碳酸酐酶9对低氧诱导未足月胎儿视网膜微血管内皮细胞增殖影响的研究
Effect of carbonic anhydrase 9 on hypoxia-induced proliferation of retinal microvascular endothelial cells in preterm fetus
罗先琼 1范弯弯 2王宁 2陈娟 2马健3
作者信息
- 1. 广东省妇幼保健院遗传代谢与内分泌科,广州 511442
- 2. 广州医科大学研究生院,广州 510180
- 3. 广东省妇幼保健院转化医学中心,广州 511442
- 折叠
摘要
目的 探讨碳酸酐酶9(carbonic anhydrase 9,CA9)对低氧诱导未足月胎儿视网膜微血管内皮细胞(retinal microvascular endothelial cell,RMEC)增殖的影响.方法 获取广东省妇幼保健院自然流产未足月胎儿眼球,分离视网膜并得到RMEC,使用CD34进行内皮细胞鉴定.将得到的胎儿RMEC及购买的成人RMEC置于常氧及低氧培养箱(1％O2+5％CO2+94％N2)培养,使用实时荧光定量聚合酶链反应及蛋白质印迹法检测CA9的差异表达.使用小干扰RNA技术敲降CA9基因后,CCK-8法检测细胞增殖情况,加入CA9抑制剂U-104后CCK-8法检测细胞活率.结果 成功提取原代RMEC,第三代细胞CD34免疫荧光染色阳性细胞计数近100％.未足月胎儿及成人RMEC低氧组CA9 mRNA 表达量均高于常氧组(胎儿 67.80±10.31 比 1.00±0.04,P＜0.001;成人 1.72±0.22 比 1.00± 0.02,P=0.014);蛋白质印迹法检测可见未足月胎儿RMEC低氧组CA9表达量明显升高,与CA9 mRNA表达量相对应.在未足月胎儿RMEC转染siCA9 20 nM时,敲降效率可达95％(P＜0.001).CCK-8法检测敲降CA9基因48h后对未足月胎儿RMEC增殖的影响,可见siCA9组OD值低于阴性对照siNC组(0.57±0.05 比0.90±0.03,P＜0.001).加入 100 μM CA9抑制剂U-104,低氧+100 µM U-104组未足月胎儿RMEC细胞活率低于低氧组(99.16％±3.82％比119.10％±1.72％,P=0.002).结论 在低氧诱导模型中,CA9在成人及未足月胎儿的RMEC表达不同,抑制CA9可抑制未足月胎儿RMEC增殖.
Abstract
Objective We applied a hypoxia-induced model of human fetal retinal microvascular endothelial cell(RMEC)to study the effect of carbonic anhydrase 9(CA9)on cell proliferation.Methods The eyeballs of spontaneously aborted fetuses in Guangdong Women and Children's Hospital were obtained,and the retinas were isolated.RMEC was obtained by trypsin and collagenase two-step enzyme digestion,and endothelial cells were identified by CD34.The fetal RMEC and the purchased adult RMEC were cultured in normoxic and hypoxic incubators(1％O2+5％CO2+94％N2),and the expression of CA9 was detected by qPCR and Western blot.After knocking down the CA9 by small interference RNA technique,the cell proliferation was detected by CCK-8 method,and the cell viability was detected by CCK-8 after adding CA9 inhibitor U-104.Results The primary RMEC was extracted successfully.Immunofluorescence staining showed the percentage of CD34 positive cells in the third-generation cells was nearly 100％.The expression of CA9 mRNA in immature fetus and adult RMEC under hypoxia culture was higher than that under normoxic culture(fetal 1％O2 group vs.fetal 21％O2 group:67.80±10.31 vs.1.00±0.04,P＜0.001;adult 1％O2 group vs.adult 21％O2 group:1.72±0.22 vs.1.00±0.02,P=0.014).Western blot analysis showed significantly increased expression of CA9 in the fetal RMEC exposed to hypoxia,which aligned with the expression of CA9 mRNA.When fetal RMEC was transfected with siCA9 20 nM,the knockdown rate of CA9 was 95％(P＜0.001).CCK-8 assay showed significantly lower proliferation of fetal RMEC cells in siCA9 group compared to siNC group(0.57±0.05 vs.0.90±0.03,P＜0.001),which was reflected by the OD value.With the addition of 100 μM CA9 inhibitor U-104,the viability of fetal RMEC in the treated groupwas significantly lower than that in the untreated group(99.16％±3.82％vs.119.10％±1.72％,P=0.002).Conclusions The expression of CA9 differed between adult and preterm fetus in our hypoxia-induced RMEC model.Inhibiting CA9 can inhibit the proliferation of retinal microvascular endothelial cells of preterm fetus.
关键词
早产儿视网膜病/碳酸酐酶9/视网膜微血管内皮细胞/低氧/细胞增殖Key words
Retinopathy of prematurity/Carbonic anhydrase 9/Retinal microvascular endothelial cell/Hypoxia/Cell proliferation引用本文复制引用
基金项目
广东省自然科学基金项目(2019A1515011417)
广东省高水平医院建设专项(2019A1515011417)
出版年
2024
NETL
NSTL
维普
万方数据