Zhang Lifang, Qin Zhenwei, Lyu Danni, Lu Bing, Chen Zhijian, Fu Qiuli, Yao Ke
Eye Center of the 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou, China.
Department of Ophthalmology, The Affiliated People's Hospital of Ningbo University, Ningbo, China.
Front Pharmacol. 2022 Aug 19;13:959978. doi: 10.3389/fphar.2022.959978. eCollection 2022.
Our previous study observed that human induced pluripotent stem cell (HiPSC)-derived lentoid bodies (LBs) became cloudy with extended culture time, partially mimicking the progress of human age-related cataracts (ARCs) in a dish. In the present study, lanosterol, a potential anticataract drug, was used to further verify the value of this model in drug screening for cataract treatment. Mature LBs on day 25, which were differentiated from HiPSCs using the "fried egg" method, were continually cultured and treated with either dimethyl sulfoxide (control) or lanosterol. The LBs' shape and opacity alterations were examined using light microscopy and mean gray value evaluation. The soluble and insoluble proteins were examined through SDS-PAGE gel electrophoresis combined with Coomassie blue staining. The protein aggregations were examined with immunofluorescence. The mature LBs became cloudy with an extended culture time, and the opacification of the LBs was partially prevented by lanosterol treatment. There was less increase in insoluble proteins in the lanosterol-treated LBs than in the control group. There were also fewer cells containing aggregated protein (αA-crystallin and αB-crystallin) puncta in the lanosterol-treated LBs than in the control LBs. It was found that the opacification of LBs could be delayed by lanosterol treatment, which could be achieved by reducing protein aggregation, suggesting a promising HiPSC-derived drug-screening model for Age-related cataract.
我们之前的研究观察到，人诱导多能干细胞（HiPSC）衍生的晶状体样小体（LB）随着培养时间的延长会变得浑浊，在培养皿中部分模拟了人类年龄相关性白内障（ARC）的进展。在本研究中，羊毛甾醇作为一种潜在的抗白内障药物，被用于进一步验证该模型在白内障治疗药物筛选中的价值。使用“煎蛋”方法从HiPSC分化而来的第25天的成熟LB，持续培养并用二甲基亚砜（对照）或羊毛甾醇处理。使用光学显微镜和平均灰度值评估来检查LB的形状和不透明度变化。通过SDS-PAGE凝胶电泳结合考马斯亮蓝染色来检测可溶性和不溶性蛋白质。用免疫荧光检查蛋白质聚集情况。随着培养时间延长，成熟LB变得浑浊，而羊毛甾醇处理可部分阻止LB的浑浊化。与对照组相比，羊毛甾醇处理的LB中不溶性蛋白质的增加较少。在羊毛甾醇处理的LB中，含有聚集蛋白（αA-晶状体蛋白和αB-晶状体蛋白）斑点的细胞也比对照LB中的少。研究发现，羊毛甾醇处理可延迟LB的浑浊化，这可通过减少蛋白质聚集来实现，这表明该HiPSC衍生模型在年龄相关性白内障药物筛选方面具有前景。
