Establishment of a human corneal epithelial cell line lacking the functional TACSTD2 gene as an in vitro model for gelatinous drop-like dystrophy.
Establishment of a human corneal epithelial cell line lacking the functional TACSTD2 gene as an in vitro model for gelatinous drop-like dystrophy.
复制标题
建立缺乏功能性 TACSTD2 基因的人角膜上皮细胞系作为凝胶状水滴样营养不良的体外模型。
DOI：
10.1167/iovs.12-11043
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
Kinoshita S.
中科院分区：
文献类型：
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作者：
Kitazawa K;Kawasaki S;Shinomiya K;Aoi K;Matsuda A;Funaki T;Yamasaki K;Nakatsukasa M;Ebihara N;Murakami A;Hamuro J;Kinoshita S.
目的。凝胶滴状角膜营养不良（GDLD）的特征是上皮下淀粉样蛋白沉积，可导致严重的视力丧失。这种疾病的确切发病机制还有待阐明。没有根本的治疗方法。本研究旨在建立永活角膜上皮细胞系，用于GDLD疾病模型。方法：从GDLD患者手术中获得角膜组织标本。用酶将角膜上皮细胞从角膜中分离出来，并进一步分离成单个细胞。通过慢病毒转导类人猿病毒40 （SV40）大T抗原和人端粒酶逆转录酶（hTERT）基因使上皮细胞永生化。对永生化细胞进行了增殖动力学、基因表达和功能分析。结果：尽管累积数量超过100倍，永生化角膜上皮细胞仍能继续增殖。细胞几乎没有衰老的迹象，并表现出很强的集落形成活性。这些细胞表现出较低的上皮屏障功能，以及紧密连接相关蛋白claudin 1和7的表达减少。利用来自GDLD患者的永生性角膜上皮细胞，我们测试了基因治疗的可能性。结论：我们建立了来自GDLD患者的永生性角膜上皮细胞系。永生化细胞表现出与体内GDLD相似的细胞表型。这些永生化细胞被认为对开发治疗GDLD角膜的新疗法和阐明GDLD的病理生理学是有用的。
Purpose.: Gelatinous drop-like corneal dystrophy (GDLD) is characterized by subepithelial amyloid deposition that engenders severe vision loss. The exact mechanism of this disease has yet to be elucidated. No fundamental treatment exists. This study was conducted to establish an immortalized corneal epithelial cell line to be used as a GDLD disease model.Methods.: A corneal tissue specimen was obtained from a GDLD patient during surgery. Corneal epithelial cells were enzymatically separated from the cornea and were dissociated further into single cells. The epithelial cells were immortalized by the lentiviral transduction of the simian virus 40 (SV40) large T antigen and human telomerase reverse transcriptase (hTERT) genes. For the immortalized cells, proliferative kinetics, gene expressions, and functional analyses were performed.Results.: The immortalized corneal epithelial cells continued to proliferate despite cumulative population doubling that exceeded 100. The cells showed almost no sign of senescence and displayed strong colony-forming activity. The cells exhibited a low epithelial barrier function as well as decreased expression of tight-junction–related proteins claudin 1 and 7. Using the immortalized corneal epithelial cells derived from a GDLD patient, we tested the possibility of gene therapy.Conclusions.: We established an immortalized corneal epithelial cell line from a GDLD patient. The immortalized cells exhibited cellular phenotypes similar to those of in vivo GDLD. The immortalized cells are thought to be useful for the development of new therapies for treating GDLD corneas and for elucidation of the pathophysiology of GDLD.