Eye Program, Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, California.
Invest Ophthalmol Vis Sci. 2013 Dec 17;54(13):8172-80. doi: 10.1167/iovs.13-13233.
Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression.
Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections.
Proteinase silencing reduced epithelial wound healing time versus rAV-enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α₃β₁ integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain.
Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.
糖尿病角膜过度表达包括基质金属蛋白酶-10（M10）和组织蛋白酶 F（CF）在内的蛋白水解酶。我们的目的是评估使用重组腺病毒（rAV）驱动的短发夹 RNA（rAV-sh）在器官培养的糖尿病角膜中沉默 M10 和 CF 是否会使缓慢的伤口愈合以及糖尿病和干细胞标志物表达正常化。
将 16 对器官培养的尸检人类糖尿病角膜（每组 4 对）用 rAV-sh 处理。单独或联合（组合）使用 rAV 驱动的 c-met 基因过表达，分别或同时沉默蛋白酶基因。对照角膜接受 rAV-EGFP。定量 RT-PCR 证实了短发夹 RNA（shRNA）沉默效果。转染后 10 天，用正庚烷制作 5-mm 上皮伤口，并记录愈合时间。通过角膜冷冻切片的免疫荧光研究糖尿病、信号和推定的干细胞标志物。
与 rAV-增强型绿色荧光蛋白（EGFP）对照相比，蛋白酶沉默减少了上皮伤口愈合时间（rAV-shM10 为 23%，rAV-shCF 为 31%，rAV-shM10+rAV-shCF 为 36%）。组合治疗更有效（减少 55%）。rAV-sh 处理后，糖尿病标志物（α₃β₁整合素和 nidogen-1）以及激活的表皮生长因子受体及其信号靶标激活的 Akt 的染色模式正常化。组合治疗还恢复了正常的激活 p38 染色。所有治疗方法，尤其是联合治疗方法，增加了糖尿病改变的推测性角膜缘干细胞标志物ΔNp63α、ABCG2、角蛋白 15 和 17 以及层粘连蛋白γ3 链的染色。
在糖尿病角膜中过度表达的蛋白酶的短发夹 RNA 沉默增强了角膜上皮和干细胞标志物的染色，并加速了伤口愈合。与 c-met 过表达联合治疗更为有效。特定的角膜基因治疗具有治疗糖尿病角膜病变的潜力。
