Regeneration from human cord blood in SCID mice

为了检验人脐带血细胞向视网膜神经细胞的反式分化，我们使用了人脐带血的谱系阴性部分。首先，利用淋巴细胞提取人脐带血的低密度部分。低密度细胞用生物素标记的抗cd3、CD19、CD14、CD15、CD16、CD56、CD11c抗体孵育，然后用亲和素标记的磁珠孵育。用磁铁从低密度细胞中删除谱系细胞。剩余细胞作为人脐带血谱系阴性细胞。用PKH26标记谱系阴性细胞，将其注射到SCID小鼠视网膜下间隙，使用抗asialogm1抗体耗尽NK细胞。注射2周后处死小鼠，取眼。在组织学检查中，我们使用激光扫描共聚焦显微镜和实时RT-PCR检测SCID小鼠眼中注射的人细胞。注射细胞在组织学上表达人巢蛋白、人MAP2、人神经元特异性烯醇化酶、β-III微管蛋白和视紫红质，并通过实时RT-PCR表达人视紫红质mRNA。这些结果表明人类脐带血细胞可以分化为视网膜神经细胞。

To examine trans-differentiation from human cord blood cells to retinal nerve cells, we used lineage negative fraction from human cord blood. First, low density fraction of human cord blood was obtained using lymphoprep. The low density cells were incubated with biotin-labeled anti-CD3, CD19, CD14, CD15, CD16, CD56, CD11c antibodies, followed by incubation with avidin-labeled magnetic beads. Lineage cells were deleted using a magnet from the low density cells. Residual cells were used as lineage negative cells of human cord blood. The lineage negative cells were labeled with PKH26 and they were injected into subretinal space of SCID mice, in which NK cells had been depleted using anti-asialoGM1 antibody. Two weeks after injection, we sacrificed the mice and obtained the eyes. In histological examination, we detected injected human cells in the eyes of the SCID mice, using a laser-scanning confocal microscope and real time RT-PCR. And the injected cells express human nestin, human MAP2, human neuron specific enolase,β-III tubulin and rhodopsin histologically and they expressed mRNA of human rhodopsin in real time RT-PCR. These results suggest that human cord blood cells can differentiate into retinal nerve cells.