Guo Xiaoqing, Hutcheon Audrey E K, Zieske James D
Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.
Exp Eye Res. 2004 May;78(5):997-1005. doi: 10.1016/j.exer.2003.12.010.
A technology has recently been developed that allows for the rapid transduction of full-length functionally active proteins into intact tissue through intravenous injection and into cultured cells. This technology involves the fusion of an 11 amino acid sequence of the HIV TAT protein to the protein of interest. In the current investigation, we determined whether functionally active TAT fusion proteins could be transduced into intact corneas by topical application. TAT-beta-galactosidase was purified from bacterial cells and applied in serial dilutions (12.5-250 nm) to cultured epithelial cells for 5 or 15 min. In addition, enucleated globes and excised corneas with or without a central 3-mm epithelial debridement were incubated with TAT-beta-galactosidase for 1 or 2 hr. Excised corneas were allowed to heal in organ culture. Transduction of active beta-galactosidase was detected by incubating the cells or corneas with X-gal. TAT-beta-galactosidase was transduced into nearly all cultured cells in a concentration-dependent manner. When TAT-beta-galactosidase was topically applied to intact corneas, only the most superficial layer of epithelium was highly transduced. When the superficial layer was removed with nitrocellulose, two to four layers of cells were transduced. In corneas with a central debridement, epithelial cells at the edge of the debridement were transduced as well as the stromal cells subjacent to the debridement. Active beta-galactosidase was maintained at least 1 day in organ culture. No X-gal reaction was seen in either cells or corneas not incubated with TAT-beta-galactosidase. Functionally active proteins can be efficiently transduced into corneal epithelial and stromal cells using TAT fusion protein technology. The intact epithelium provides a barrier to penetration of TAT proteins. This barrier can be overcome by disrupting the epithelium. TAT-mediated protein transduction may be extremely useful in studies of corneal wound healing and homeostasis.
最近开发了一种技术，该技术可通过静脉注射将全长功能活性蛋白快速转导至完整组织，并转导至培养细胞中。这项技术涉及将HIV TAT蛋白的11个氨基酸序列与目标蛋白融合。在当前的研究中，我们确定了功能活性TAT融合蛋白是否可以通过局部应用转导至完整角膜中。从细菌细胞中纯化出TAT-β-半乳糖苷酶，并以系列稀释液（12.5 - 250纳米）应用于培养的上皮细胞5或15分钟。此外，将摘除眼球和切除的角膜（有或没有中央3毫米上皮清创术）与TAT-β-半乳糖苷酶孵育1或2小时。切除的角膜在器官培养中愈合。通过用X - gal孵育细胞或角膜来检测活性β-半乳糖苷酶的转导。TAT-β-半乳糖苷酶以浓度依赖的方式转导至几乎所有培养细胞中。当将TAT-β-半乳糖苷酶局部应用于完整角膜时，只有最表层的上皮细胞被高度转导。当用硝酸纤维素去除表层时，两到四层细胞被转导。在有中央清创术的角膜中，清创边缘的上皮细胞以及清创下方的基质细胞都被转导。活性β-半乳糖苷酶在器官培养中至少维持1天。在未与TAT-β-半乳糖苷酶孵育的细胞或角膜中均未观察到X - gal反应。使用TAT融合蛋白技术可将功能活性蛋白有效地转导至角膜上皮细胞和基质细胞中。完整的上皮细胞对TAT蛋白的渗透形成屏障。通过破坏上皮细胞可以克服这一屏障。TAT介导的蛋白转导在角膜伤口愈合和内环境稳定的研究中可能极其有用。
