2021 年第 9 期 第 16 卷

敲减SRY相关高迁移率族盒蛋白9基因下调Wnt/β-连环蛋白信号对气道肉芽组织成纤维细胞增殖和凋亡的影响

The effect of knocking down SRY-related high mobility group-box 9 gene to down-regulating Wnt/β-catenin signal on proliferation and apoptosis of airway granulation tissue fibroblasts

作者：罗戈雯张维何杰

英文作者：Luo Gewen Zhang Wei He Jie

单位：成都医学院临床医学院成都医学院第一附属医院呼吸与危重症医学科610500

英文单位：Department of Pulmonary and Critical Care Medicine Clinical Medical College and The First Affiliated Hospital of Chengdu Medical College Chengdu 610500 China

关键词：气道狭窄；SRY相关高迁移率族盒蛋白9基因；Wnt/β-连环蛋白信号通路；增殖；凋亡

英文关键词：Airwaystenosis;SRY-relatedhighmobilitygroup-box9gene;Wnt/β-cateninsignalingpathway;Proliferation;Apoptosis

• 摘要：

• 目的 探讨敲减SRY相关高迁移率族盒蛋白9（SOX9）基因下调Wnt/β-连环蛋白信号对气道肉芽组织成纤维细胞增殖和凋亡的影响。方法 应用基因芯片筛选出气道肉芽组织和气道正常黏膜组织差异表达的基因，用R软件进行分析，挑选最显著的差异基因SOX9作为研究对象，并借助逆转录聚合酶链反应技术验证气道肉芽组织及气道正常黏膜组织SOX9的表达。提取气道肉芽组织成纤维细胞，通过构建shRNA-SOX9慢病毒载体转染气道肉芽组织成纤维细胞，稳定感染shRNA-SOX9慢病毒的细胞标记为shRNA-SOX9（shRNA-SOX9组），稳定感染shRNA阴性对照的慢病毒的细胞标记为阴性对照组，无质粒载体加入的细胞设定为空白对照组。利用实时定量逆转录聚合酶链反应、蛋白质印迹法检测细胞内SOX9和Wnt/β-连环蛋白信号通路上下游基因表达情况；使用细胞计数试剂盒8检测成纤维细胞的活力；流式细胞术检测细胞凋亡率，组间做比较。结果 SOX9在气道肉芽组织中呈高表达，shRNA-SOX9慢病毒转染气道肉芽组织成纤维细胞后，SOX9和Wnt/β-连环蛋白信号通路相关基因表达均低于阴性对照组和空白对照组；敲减SOX9后，气道肉芽组织成纤维细胞的增殖能力下降，转染24、48、72 h后，shRNA-SOX9组气道肉芽组织成纤维细胞增殖抑制率明显高于阴性对照组和空白对照组，差异均有统计学意义（均P＜0.05）。shRNA-SOX9组细胞凋亡率高于空白对照组和阴性对照组［（16.9±2.0）%比（9.1±1.2）%、（10.1±1.8）%］，3组间差异有统计学意义（F=3.854，P=0.021）。结论 敲减SOX9可以通过调节Wnt/β-连环蛋白信号通路抑制气道肉芽组织成纤维细胞的增殖、促进凋亡。

• Objective To discuss the effect of knocking down SRY-related high mobility group-box 9(SOX9) gene to down-regulating Wnt/β-catenin signal on proliferation and apoptosis of airway granulation tissue fibroblasts. Methods Gene chips was used to screen differential gene expression in airway granulation tissue and normal airway mucosa tissue. The results of gene chips were analyzed by R software. The most significantly differentially expressed gene, SOX9, was selected and the expression of SOX9 in those two tissues was validated by reverse transcriptase-polymerase chain reaction. Airway granulation tissue fibroblasts were extracted and were transduced with shRNA-SOX9 lentiviral vector. The cells stably infected with shRNA-SOX9 were labeled as shRNA-SOX9(shRNA-SOX9 group), the cells stably infected with shRNA negative control were labeled as negative control group, and the cells without plasmid vector were set as blank control group. Real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction and western blotting were used to evaluated the expressions of SOX9 and Wnt/β-catenin signaling pathway upstream and downstream genes. The viability of fibroblasts was evaluated by cell counting kit-8. Apoptosis rate was detected by flow cytometry, and the results were compared among the groups. Results SOX9 was highly expressed in airway granulation tissue. The expressions of SOX9 and Wnt/β-catenin signaling pathway related genes of shRNA-SOX9 group were lower than those of negative control group and blank control group after the transfection of shRNA-SOX9 lentivirus; after knocking down SOX9, the proliferation of airway granulation tissue fibroblasts decreased, and the proliferation inhibition rate of airway granulation fibroblasts of shRNA-SOX9 group was significantly higher than that of negative control group and blank control group after 24, 48 and 72 h of transfection (all P<0.05). The apoptosis rate of shRNA-SOX9 group was significantly higher than that of negative control group and blank control group［（16.9±2.0）% vs （9.1±1.2）%,（10.1±1.8）%］(F=3.854, P=0.021). Conclusion knocking down SOX9 gene can inhibit proliferation of airway granulation tissue fibroblasts and promote apoptosis through modulation of Wnt/β-catenin signaling pathway.