目的建立 Grx2基因敲除(KO)和基因敲入(KI)小鼠模型，探讨 Grx2基因在白内障发病机制中的作用。
方法选取黑色C57BL/6J鼠各5只，利用CRISPR/Cas9系统分别制作 Grx2 KO小鼠模型和 Grx2 KI小鼠模型，后代小鼠剪尾测序后根据基因型纳入对应分型，观察并比较各基因型小鼠一般情况和晶状体混浊情况。处死各型小鼠，采用苏木精－伊红染色法观察小鼠晶状体病理学改变；采用ELISA法测定小鼠晶状体活性氧簇(ROS)和8-羟基脱氧鸟苷(8-OHdG)含量；采用Western blot法测定小鼠晶状体中Grx2、谷胱甘肽(GSH)、B淋巴细胞瘤-2(Bcl-2)、二硫化谷胱甘肽(GSSG)和Bcl-2相关X蛋白(Bax)蛋白相对表达量。
结果剪尾巢式PCR及基因测序证实获得 Grx2 KO和 Grx2 KI纯合子及杂合子小鼠后代。与同龄野生型(WT)小鼠相比， Grx2 KO纯合子小鼠晶状体混浊提前，而 Grx2 KI纯合子小鼠晶状体一直维持透明。苏木精－伊红染色结果显示，5月龄 Grx2 KO小鼠晶状体纤维出现大量缝隙和空泡。5月龄 Grx2 KO小鼠晶状体8-OHdG含量为(3.886±0.326)ng/ml，高于WT小鼠的(3.531±0.250)ng/ml，差异有统计学意义( t＝2.711， P＝0.033)； Grx2 KO小鼠晶状体ROS荧光强度为1 594±132，高于WT小鼠的1 157±123，差异有统计学意义( t＝3.384， P＝0.028)。Western blot结果显示，5月龄 Grx2 KO小鼠晶状体Grx2、GSH和Bcl-2相对表达量分别为0.23±0.01、0.70±0.06和0.32±0.03，较WT小鼠的0.52±0.02、1.04±0.08和0.49±0.04降低，差异均有统计学意义( t＝2.815， P＝0.020； t＝2.457， P＝0.033； t＝2.279， P＝0.041)。
结论本实验成功制作 Grx2 KO和 Grx2 KI小鼠模型， Grx2 KO小鼠年龄相关性白内障的发生和发展加速。
ObjectiveTo explore the role of Grx2 in the pathogenesis of cataract by establishing Grx2 knockout (KO) and knockin (KI) mouse models.
MethodsTen black C57BL/6J mice were selected to make Grx2 KO model ( n=5) and Grx2 KI model ( n=5) using CRISPR/Cas9 system.The offspring mice were sequenced by tail clipping and included in the corresponding experimental group according to the genotype.The general condition and lens opacity was recorded.After the mice were sacrificed, the pathological changes of lens were observed by hematoxylin-eosin staining.The contents of reactive oxygen species (ROS) and 8-hydroxy-desoxyguanosine (8-OHdG) were analyzed by enzyme-linked immunosorbent assay (ELISA).The relative expression levels of Grx2, glutathione (GSH), B-cell lymphoma-2 (Bcl-2) , glutathione disulfide (GSSG) and Bcl-2-associated X protein (Bax) in mice lens were assayed.The use and feeding of experimental animals were in accordance with the Regulations on the Management of Experimental Animals issued by the State Science and Technology Commission.The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University (No.2020-125).
ResultsThe offspring of Grx2 KO and Grx2 KI homozygous and heterozygous mice were confirmed by tail cutting nested PCR and gene sequencing.Compared with the wild type (WT) mice of same age, the lens opacity of Grx2 KO heterozygous mice occurred earlier, while the lens of Grx2 KI homozygous mice remained transparent all the time.A large number of gaps and vacuoles were found in the lens fibers of 5-month-old Grx2 KO mice.The 8-OHdG content and ROS fluorescence intensity in the lens of 5-month-old Grx2 KO mice were (3.886±0.326)ng/ml and 1 594±132, which were significantly higher than (3.531±0.250)ng/ml and 1 157±123 in WT mice ( t=2.711, P=0.033; t=3.384, P=0.028).The relative expression levels of Grx2, GSH and Bcl-2 in the lens of 5-month-old Grx2 KO mice were 0.23±0.01, 0.70±0.06 and 0.32±0.03, which were significantly lower than 0.52±0.02, 1.04±0.08 and 0.49±0.04 of WT mice ( t=2.815, P=0.020; t=2.457, P=0.033; t=2.279, P=0.041).
Conclusions Grx2 KO and Grx2 KI mouse models are successfully established in this study.The occurrence and development of age-related cataract are accelerated in Grx2 KO mice.
郭勇,郭辰峻,张婕,等. Grx2基因敲除小鼠晶状体混浊模型的建立及 Grx2在白内障发病机制中的作用 [J]. 中华实验眼科杂志,2022,40(10)：894-901.
DOI:10.3760/cma.j.cn115989-20210308-00149版权归中华医学会所有。
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郭勇：参与实验设计、实施研究、论文撰写；郭辰峻：实验操作、论文修改；张婕：论文修改；宁小娜：统计数据；严宏：实验设计、论文修改及定稿；陈曦：动物饲养
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