早发性脊柱侧凸合并胸廓发育不良综合征幼猪模型的肺发育相关分子分析  

Molecular Analysis on the Molecular Mechanism of Lung Development in the Piglet Model of Early Onset Scoliosis Complicated with Thoracic Insufficiency Syndrome

作　　者：代兴飞 李啟堂 严荣爽 张清东 潘呈 费德锐 马裕俭 徐世鑫 张颖[1] DAI Xingfei;LI Qitang;YAN Rongshuang;ZHANG Qingdong;PAN Cheng;FEI Derui;MA Yujian;XU Shixin;ZHANG Ying(Dept.of Orthopedics,The 2nd Affiliated Hospital of Kunming Medical University,Kunming Yunnan 650101;Dept.of Orthopedics,Yuxi Hospital Affiliated to Kunming University of Science and Technology/The 2nd People’s Hospital of Yuxi,Yuxi Yunnan 653100,China)

机构地区：[1]昆明医科大学第二附属医院骨科,云南昆明650101 [2]昆明理工大学附属玉溪医院/玉溪市第二人民医院骨科,云南玉溪653100

出　　处：《昆明医科大学学报》2024年第5期49-59,共11页Journal of Kunming Medical University

基　　金：国家自然科学基金资助项目(82060414);云南省科技厅-昆明医科大学应用基础研究联合专项基金资助项目(02101AY070001-150);云南省“兴滇英才支持计划”基金资助项目(XDYC-MY-2022-0026)。

摘　　要：目的基于已建立的早发性脊柱侧凸合并胸廓发育不良综合征(EOS+TIS)幼猪模型及治疗模型获取组织标本,进行转录组测序,生物信息学分析。筛选出影响肺发育相关的部分HUB基因。方法建立EOS+TIS及治疗动物模型,进行HE及Masson染色观察肺组织形态及纤维化程度,并对3组(对照组、模型组、治疗组)肺组织测序。利用R软件的DESeq2进行差异分析,运用DAVID数据库进行差异基因的GO/KEGG富集分析,筛选核心基因,预测相关通路,并通过PCR和免疫印迹实验进行验证。结果(1)HE染色结果:模型组肺组织体现了显著的支气管肺发育不良,治疗组获得明显改善;Masson染色结果:模型组肺纤维化程度较重,治疗组减轻;(2)DESeq2分析表明,正常组与模型组有170个上调和262个下调基因,而模型组与治疗组有323个上调和467个下调基因;(3)GO功能注释显示差异基因主要富集在细胞外基质、质膜组成、免疫应答、炎症反应、钙离子结合、细胞因子活性等功能。KEGG显示差异基因主要富集在神经活性配体-受体相互作用、细胞因子-细胞因子受体相互作用等通路;(4)筛选出共同基因THBS1;(5)PCR和Western Blot实验验证,THBS1在模型组中下调,治疗后上调(P<0.05),使用Western Blot实验检测TGF-β在3组中的表达量,模型组下降,治疗后上升(P<0.05)。结论THBS1与TGF-β参与了早发性脊柱侧凸合并胸廓发育不良综合征幼猪模型的肺发育变化过程。Objective To identify key HUB genes involved in lung development through transcriptomic sequencing and bioinformatics analysis using tissue samples collected from an established piglet model of early-onset scoliosis with thoracic insufficiency syndrome(EOS+TIS)and its treatment model.Methods EOS+TIS and treatment animal models were established,followed by histological analysis using HE and Masson staining to observe lung tissue morphology and fibrosis severity.Lung tissue samples from three groups(control,model,treatment)were sequenced.Differential analysis was performed using the DESeq2 package in R,and differential gene GO/KEGG enrichment analysis was conducted using the DAVID database.Core genes were identified,relevant pathways were predicted,and validation was done via PCR and Western blot experiments.Results(1)HE staining results:The model group displayed significant bronchopulmonary dysplasia,which was notably improved in the treatment group.(2)Masson staining results:The model group showed severe lung fibrosis,which was alleviated in the treatment group.(3)DESeq2 analysis:The normal vs.model group comparison identified 170 upregulated and 262 downregulated genes,while the model vs.treatment group comparison identified 323 upregulated and 467 downregulated genes.(4)GO functional annotation:Differential genes were mainly enriched in functions like extracellular matrix,plasma membrane composition,immune response,inflammatory response,calcium ion binding,and cytokine activity.KEGG enrichment:Differential genes were primarily enriched in pathways like neuroactive ligand-receptor interaction and cytokine-cytokine receptor interaction.(5)Common gene identification:THBS1 was identified as a common gene.(6)PCR and Western Blot validation:THBS1 was downregulated in the model group and upregulated post-treatment(P<0.05).Western Blot analysis revealed that TGF-βexpression was reduced in the model group and increased post-treatment(P<0.05).Conclusion THBS1 and TGF-βare involved in the process of lung development in

关 键 词：早发性脊柱侧凸 胸廓发育不良综合征 生物信息分析 THBS1 TGF-Β 

分 类 号：R682.13[医药卫生—骨科学]