敲低LOXL1基因对HLECs中弹性蛋白表达及细胞生物学行为的抑制作用  

Inhibitory effect of knocking down LOXL1 gene on elastin expression and cell biological behavior in HLECs

作　　者：董月 玛依努[1] 易湘龙[1] Dong Yue;Ma Yinu;Yi Xianglong(Department of Ophthalmology,The First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China)

机构地区：[1]新疆医科大学第一附属医院眼科,乌鲁木齐830054

出　　处：《中华实验眼科杂志》2024年第9期807-813,共7页Chinese Journal Of Experimental Ophthalmology

基　　金：新疆维吾尔自治区自然科学基金(2023D01C102)。

摘　　要：目的探讨敲低赖氨酰氧化酶样蛋白1(LOXL1)基因对人晶状体上皮细胞(HLECs)中弹性蛋白的表达、聚集程度以及对HLECs增生活力及迁移能力的影响。方法体外培养人晶状体上皮细胞系HLE-B3,将细胞分为shLOXL1-1组、shLOXL1-2组、shLOXL1-3组和正常对照组,通过不同序列的慢病毒转染方法对shLOXL1-1组、shLOXL1-2组和shLOXL1-3组细胞进行LOXL1基因敲低干预,对正常对照组进行无意义序列慢病毒转染干预。采用实时荧光定量PCR法检测各转染组细胞的LOXL1 mRNA相对表达量;采用免疫荧光法检测HLE-B3中弹性蛋白的荧光强度;采用Western blot法检测HLE-B3中弹性蛋白的表达;采用电子显微镜扫描法检测HLE-B3中弹性蛋白的含量及聚集程度;采用细胞划痕实验检测HLE-B3的迁移率;采用细胞计数试剂盒8法检测HLE-B3的增生活力。结果敲低LOXL1基因后,shLOXL1-1、shLOXL1-2、shLOXL1-3组LOXL1 mRNA相对表达量均低于正常对照组,差异均有统计学意义(均P<0.001),其中shLOXL1-3组敲低效果最佳,故选取shLOXL1-3组与正常对照组进行后续实验并对比。免疫荧光染色后可见弹性蛋白稳定表达于HLE-B3细胞中,shLOXL1-3组弹性蛋白的平均荧光强度为56.96±5.56,明显低于正常对照组的80.52±4.78,差异有统计学意义(t=5.572,P<0.001)。shLOXL1-3组弹性蛋白相对表达量为0.807±0.002,明显低于正常对照组的1.185±0.064,差异有统计学意义(t=5.802,P<0.01)。电子显微镜下可见shLOXL1-3组弹性蛋白密度低于正常对照组,但其形态、大小、聚集程度未见明显改变。培养24 h和48 h时shLOXL1-3组细胞相对迁移率为0.292±0.041和0.439±0.032,均低于正常对照组的0.463±0.017和0.719±0.007,差异均有统计学意义(t=8.178、2.611,均P<0.05)。培养24、48、72、96 h,shLOXL1-3组细胞活力值均低于正常对照组,差异均有统计学意义(t=2.555、2.704、6.695、7.266,均P<0.05)。结论在HLECs中,敲低LOXL1基因可引起弹性蛋白ObjectiveTo investigate the effect of knocking down the lysyl oxidase-like protein 1(LOXL1)gene on the expression and aggregation of elastin in the human lens epithelial cells(HLECs),as well as its impact on the proliferation activity and migration ability of HLECs.MethodsThe human lens epithelial cell line HLE-B3 was cultured in vitro and divided into shLOXL1-1 group,shLOXL1-2 group,shLOXL1-3 group,and normal control group.The shLOXL1-1 group,shLOXL1-2 group,and shLOXL1-3 group cells were subjected to LOXL1 gene knockdown intervention using different sequences of lentiviral transfection methods,while the normal control group was subjected to meaningless sequence lentiviral transfection intervention.The relative expression level of LOXL1 mRNA in different groups was detected by real-time fluorescence quantitative PCR.The fluorescence intensity of elastin in HLE-B3 was determined by immunofluorescence.The expression of elastin in HLE-B3 was detected by Western blot.The content and aggregation degree of elastin in HLE-B3 was detected by electron microscopy scanning.The migration rate of HLE-B3 was detected by cell scratch assay.The proliferation activity of HLE-B3 was detected using the cell counting kit 8.ResultsAfter knocking down the LOXL1 gene,the relative expression levels of LOXL1 mRNA were lower in the shLOXL1-1,shLOXL1-2,and shLOXL1-3 groups than those in the normal control group,and the differences were statistically significant(all at P<0.001).The shLOXL1-3 group had the best knockdown effect,so the shLOXL1-3 group was selected for subsequent experiments and compared with the normal control group.After immunofluorescence staining,stable expression of elastin was observed in HLE-B3 cells.The average fluorescence intensity of elastin in the shLOXL1-3 group was 56.96±5.56,significantly lower than 80.52±4.78 in the normal control group(t=5.572,P<0.001).The relative expression level of elastin in the shLOXL1-3 group was 0.807±0.002,significantly lower than 1.185±0.064 in the normal control group(t=5.802,P<

关 键 词：赖氨酰氧化酶样蛋白1 剥脱综合征 弹性蛋白 迁移 增生 

分 类 号：R77[医药卫生—眼科]