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

通过同源盒基因转移从眼睛中分化和移植色素上皮细胞

基本信息

批准号：
11671736
负责人：
TAKAHASHI Masayo
金额：
$ 2.5万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671736/
关键词：
pigmented epithelial cell basic fibroblast fgrowth factor epithelial growth factor homeobos gene Rax Crx CHX10 adenovirus vector アデノウィルスベクター

项目摘要

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.

为了评估睫状体是否具有转分化为神经视网膜的能力，我们培养了成年大鼠睫状体的色素上皮细胞。将睫状体组织与眼睛分离，并以上皮侧向下的方向放置在培养皿上。在含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，并增殖为单层细胞。免疫细胞化学分析显示，大部分细胞表达未分化神经细胞的标志物nestin，部分细胞在促分化培养基中诱导分化后呈神经元样，突起较薄。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胶质细胞标记物的表达，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，但不含有视蛋白，感光标记物。为了确定这些睫状细胞是否可以获得感光细胞表型时，基因工程，我们构建了携带Crx或GFP（绿色荧光素蛋白）基因的腺病毒。当睫状体的色素上皮细胞被携带Crx基因的腺病毒感染并置于已知促进神经分化的条件下时，一些细胞获得了针对视蛋白的免疫反应性。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有可能转分化为感光细胞的Crx同源框基因的诱导。