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

本文报告了培养的人角膜内皮细胞（HCEC）联合后弹力层剥脱式自动角膜内皮移植术（DSAEK）的远期疗效。DSAEK移植物与培养的HCEC的间质板层结构和内皮细胞层与细胞间粘附结构正常。术后1年，培养的角膜内皮细胞比对照组薄，透明。结果表明，应用培养的角膜内皮细胞进行DSAEK是有效的，临床上是可行的;另一种方法是将培养的角膜内皮细胞接种于角膜板层后基质上，该方法是有效和安全的。结果表明，温度敏感培养皿在无血清、无饲养层条件下培养，可获得含有大量前体细胞的角膜上皮层。本研究旨在利用这种新型温度敏感培养皿制备角膜内皮细胞片层，并探索一种新的以二磷酸抗坏血酸（Asc 2 P）为底物的培养方法。结果表明，在以胶原为载体，加入Asc-2 P和bGFGF的培养条件下，所有病例的HCEC原代培养均获得成功。即使经过几次传代，HCEC仍显示出高增殖能力和鹅卵石样外观。ZO-1和NA+/K+-ATP酶在体外培养的人脐静脉内皮细胞中的表达水平和细胞内的分布模式与在体人脐静脉内皮细胞相似。8-在无Asc-2 P培养的HCEC中，随着传代次数的增加，OHdG和MDA增加，而在有Asc-2 P培养的HCEC中，OHdG和MDA被抑制。与Asc-2 P培养的HCEC显示出高增殖能力和类似于体内HCEC的外观。这些结果表明，Asc-2 P通过降低细胞内的氧化应激延长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.