Zhang Wenlin, Ogando Diego G, Kim Edward T, Choi Moon-Jung, Li Hongde, Tenessen Jason M, Bonanno Joseph A
School of Optometry, Indiana University, Bloomington, Indiana, United States.
Department of Biology, Indiana University, Bloomington, Indiana, United States.
Invest Ophthalmol Vis Sci. 2017 Jul 1;58(9):3723-3731. doi: 10.1167/iovs.17-21781.
To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched Slc4a11+/+ and Slc4a11-/- mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy.
We intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with Slc4a11+/+ and Slc4a11-/- C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na+/H+ exchange, bicarbonate, lactate, and Slc4a11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS).
The immortalized Slc4a11+/+ and Slc4a11-/- mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. Slc4a11-/- MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the Slc4a11-/- mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with Slc4a11+/+ MCECs.
This is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH3:H+ transporter activity in Slc4a11-/- MCECs. Furthermore, Slc4a11-/- MCECs recapitulate the glutaminolysis defects observed in Slc4a11-/- mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.
以基因匹配的Slc4a11+/+和Slc4a11-/-小鼠为模型，建立条件性永生化小鼠角膜内皮细胞系，用于研究与SLC4A11相关的先天性遗传性内皮营养不良（CHED）和Fuchs内皮角膜营养不良的病理及治疗方法。
我们将表达SV40大T抗原（tsTAg）的IFN-γ依赖性和温度敏感性突变体的H-2Kb-tsA58小鼠（永生化小鼠）与Slc4a11+/+和Slc4a11-/- C57BL/6小鼠进行杂交。通过倍增时间评估细胞系的生长特性。通过细胞内pH测量分析离子转运活性（Na+/H+交换、碳酸氢盐、乳酸和Slc4a11氨转运）。通过气相色谱质谱联用（GC-MS）分析三羧酸循环中间体来评估细胞系的代谢状态。
永生化的Slc4a11+/+和Slc4a11-/-小鼠角膜内皮细胞（MCECs）在传代49次时仍保持增殖，并维持相似的活性离子转运活性。正如预期的那样，增殖对温度敏感且依赖IFN-γ。与Slc4a11+/+ MCECs相比，Slc4a11-/- MCECs表现出增殖能力下降、NH3:H+转运减少、谷氨酰胺分解酶表达改变，类似于Slc4a11-/-小鼠，并且源自谷氨酰胺的三羧酸循环中间体比例降低，同时葡萄糖通量代偿性增加。
这是关于MCECs永生化的首次报道。除了Slc4a11-/- MCECs中的NH3:H+转运体活性外，永生化内皮细胞的离子转运仍保持活跃。此外，Slc4a11-/- MCECs重现了在Slc4a11-/-小鼠角膜内皮中观察到的谷氨酰胺分解缺陷，为研究与角膜内皮营养不良相关的SLC4A11突变的发病机制和筛选潜在治疗药物提供了一个极好的工具。
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