Department of Ophthalmology, Affiliated First Hospital of Jinan University, 613 Huangpu Road, Guangzhou, Guangdong, China.
Biomedical Institute of Jinan University, Jinan University, Guangzhou, Guangdong, China.
Exp Eye Res. 2014 Mar;120:36-42. doi: 10.1016/j.exer.2013.12.016. Epub 2014 Jan 17.
PTD-fusion protein technology was used to transduce heat shock protein 27 (HSP27), an anti-apoptotic protein, into human lens epithelial cells (HLECs) (SRA01/04). The protein transduction domain (PTD) of the 11-amino acid YGRKKRRQRRR was tagged at the N-terminus of HSP27. The fusion protein was purified from bacteria transformed with a pKYB-PTD-HSP27 construct. The HLECs were incubated with PTD-HSP27-FITC and the fluorescence inside HLECs was found by fluorescence microscopic examination. To test the ability of PTD-HSP27 to pass through the corneas, PTD-HSP27-FITC was dropped onto the conjunctival sacs of rabbits; fluorescent labeled PTD-HSP27 was then observed in the rabbit aqueous humor. After being incubated with the PTD-HSP27 protein and irradiated with ultraviolet-B (UVB) light, HLECs was analyzed by flow cytometry, Hoechst 33258 staining and measurement of the potential of the mitochondrial transmembrane. HLECs incubated with PTD-HSP27 had a lower apoptotic rate and a higher mitochondrial membrane potential than the control cells. PTD-HSP27 appears to be sufficient to protect HLECs against UVB-induced apoptosis.
采用 PTD-融合蛋白技术将抗凋亡蛋白热休克蛋白 27（HSP27）转导至人晶状体上皮细胞（HLECs）（SRA01/04）。将 11 个氨基酸 YGRKKRRQRRR 的蛋白转导结构域（PTD）标记在 HSP27 的 N 端。融合蛋白从转化了 pKYB-PTD-HSP27 构建体的细菌中纯化。将 HLEC 与 PTD-HSP27-FITC 孵育，并通过荧光显微镜检查发现 HLEC 内的荧光。为了测试 PTD-HSP27 通过角膜的能力，将 PTD-HSP27-FITC 滴入兔子的结膜囊中；然后观察兔子房水中的荧光标记的 PTD-HSP27。在用 PTD-HSP27 蛋白孵育并用紫外线-B（UVB）光照射后，通过流式细胞术、Hoechst 33258 染色和测量线粒体跨膜电位分析 HLEC。与对照细胞相比，用 PTD-HSP27 孵育的 HLEC 具有较低的凋亡率和较高的线粒体膜电位。PTD-HSP27 似乎足以保护 HLEC 免受 UVB 诱导的凋亡。
