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

观察Descemet剥离式全自动角膜内皮移植(DSAEK)移植培养的人角膜内皮细胞(HCEC)的远期疗效。DSAEK与培养的人脐静脉内皮细胞复合后，移植物具有正常的基质板层结构和细胞间黏附结构的内皮细胞层。术后1年，培养的HCEC较对照组角膜变薄，透明。结果表明，用培养的人角膜上皮细胞移植DSAEK是有效的，临床上是可行的。另一种方法是将培养的人角膜上皮细胞种植在厚厚的角膜瓣后的角膜基质上，该方法是安全有效的。结果表明，该温度敏感型培养皿在无血清和非饲养层细胞培养条件下，可获得含有大量祖细胞的角膜上皮细胞。我们计划使用这种新型的温度响应型培养皿来生产角膜内皮细胞的细胞片。研究了一种使用二磷酸抗坏血酸(ASC-2P)培养人角膜内皮细胞的新方法。结果表明，在以硫代胶原为载体、ASC-2P和bGFGF为基质的培养条件下，人脐静脉内皮细胞原代培养均获得成功。即使经过多次传代，人脐静脉内皮细胞仍表现出高增殖能力和鹅卵石样外观。ZO-1和Na+/K+-ATPase在培养的人脐静脉内皮细胞中的表达水平和细胞内分布模式与体内的相似。不加ASC-2P的HCEC随着传代次数的增加，8-OHdG和MDA含量增加，而加入ASC-2P的HCEC则受到抑制。用ASC-2P培养的人脐静脉内皮细胞具有较高的增殖能力，其形态与体内人脐静脉内皮细胞相似。这些结果表明，ASC-2P通过减少细胞内氧化应激而延长了HCEC的寿命。

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.