体TrkB的表达，因为受体可能控制BDNF的作用。到目前为止，已经报道了许多TrkB的异构体，我们研究了TrkB的两个主要异构体；TrkB-FL在细胞中有酪氨酸激酶活性而TrkB-T1则没有。免疫组织化学显示，这些亚型的表达并不总是完全相同。TrkB-FL主要表达于神经纤维层、神经节细胞层和内核层（INL）， TrkB-T1表达于INL和RPE。这两种异构体在ONL和光感受器层均不表达。S100β双免疫染色结果显示，这些异构体在Muller细胞上均有表达。此外，尽管AAV-BDNF-IPE移植增强了这些异构体在视网膜中的表达，但这些异构体在空间和时间上的表达方式都不同。当我们进行原位杂交时，也证实了相同的表达模式。siRNA实验显示，当我们注射TrkB-T1的siRNA时，光感受器的保护作用明显减弱。当我们通过Muller细胞系的DNA微阵列检测BDNF刺激和未刺激的rMC-1之间的差异基因表达时，迄今为止报道的神经营养因子表达不多。另一种机制，如清除周围环境中的离子可能很重要。

We report a subretinal iris pigment epithelial cell (IPE) transplantation with safely. When these cells were genetically engineered that expressing neurotrophic factors, the transplantation rescued the photoreceptor cells from phototoxicity. We used adeno-associate virus (AAV2) for delivering neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) to the transplanted cells. If we used more than 1x107 capsides/ml AAV-BDNF for transfection, statistically significant photoreceptor protection was observed in the AAV-BDNF-IPE transplantation from phototoxicity. However, the rescue effect was not dose-dependent. Then, we examined the BDNF receptor TrkB expression, because the receptor may control the effect of BDNF. So far many isoforms of TrkB have reported and we examined two of the major isoforms of TrkB ; TrkB-FL which has tyrosine kinase activity in the cell and TrkB-T1 which has not. These isoforms showed not always completely same expression by immunohistochemistry. TrkB-FL was expressed mainly on nerve fiber layer, ganglion cells layer, and inner nuclear layer (INL), conversely TrkB-T1 was on INL and RPE. Both isoforms were not expressed in ONL and photoreceptor layer. From the results of double immunostaining with S100β, These isforms were expressed on Muller cells. Further these isoforms were expressed in the retina not only spatially but also temporally different way, although the expression was enhanced by the AAV-BDNF-IPE transplantation. When we performed in situ hybridization, same expression pattern were also confirmed. siRNA experiments showed significant less photoreceptor protection when we injected siRNA of TrkB-T1. When we examined differential gene expression by DNA microarray of Muller cell line, rMC-1 between BDNF stimulated and non-stimulated, neurotrophic factors that were so far reported were not many expressed. Another mechanism such as scavenging the ions in the surrounding circumstances may be important.