敲减烟酰胺磷酸核糖转移酶基因促进转化生长因子β_(1)诱导的人胚肺成纤维细胞自噬功能的观察

Observation of knockdown of nicotinamide phosphoribosyl transferase gene promoting human embryonic lung fibroblast autophagy function induced by transforming growth factor-β_(1)

目的探讨烟酰胺磷酸核糖转移酶(NAMPT)基因的表达对转化生长因子β_(1)(TGF-β_(1))诱导的人胚肺成纤维细胞系MRC-5细胞自噬功能的影响。方法基于基因表达综合数据库和人类自噬基因数据库,运用R软件筛选出GSE53845、GSE10667数据集中差异表达的自噬相关基因NAMPT。选择2018年1月至2020年3月于成都医学院第一附属医院就诊的50例经纤维支气管镜冷冻肺活检确诊的特发性肺纤维化(IPF)患者的病变肺组织标本作为IPF组,选择同期50例经外科手术治疗的早期肺腺癌患者的正常肺组织样本作为对照组,验证组间NAMPT的表达差异。将人胚肺成纤维细胞系MRC-5细胞分为空白对照组、TGF-β_(1)组、阴性对照组以及运用小干扰RNA敲减的NAMPT(si-NAMPT)组,应用蛋白质印迹法检测各组自噬相关蛋白表达,噻唑蓝比色法检测各组细胞活力,并应用单丹磺酰尸胺(MDC)染色对转染后细胞自噬体进行测定。结果IPF肺组织中NAMPT mRNA的相对表达量高于正常肺组织[(3.7±2.3)比(1.4±0.7)],差异有统计学意义(t=6.804,P=0.001)。TGF-β_(1)组MRC-5细胞NAMPT蛋白表达量明显高于空白对照组和si-NAMPT组[(1.178±0.087)比(0.461±0.070)、(0.564±0.066)](均P<0.05),与阴性对照组比较差异无统计学意义(P>0.05)。TGF-β_(1)组自噬相关蛋白表达水平明显低于空白对照组,si-NAMPT组明显高于TGF-β_(1)组,差异均有统计学意义(均P<0.05)。TGF-β_(1)组MRC-5细胞活力明显高于空白对照组和si-NAMPT组[(1.69±0.18)比(0.66±0.04)、(0.94±0.06)](均P<0.05)。MDC染色结果显示TGF-β_(1)组MRC-5细胞内自噬体荧光斑点基本消失,si-NAMPT组细胞内见大量自噬体荧光斑点。结论IPF组织中NAMPT呈现高表达,敲减NAMPT基因可以促进TGF-β_(1)诱导的MRC-5细胞的自噬功能并抑制细胞生长。

Objective To investigate the effectiveness of nicotinamide phosphoribosyl transferase(NAMPT)gene expression on the autophagy of human embryonic lung fibroblasts MRC-5 cells induced by transforming growth factor-β_(1)(TGF-β_(1)).Methods R software was used to screen out differentially expressed autophagy-related gene NAMPT in GSE53845 and GSE10667 datasets based on gene expression omnibus and human autophagy database.From January 2018 to March 2020,50 samples from lung tissue of patients with idiopathic pulmonary fibrosis(IPF)diagnosed by fiberoptic bronchoscope lung cryo biopsy in the First Affiliated Hospital of Chengdu Medical College were selected as the IPF group.The normal lung tissue samples of 50 patients with early lung adenocarcinoma treated by surgery were selected as the control group.Expression of NAMPT between the two groups were compared.The MRC-5 cells were divided into blank control group,TGF-β_(1)group,negative control(NC)group and small interfering RNA knocking down NAMPT(si-NAMPT)group.Western blotting was used to detected autophagy-related proteins,methyl thiazolyl tetrazolium assay was used to detected cell viability,and monodansylcadaverin(MDC)staining was used to detected autophagosome.Results The expression of NAMPT mRNA in lung tissue of IPF was higher than that in normal lung tissue[(3.7±2.3)vs(1.4±0.7)](t=6.804,P=0.001).The expression of NAMPT protein in MRC-5 cells in TGF-β_(1)group was higher than that in blank control group and si-NAMPT group[(1.178±0.087)vs(0.461±0.070),(0.564±0.066)](all P<0.05),and there was no significant difference between NC group and TGF-β_(1)group(P>0.05).The level of autophagy-related protein in TGF-β_(1)group was lower than that in blank control group,and that in si-NAMPT group was higher than that in TGF-β_(1)group(both P<0.05).MRC-5 cells viability in TGF-β_(1)group was higher than that in blank control group and si-NAMPT group[(1.69±0.18)vs(0.66±0.04),(0.94±0.06)](both P<0.05).MDC staining showed that autophagosome fluorescence spots in MRC-5 cells in TGF-β_(1)group almost disappeared,and a large number of autophagosome fluorescence spots were found in si-NAMPT group.Conclusions NAMPT is highly expressed in IPF tissue.Knockdown of NAMPT gene can promote autophagy and inhibit the growth of MRC-5 cells induced by TGF-β_(1).