Establishment of fundamental technology for clinically applicable regenerative medicine in corneal endothelium and stroma.

建立临床适用的角膜内皮和基质再生医学基础技术。

基本信息

批准号：
21390465
负责人：
AMANO Shiro
金额：
$ 11.23万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21390465/
关键词：
角膜再生医療角膜内皮角膜実質

项目摘要

Long-term efficacy of transplantation of cultured human corneal endothelial cells(HCEC) with Descemet's stripping automated endothelial keratoplasty(DSAEK) was investigated. DSAEK grafts with cultured HCEC had normal stromal lamellar structure and endothelial cell layer with intercellular adhesion structure. The corneas in which cultured HCEC were thinner than those in the control group and were transparent at 1 year after the surgery. These results indicated that DSAEK using cultured HCEC is effective and clinically feasible.The other method of seeding cultured HCEC on the posterior corneal stroma which was obtained after a thick corneal flap was made using a microkeratome, was confirmed to be effective and safe.A new temperature-responsive culture dish was developed and evaluated. The results indicated that the temperature-responsive culture dish can produce a sheet of corneal epithelium containing a lot of progenitor cells after culture with non-serum and non-feeder cells. We plan to use this new temperature-responsive culture dish to produce a cell sheet of corneal endothelial cells.A new culture method of HCEC using bi-phosphate ascorbic acid(Asc-2P) was investigated. The results indicated that the primary culture of HCEC was successful in all cases under the culture condition with athello-collagen as a carrier and Asc-2P and bGFGF in the media. Even after several passage, HCEC showed a high proliferative capacity and a cobble-stone like appearance. The expression level and intrecellular distribution pattern of ZO-1 and NA+/K+-ATPase in cultured HCEC were similar to those in in vivo HCEC. 8-OHdG and MDA increased in HCEC cultured without Asc-2P as passage increased whereas they were suppressed in HCEC cultured with Asc-2P. HCEC cultured with Asc-2P showed high proliferative capacity and appearance similar to in vivo HCEC. These results suggested that Asc-2P elongated the lifespan of HCEC by reducing intracellular oxidative stress.

研究了培养的人角膜内皮细胞（HCEC）的长期疗效，该细胞具有Descemet剥离自动内皮角膜置换术（DSAEK）的长期疗效。具有培养的HCEC的DSAEK移植物具有正常的基质层状结构和具有细胞间粘附结构的内皮细胞层。培养的HCEC的角膜比对照组较薄，并且在手术后1年透明。这些结果表明，使用培养的HCEC的DSAEK有效且在临床上是可行的。将培养的HCEC播种在后角膜基质上的另一种方法是在使用微型培训组制成厚的角膜皮瓣后获得的，该方法被证实是有效且安全的。一种新的温度响应性培养菜肴是开发和评估的。结果表明，温度响应性培养皿可以产生一片角膜上皮，这些角膜上皮含有大量祖细胞，并在培养了非副细胞和非喂养细胞后。我们计划使用这种新的温度响应性培养皿生成角膜内皮细胞的细胞片。研究了使用双磷酸抗坏血酸（ASC-2P）的HCEC的新培养方法。结果表明，HCEC的主要培养在培养条件下的所有情况下都是成功的，而Athello-Collagen在媒体中是载体，ASC-2P和BGFGF。即使经过了几次通过，HCEC也表现出很高的增殖能力和像鹅卵石一样的外观。培养的HCEC中ZO-1和Na+/K+-ATPase的表达水平和细胞内分布模式与体内HCEC中的表达水平和na+/K+-ATPase相似。在没有ASC-2P的HCEC培养的HCEC中增加了8-OHDG和MDA，随着通过的增加，在用ASC-2P培养的HCEC中抑制它们。用ASC-2P培养的HCEC表现出很高的增殖能力和外观，类似于体内HCEC。这些结果表明，ASC-2P通过减少细胞内氧化应激来延长HCEC的寿命。