Recovery of rerinal clgeneration of Rus rat using gene introduction

使用基因导入恢复 Rus 大鼠肾再生

• 批准号: 14571677
• 负责人: KOBAYASHI Hiroshi
• 金额: $ 2.56万
• 依托单位: Saga University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571677/
• 关键词: Retinal pigment epithelium; siRNA; Silencing; pP344; セリンプロテアーゼインヒビター; 免疫組織化学法; ウエスタンブロット法

It is appropriate to use non-pathogenic virus with the promoter of pP344 gene to develop the delivery system which provides the specific expression of a target gene in the retinal pigment epithelium. We thought that not only isolation of pP344 promotor but also study of distribution of pP344 product was important. We raised antibodies against PP344 protein to investigate its ocular distribution. lmmunohistochemical study showed. the significant staining in the RPE and the entire neural retina in chick embiyo eyes and mouse eyes. Western blotting analysis revealed the 65 kDa band of cell extract of RPE and neural retina, Therefore, the PP344 protein was secreted to the apical side and be distributed in the neural retina, and may play an important role in the functions of neural retina. We also addressed on the way of the control of gene expression. The use of small interfering RNAs (siRNA) has become a powerful tool to knock down specific gene expression in a wide range of cells. To identify the functional rnechanism.of Mitf (microphthalmia-inducing transcription factor) in differentiation, we used siRNA to specifically suppress Mitf expression in RPE and investigate the expression of MMP 115 (melanosomal matrix,protein 115 kDa) in the transdiffei鋲ntiation process. The expression of MMP 115 was extremely reduced by 75 %. The expression of MMP 115 was extremely reduced and the number of differentiated cell was decreased. Mitf expression in RPE cells was efficiently inhibited using RNA interference technology. Knockdown of Mitf in RPE cells results in their, promotion of transdifferentiation.

It is appropriate to use non-pathogenic virus with the promoter of pP344 gene to develop the delivery system which provides the specific expression of a target gene in the retinal pigment epithelium. We thought that not only isolation of pP344 promotor but also study of distribution of pP344 product was important. We raised antibodies against PP344 protein to investigate its ocular distribution. lmmunohistochemical study showed. the significant staining in the RPE and the entire neural retina in chick embiyo eyes and mouse eyes. Western blotting analysis revealed the 65 kDa band of cell extract of RPE and neural retina, Therefore, the PP344 protein was secreted to the apical side and be distributed in the neural retina, and may play an important role in the functions of neural retina. We also addressed on the way of the control of gene expression. The use of small interfering RNAs (siRNA) has become a powerful tool to knock down specific gene expression in a wide range of cells. To identify the functional rnechanism.of Mitf (microphthalmia-inducing transcription factor) in differentiation, we used siRNA to specifically suppress Mitf expression in RPE and investigate the expression of MMP 115 (melanosomal matrix,protein 115 kDa) in the transdiffei鋲ntiation process. The expression of MMP 115 was extremely reduced by 75 %. The expression of MMP 115 was extremely reduced and the number of differentiated cell was decreased. Mitf expression in RPE cells was efficiently inhibited using RNA interference technology. Knockdown of Mitf in RPE cells results in their, promotion of transdifferentiation.

项目成果

Kobayashi K, Iwakiri R, Kobayashi H, Okinami S: "Expression of estrogen receptor alpha and 17beta-hydroxysteroid dehydrogenase 4 in the ciliary body"Graefes Arch Clin Exp Ophthalmol.. 242. 172-176 (2004)

Kobayashi K、Iwakiri R、Kobayashi H、Okinami S：“睫状体中雌激素受体 α 和 17β-羟基类固醇脱氢酶 4 的表达”Graefes Arch Clin Exp Ophthalmol.. 242. 172-176 (2004)

Iwakiri R, Kobayashi K, Kobayashi H, Okiami S: "Distribution of a retinal pigment epithelium-specific gene product in chick embryo and mouse eyes"Invest Ophthalmol Vis Sci. 44. s58 (2003)

Iwakiri R、Kobayashi K、Kobayashi H、Okiami S：“鸡胚和小鼠眼睛中视网膜色素上皮特异性基因产物的分布”Invest Ophasemol Vis Sci。

Kobayashi K, Iwakiri R, Kobayashi H, Okinami S.: "Expression of matrix metalloproteinase-3 in the sclera an trabecular meshwork of glaucoma patients with uveits."Invest Ophthalnmol Vis Sci. 44. s16 (2003)

Kobayashi K、Iwakiri R、Kobayashi H、Okinami S.：“青光眼患者巩膜和小梁网中基质金属蛋白酶 3 的表达。”Invest Olookingnmol Vis Sci。

Kobayashi K, Iwakiri R, Kobayashi H, Okinami S: "Expression of matrix metalloproteinase-3 in the sclera an trabecular meshwork of glaucoma patients with uveitis"Invest Ophthalmol Vis Sci. 44. S16 (2003)

Kobayashi K、Iwakiri R、Kobayashi H、Okinami S：“基质金属蛋白酶 3 在患有葡萄膜炎的青光眼患者巩膜和小梁网中的表达”投资眼科 Vis Sci。

Kobayashi K, Iwakiri R, Kobayashi H: "Functional inhibition of Mitf in retinal pigment epithelium by small interfering RNA"Invest Ophthalmol Vis Sci. 45. S199 (2004)

Kobayashi K、Iwakiri R、Kobayashi H：“小干扰 RNA 对视网膜色素上皮中 Mitf 的功能抑制”Invest Ophasemol Vis Sci。