Differentiation and transplantation of pigmented epithelial cells from the eye with homebox gene transfer

为了评估睫状体是否具有向神经视网膜转分化的能力，我们培养了成年大鼠睫状体的色素上皮细胞。睫状体组织从眼睛分离出来，以上皮侧向下的方向放置在培养皿上。在含有碱性成纤维细胞生长因子的无血清培养液中，许多细胞从睫状体组织中迁移出来，并以单层细胞的形式增殖。免疫细胞化学分析显示，大多数细胞表达未分化神经细胞的标志物巢蛋白，部分细胞在已知促进神经分化的介质中诱导分化后，呈神经元状，突起细小。为了证实这些细胞确实获得了神经表型，我们检测了成熟神经元或神经胶质标记的表达。免疫细胞化学分析显示，这些细胞含有神经丝200或胶质纤维酸性蛋白的免疫反应细胞，但不含光感受器标记视蛋白的免疫反应细胞。为了确定这些睫状体细胞在基因工程中是否能够获得光感受器表型，我们构建了携带CRX或GFP(绿色荧光素蛋白)基因的腺病毒。当睫状体色素上皮细胞感染携带CRX基因的腺病毒并置于促进神经分化的条件下时，部分细胞对视蛋白产生免疫反应。而携带GFP基因的腺病毒感染不能获得视蛋白阳性细胞。这一发现表明，成年大鼠睫状体上皮细胞在CRX同源框基因的诱导下，具有向光感受器细胞转化分化的潜力。

To evaluate if the ciliary body possesses any ability to transdifferentiate into neural retina, we cultured the pigment epithelial cells of ciliary bodies of adult rats. The ciliary tissues were separated from the eyes and were placed onto the dishes in an orientation that the epithelial side was down. Cultured in the serum free medium containing basic fibroblast growth factor, many cells migrated out from the ciliary tissues and proliferated as monolayered cells. Immunocytochemical analysis revealed that majority of these cells expressed the nestin, the marker for undifferentiated neural cells.Some of the cells showed the neuronal shape with thin processes after they were induced to differentiate in the medium known to promote neural differentiation. To confirm that these cells really acquired the neural phenotypes, we examined the expression of the mature neuronal or glial markers.Immunocytochemical analysis revealed that these cells contained immunoreactive cells for the neurofilament 200 or the glial fibrilary acidic protein, but not for the opsin, the photoreceptor marker. To determine if these ciliary cells could acquire the photoreceptor phenotypes when genetically engineered, we constructed adenovirus carrying either Crx or GFP(Green Fluorescein Protein)gene. When the pigmented cpithelial cells of ciliary bodies were infected with adenovirus carrying Crx gene and were put under the condition that is known to promote neural differentiation, some of the cells acquired immunoreactivity against the opsin. However, infection with adenovirus carrying GFP gene could not acquire the opsin positive cells. This finding shows that the epithelial cells of adult rat ciliary bodies have potential to transdifferentiate into the photoreceptor cells with the induction of Crx homeobox gene.