Molecular Analysis on the Molecular Mechanism of Lung Development in the Piglet Model of Early Onset Scoliosis Complicated with Thoracic Insufficiency Syndrome
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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。 Abstract: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-β expression was reduced in the model group and increased post-treatment （P < 0.05）. Conclusion THBS1 and TGF-β are involved in the process of lung development in the early onset scoliosis with thoracic insufficiency syndrome pig models. -
Key words:
- Early-onset scoliosis /
- Thoracic insufficiency syndrome /
- Bioinformatics analysis /
- THBS1 /
- TGF-β
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图 1 HE及Masson染色结果（标尺：100 μm）
A：对照组肺组织HE染色；B：模型组肺组织HE染色；C：治疗组肺组织HE染色；A1：对照组肺组织Masson染色；B1：模型组肺组织Masson染色；C1：治疗组肺组织Masson染色；红色代表肌纤维，蓝色为胶原纤维。
Figure 1. Results of HE and Masson staining （ scale bar 100 μm ）
图 2 3组肺泡计数、肺泡间隔厚度、平均胶原纤维含量测量结果
A：3组放射状肺泡计数、肺泡间隔厚度；B：3组平均胶原纤维含量。
Figure 2. Measurement results of three groups of alveolar count，alveolar septal thickness，and average collagen fiber content
图 3 差异基因的火山图和热图
A：模型组与对照组差异基因的火山图，绿色代表下调基因，红色代表上调基因；B：差异基因在模型组与对照组中的热图，蓝色为对照组（CON），红色为模型组（MX）；C：治疗组与模型组差异基因的火山图；D：差异基因在治疗组与模型组中的热图，蓝色为模型组（MX），红色为治疗组（ZL）。
Figure 3. Volcanic map and heat map of differentially expressed genes
图 4 模型组与对照组差异基因的GO和KEGG情况
A：生物学过程（BP）；B：细胞组分（CC）；C：分子功能（MF）；D：KEGG富集结果。
Figure 4. GO and KEGG enrichment analysis of differential genes between the model group and the control group
图 5 粉色为钙离子相关基因、紫色区域代表免疫相关基因、绿色区域代表细胞外基质相关基因
Figure 5. Pink represents calcium-related genes，purple area represents immune-related genes，and green area represents extracellular matrix-related genes
图 6 THBS1在3组中的基因相对表达量情况
Figure 6. Relative gene expression levels of THBS1 among the three groups
图 8 THBS1在3组中的蛋白相对表达量情况
Figure 8. Relative protein expression levels of THBS1 among the three groups
图 10 TGF-β在3组中的蛋白相对表达量情况
Figure 10. Relative protein expression levels of TGF-β among the three groups
表 1 THBS1及内参的引物序列
Table 1. Primer sequences of THBS1 and internal reference
序列 名称 长度（bp） CATCCAAAGCATCTTCAC THBS1（pig）-F 77 GTGAGAAGGACATTGGTA THBS1（pig）-R GTTCCAGTATGATTCCAC GAPDH（pig）-F 56 GACTCCACAACATACGTA GAPDH（pig）-R 
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表 2 3组幼猪肺组织HE和Masson染色的测量结果（$\bar x \pm s$）
Table 2. Results of HE and Masson staining in lung tissue of three groups of piglets （$\bar x \pm s$）
参数 对照组 模型组 治疗组 F P 放射状肺泡计数（个） 9.24±2.56 6.53±2.08*** 7.88±3.34# 19.25 <0.001※ 肺泡壁厚度（μm） 5.81±2.78 12.04±3.10*** 9.44±2.63## 88.37 <0.001※ 平均胶原纤维量 0.02±0.01 0.18±0.02*** 0.13±0.02### 1834.28 <0.001※ 与对照组比较，***P < 0.001；与模型组比较，#P < 0.05，##P < 0.01，###P < 0.001；※P < 0.001。
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表 3 钙离子相关基因
Table 3. Calcium ion-related genes
基因名称 LogFC P 校正后P FBN2 1.976806186 5.53E-47 1.71E-44 RCVRN −1.699843423 7.65E-10 1.89E-08 TENM2 −2.236357095 2.22E-07 3.50E-06 CLSTN2 −1.324235347 0.01174761 0.04453978 TNFAIP6 1.061241719 0.000194661 0.001447986 ACTN2 1.683023897 5.87E-08 1.04E-06 DGKB −2.383519856 1.67E-07 2.70E-06 PON1 2.571409628 6.39E-45 1.74E-42 VWDE 3.543226537 0.000610734 0.003897322 TUBB4A 1.433642182 0.007848305 0.032416471 THBS1 −1.256610433 6.47E-86 4.87E-83 THBS4 −3.280684277 0.000147676 0.001142492 MYL4 −4.865802484 6.08E-21 4.71E-19 SCGN 3.150720501 0.00280126 0.014003149 EGFLAM −1.048561742 0.000400331 0.002700883 VSNL1 1.482799905 6.29E-09 1.33E-07 SYT10 −3.851326165 0.006301379 0.027241397 EFHD1 −1.084242649 1.75E-05 0.000178469 ANXA9 −1.111630395 2.47E-12 8.42E-11 ASTN2 −1.202867655 6.21E-05 0.000542506 LOC100522787 1.087678946 0.009057674 0.036435259 PPEF2 1.557961567 0.003120276 0.015314764 UMODL1 1.277593564 0.006711926 0.028675953
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表 5 免疫应答相关基因
Table 5. Genes related to immune response
基因名称 LogFC P 校正后P CD274 −1.171256037 2.60E-17 1.49E-15 SLA-DRB1 1.337022797 3.35E-38 6.62E-36 JUN −1.850222012 6.44E-158 3.44E-154 CXCL8 −1.561364145 2.25E-38 4.50E-36 OSM −1.138466837 1.79E-05 0.000182168 CXCL14 −1.021217549 2.23E-09 5.16E-08 CXCL2 −1.789799638 1.19E-37 2.27E-35 THBS1 −1.256610433 5.47E-86 4.87E-83 IL1A −1.242147858 2.09E-21 1.64E-19 CCL8 −1.300951991 4.26E-26 4.32E-24 CTSL −2.367477476 5.27E-05 0.000470351 IL1B −1.733900902 8.48E-19 5.37E-17 SLA-2 1.181319808 3.31E-28 3.87E-26
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表 4 细胞外基质相关基因
Table 4. Genes related to extracellular matrix
基因名称 LogFC P 校正后P ASAH2 −1.681188858 0.010202271 0.039995696 TNFAIP6 1.061241719 0.000194661 0.001447986 PON1 2.571409628 6.39E-45 1.74E-42 WFIKKN2 1.000982632 6.38E-10 1.60E-08 KLK5 3.198981333 0.011602127 0.04414435 CD1E 1.412361441 1.11E-07 1.86E-06 CXCL14 −1.021217549 2.23E-09 5.16E-08 THBS1 −1.256610433 5.47E-86 4.87E-83 AREG −2.393896705 1.88E-06 2.43E-05 CXCL2 −1.789799638 1.19E-37 2.27E-35 THBS4 −3.280684277 0.000147676 0.001142492 ADAMTS4 −1.020844602 9.62E-09 1.97E-07 CCL8 −1.300951991 4.26E-26 4.32E-24 CTSL −2.367477476 5.27E-05 0.000470351 FLRT1 3.028606366 0.001234795 0.007069199 OTOG 1.419656249 0.001549784 0.008528336 CCL2 −1.003309962 1.86E-06 2.40E-05 NDP 1.297577098 0.001027728 0.006056795 APOB −1.67037521 0.000425658 0.002851356 XDH 4.945718658 2.01E-70 1.11E-67 COL22A1 −3.012702444 0.001677249 0.009126374 BMP8A −1.131393016 1.58E-09 3.74E-08 DKK1 −1.3841467 2.94E-10 7.75E-09 IL1A −1.242147858 2.09E-21 1.64E-19 IL6 −1.828458638 6.10E-07 8.74E-06 SFRP2 −2.214720482 0.012270295 0.046204564 TG 2.389166701 0.000128918 0.001023048 CILP −1.030855274 2.52E-12 8.58E-11 IL1B −1.733900902 8.48E-19 5.37E-17 IFNE −1.147986534 0.002583079 0.013078573 UPTI 1.310012634 0.00120919 0.006944936 DPEP1 −1.726138591 0.002018831 0.01068401 LOC100521998 −4.045174213 0.006775299 0.028877436 ACPP 1.016963504 0.00127865 0.007273515 UMODL1 1.277593564 0.006711926 0.028675953 CXCL8 −1.561364145 2.25E-38 4.50E-36
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表 6 THBS1基因相对表达量（$\bar x \pm s$）
Table 6. Relative gene expression levels of THBS1 （$\bar x \pm s$）
基因 对照组 模型组 治疗组 F P THBS1 1.04±0.07 0.53±0.06*** 2.08±0.07### 755.75 P < 0.001※※※ 与对照组比较，***P < 0.001；与模型组比较，###P < 0.001；※※※P < 0.001。
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表 7 THBS1蛋白相对表达量（$\bar x \pm s$ ）
Table 7. Relative protein expression levels of THBS1 （$\bar x \pm s$）
归一值 对照组 模型组 治疗组 F P THBS1 1.00±0.08 0.62±0.08** 0.77±0.10# 15.02 0.005※※ 与对照组比较，**P < 0.01；与模型组比较，#P < 0.05；※※P < 0.01。
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表 8 TGF-β蛋白相对表达量（$\bar x \pm s$）
Table 8. Relative protein expression levels of TGF-β （$\bar x \pm s$）
归一值 对照组 模型组 治疗组 F P TGFβ归一值 1.00±0.24 0.55±0.14* 0.91±0.05# 6.07 0.036※ 与对照组比较，*P < 0.05；与模型组比较，#P < 0.05；※P < 0.05。
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[1] Campbell R M Jr,Smith M D,Mayes T C,et al. The characteristics of thoracic insufficiency syndrome associated with fused ribs and congenital scoliosis[J]. J Bone Joint Surg Am,2003,85(3):399-408. [2] Cooney T P,W M Thurlbeck. The radial alveolar count method of emery and mithal: A reappraisal 2-intrauterine and early postnatal lung growth[J]. Thorax,1982,37(8):580-583. doi: 10.1136/thx.37.8.580 [3] Dogar A M,Semplicio G,Guennewig B,et al. Multiple microRNAs derived from chemically synthesized precursors regulate thrombospondin 1 expression[J]. Nucleic Acid Ther,2014,24(2):149-159. doi: 10.1089/nat.2013.0467 [4] Harpel J G,Schultz-Cherry S,Murphy-Ullrich J E,et al. Tamoxifen and estrogen effects on TGF-beta formation: Role of thrombospondin-1,alphavbeta3,and integrin-associated protein[J]. Biochem Biophys Res Commun,2001,284(1):11-14. doi: 10.1006/bbrc.2001.4922 [5] Bissinger R,Petkova-Kirova P,Mykhailova O,et al. Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance,survival,and deformability of human red blood cells[J]. Cell Commun Signal,2020,18(1):155. doi: 10.1186/s12964-020-00651-5 [6] Grosser J A,Maes M E,Nickells R W. Characteristics of intracellular propagation of mitochondrial BAX recruitment during apoptosis[J]. Apoptosis,2021,26(1-2):132-145. doi: 10.1007/s10495-020-01654-w [7] Jiang D,Guo B,Lin F,et al. Effect of THBS1 on the biological function of hypertrophic scar fibroblasts[J]. Biomed Res Int,2020,2020(1):8605407. [8] Lai Y H,Lee P Y,Lu C Y,et al. Thrombospondin 1-induced exosomal proteins attenuate hypoxia-induced paraptosis in corneal epithelial cells and promote wound healing[J]. FASEB J,2021,35(1):e21200. [9] 陈冲,封志纯. 支气管肺发育不良分子遗传学研究进展[J]. 实用儿科临床杂志,2012,27(16):1282-1284. [10] Inui N,Sakai S,Kitagawa M. Molecular pathogenesis of pulmonary fibrosis,with focus on pathways related to TGF-β and the ubiquitin-proteasome pathway[J]. Int J Mol Sci,2021,22(11):6107. doi: 10.3390/ijms22116107 [11] Chanda D,Otoupalova E,Smith S R,et al. Developmental pathways in the pathogenesis of lung fibrosis[J]. Mol Aspects Med,2019,65(1):56-69. doi: 10.1016/j.mam.2018.08.004 [12] Lawler J,Miao W M,Duquette M,et al. Thrombospondin-1 gene expression affects survival and tumor spectrum of p53-deficient mice[J]. Am J Pathol,2001,159(5):1949-1956. doi: 10.1016/S0002-9440(10)63042-8 [13] Zippel N,Malik R A,Fromel T,et al. Transforming growth factor-beta-activated kinase 1 regulates angiogenesis via AMP-activated protein kinase-alpha1 and redox balance in endothelial cells[J]. Arterioscler Thromb Vasc Biol,2013,33(12):2792-2799. [14] Xu S,Yang S,Sun G,et al. Transforming growth factor-beta polymorphisms and serum level in the development of osteosarcoma[J]. DNA Cell Biol,2014,33(1):802-806. doi: 10.1089/dna.2014.2527 [15] Rashid A,Zeng C,Motta-Ribeiro G,et al. Proteomics of lung tissue reveals differences in inflammation and alveolar-capillary barrier response between atelectasis and aerated regions[J]. Sci Rep,2022,12(1):7065. doi: 10.1038/s41598-022-11045-7 [16] Kaneshita S,Kida T,Yoshioka M,et al. CG223,a novel BET inhibitor,exerts TGF-β1-mediated antifibrotic effects in a murine model of bleomycin-induced pulmonary fibrosis[J]. Pulm Pharmacol Ther,2021,70(2):102057. doi: 10.1016/j.pupt.2021.102057 [17] Wang I M,Stepaniants S,Boie Y,et al. Gene expression profiling in patients with chronic obstructive pulmonary disease and lung cancer[J]. Am J Respir Crit Care Med,2008,177(4):402-411. doi: 10.1164/rccm.200703-390OC -
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