神经元五环素1对腰椎间盘突出症术后瘢痕成纤维细胞增殖和迁移的影响  

Effect of neuronal pentraxin 1 gene on keloid after lumbar and back surgery

作　　者：陈啸[1] 褚言琛[1] 迟静薇[2] 张文洁[3] 刘波[1] 王志杰[1] Chen Xiao;Chu Yanchen;Chi Jingwei;Zhang Wenjie;Liu Bo;Wang Zhijie(Department of Spinal Surgery,the Affiliated Hospital of Qingdao University,Qingdao 266555,China;Department of Thyroid Lab,the Affiliated Hospital of Qingdao University,Qingdao 266555,China;Department of Hospital Infection Management,the Affiliated Hospital of Qingdao University,Qingdao 266005,China)

机构地区：[1]青岛大学附属医院脊柱外科,266555 [2]青岛大学附属医院甲状腺实验室,266555 [3]青岛大学附属医院医院感染管理部,266005

出　　处：《中华实验外科杂志》2021年第3期523-525,共3页Chinese Journal of Experimental Surgery

基　　金：山东省重点研发计划项目(2018GSF118080);青岛市科技惠民专项课题(19-6-1-16-nsh)。

摘　　要：目的观察NPTX-1基因对腰背部术后瘢痕成纤维细胞增殖及迁移的影响。方法收集2018年9月至2020年9月于青岛大学附属医院诊断为腰椎间盘突出症术后并接受取内固定的二次手术患者的瘢痕组织6例。体外分离培养瘢痕成纤维细胞;将瘢痕成纤维细胞分为实验组(A组)和对照组(B组),其中A组转染敲除NPTX-1基因的小干扰RNA(siRNA),B组转染NPTX-1基因的阴性对照序列(NC)。采用实时荧光定量聚合酶链反应(RT-qPCR)和蛋白质印迹(Western blot)检测NPTX-1和PCNA的表达。细胞计数试剂盒(CCK8)检测细胞增殖能力。细胞迁移实验(Transwell实验)检测细胞迁移能力。采用独立样本t检验比较两组的均数。结果RT-qPCR结果显示,A组细胞NPTX-1表达低于B组(0.18±0.03比1.56±0.04,t=49.691,P<0.01),差异有统计学意义。Western blot结果显示,A组细胞NPTX-1蛋白表达低于B组(0.29±0.04比0.68±0.01,t=15.302,P<0.05),差异有统计学意义。CCK-8结果显示,A组细胞在24、48、72 h的490 nm吸光度(A490 nm)值低于B组(0.93±0.04比1.06±0.05、1.42±0.05比1.66±0.08、1.65±0.06比1.99±0.07,t=4.469、4.958、6.192,P<0.01),差异有统计学意义。RT-qPCR结果显示,A组细胞PCNA表达低于B组(0.36±0.02比0.96±0.01,t=55.164,P<0.01),差异有统计学意义。Western blot结果显示,A组细胞PCNA蛋白表达低于B组(0.25±0.04比0.45±0.03,t=6.871,P<0.05),差异有统计学意义。Transwell结果显示,A组细胞迁移数量低于B组[(138.67±4.51)个比(275.00±5.00)个,t=35.072,P<0.01],差异有统计学意义。结论下调瘢痕成纤维细胞的NPTX-1基因可抑制瘢痕成纤维细胞的增殖能力和迁移能力,并抑制PCNA的表达。Objective To observe the influence of neuronal pentraxin 1(NPTX-1)gene on the proliferation and metastasis of keloid fibroblasts after lumbar back surgery.Methods From September 2018 to September 2020,6 cases of keloid tissues were collected from patients who underwent internal fixation after lumbar process in the West Coast District of Qingdao University Affiliated Hospital.The patients were all womens whose age was between 40-50 years old.The keloid fibroblasts were separated and cultured in vitro and then divided into experimental group(group A)and control group(group B),in which group A was transfected with small interfering RNA(siRNA)to knockout NPTX-1 gene and group B was transfected with negative control NC.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was performed to measure the messenger ribonucleic acid(mRNA)expression of NPTX-1 and proliferating cell nuclear antigen(PCNA)in groups A and B.Western blotting was used to measure the protein expression of NPTX-1 and PCNA in groups A and B.Cell counting kit-8(CCK-8)assay was applied to test the fibroblast proliferation ability in both groups and verify the effect of NPTX-1 gene on keloid fibroblast proliferation.Transwell assay was conducted to measure the number of migrating fibroblasts in both groups and verify the effect of NPTX-1 gene on the migration of keloid fibroblasts.The independent samples t test was used to compare the means of the two groups.Results RT-qPCR and Western blotting showed that as compared with group B,the mRNA and protein expression of NPTX-1 gene decreased significantly in group A(0.18±0.03 vs.1.56±0.04,0.29±0.04 vs.0.68±0.01,t=49.691,15.302,P<0.01).The results of CCK-8 assay revealed that the A490 nm value in group A at 24,48 and 72 hwas significantly lower than that in group B(0.93±0.04 vs.1.06±0.05,1.42±0.05 vs.1.66±0.08,1.65±0.06 vs.1.99±0.07,t=4.469,4.958,6.192,P<0.01)and the proliferation ability decreased obviously,which also showed a significant difference in statistics.RT-qPCR an

关 键 词：腰背部 术后 瘢痕成纤维细胞 基因敲除 细胞增殖 细胞迁移 细胞增殖核抗原 

分 类 号：R687.3[医药卫生—骨科学]