靶向沉默DNMT3A对小鼠肺成纤维细胞胶原沉积、增殖及迁移活性的影响  

Effect of targeted silencing of DNMT3A on collagen deposition,proliferation and migration activity of mouse lung fibroblasts

作　　者：汪先晨 尤峻柏 凌辉 范家好 陈齐[2] 陶辉[2] 沙纪名[1] Wang Xianchen;You Junbo;Ling Hui;Fan Jiahao;Chen Qi;Tao Hui;Sha Jiming(Dept of Thoracic Surgery,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601;:2 Dept of Anesthesiology and Perioperative Medicine,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601)

机构地区：[1]安徽医科大学第二附属医院胸外科,合肥230601 [2]安徽医科大学第二附属医院麻醉与围手术期医学科,合肥230601

出　　处：《安徽医科大学学报》2025年第1期66-72,共7页Acta Universitatis Medicinalis Anhui

基　　金：安徽省高校自然科学研究项目(编号:2023AH040376)。

摘　　要：目的探讨靶向沉默DNA甲基化转移酶3A(DNMT3A)对小鼠肺成纤维细胞(PFs)胶原沉积、增殖与迁移活性的作用。方法为保证原代成纤维细胞的增殖迁移活性取新生C57乳鼠肺组织,剪碎后提取PFs,在显微镜下观察并鉴定形态。细胞贴壁后使用5 ng/ml TGF-β_(1)作用24 h诱导PFs细胞活化,并用小干扰RNA构建DNMT3A沉默模型;实验分为Control组、TGF-β_(1)组、TGF-β_(1)+siRNA-NC组和TGF-β_(1)+siRNA-DNMT3A组。应用Western blot方法检测DNMT3A、α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原(CollagenⅠ)的蛋白表达变化;应用实时荧光定量逆转录聚合酶链反应(RT-qPCR)方法检测DNMT3A、α-SMA、CollagenⅠ的mRNA表达变化;CCK-8与EdU染色检测PFs增殖能力;划痕实验与Transwell迁移实验检测PFs迁移能力。结果与Control组相比,TGF-β_(1)诱导活化的PFs细胞组中DNMT3A升高(P<0.01),纤维化与增殖相关指标α-SMA、CollagenⅠ的蛋白及mRNA水平也升高(均P<0.05),同时PFs的增殖与迁移能力升高(均P<0.0001)。而与siRNA-NC组相比,在DNMT3A沉默组中通过Western blot法检测DNMT3A(P<0.0001)及相关指标α-SMA(P<0.01)、CollagenⅠ(P<0.01)的蛋白表达水平均降低,同时通过RT-qPCR法检测DNMT3A、α-SMA及CollagenⅠ的mRNA水平也均降低(P<0.001),并且PFs细胞的增殖(P<0.01)和迁移能力(P<0.05)较对照组下降。结论沉默DNMT3A能够抑制胶原的沉积及PFs的增殖,DNMT3A可以促进PFs的增殖和迁移能力,进而促进PFs的活化以及肺纤维化的发展,这一过程可能受到DNA甲基化修饰的调控。Objective To investigate the effect of targeted silencing of DNA methyltransferase 3A(DNMT3A)on collagen deposition,proliferation and migration activity of mouse lung fibroblasts(PFs).Methods In order to ensure the proliferation and migration activity of primary fibroblasts,the lung tissues of neonatal C57 suckling mice were taken,PFs were extracted after being sheared,and the morphology was observed and identified under the microscope.PFs cells were activated by 5 ng/ml TGF-β_(1)for 24 h after cell attachment,and DNMT3A silencing model was constructed by small interfering RNA;The experiment was divided into control group,TGF-β_(1)group,TGF-β_(1)+siRNA-NC group and TGF-β_(1)+siRNA-DNMT3A group.The protein expressions of DNMT3A,α-smooth muscle actin(α-SMA)and CollagenⅠwere detected by Western blot;Real time quantitative reverse transcription polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression changes of DNMT3A,α-SMA and CollagenⅠ.The proliferation ability of PFs was detected by CCK-8 and EdU staining;the migration ability of PFs was detected by scratch test and Transwell migration test.Results Compared with the control group,TGF-β_(1)induced the increase of DNMT3A in the activated PFs cell group(P<0.01),the protein and mRNA levels of fibrosis and proliferation related indicatorsα-SMA and CollagenⅠalso increased(all P<0.05),and the proliferation and migration ability of PFs increased(all P<0.0001).Compared with the siRNA-NC group,the protein expression levels of DNMT3A(P<0.0001)and related indicatorsα-SMA(P<0.01)and CollagenⅠ(P<0.01)significantly decreased in the DNMT3A silencing group by Western blot,and the mRNA levels of DNMT3A,α-SMA and CollagenⅠby RT-qPCR also decreased(all P<0.001),and the proliferation(P<0.01)and migration ability(P<0.05)of PFs cells decreased compared with the control group.Conclusion Silencing DNMT3A can inhibit the deposition of collagen and the proliferation of PFs.DNMT3A can promote the proliferation and migration of PFs,and then promote the act

关 键 词：DNMT3A DNA甲基化 肺成纤维细胞 增殖 迁移 肺纤维化 

分 类 号：R332.2[医药卫生—人体生理学] R322.3[医药卫生—基础医学] R563.9