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全综合征幼猪模型的肺发育变化过程。 Abstract:Objective To obtain the tissue specimens for transcriptome sequencing and bioinformatics analysis and screen for some HUB genes related to the lung development on the basis of an established early-onset scoliosis combined with thoracic insufficiency syndrome （EOS+TIS） piglet model and treatment model. Methods EOS+TIS and therapeutic animal models, perform HE and Masson staining were established to observe the lung tissue morphology and fibrosis degree, and the lung tissue of three groups （control group, model group, and treatment group） was sequenced. Differential analysis using DESeq2 in R software was performed and GO/KEGG enrichment analysis of differential genes using DAVID database, screen core genes were conducted so as to predict the related pathways and verify them through PCR and immunoblotting experiments. Results （1）HE staining results: The lung tissue of the model group showed the significant bronchopulmonary dysplasia, which was significantly improved in the treatment group; Masson staining results: The lung fibrosis degree of the model group was severe, and it was reduced in the treatment group. （2）DESeq2 analysis showed that there were 170 up-regulated and 262 down-regulated genes in the normal group compared with the model group, while there were 323 up-regulated and 467 down-regulated genes in the model group compared with the treatment group. （3）GO functional annotation showed that the differentially expressed genes were mainly enriched in extracellular matrix, plasma membrane composition, immune response, inflammatory response, calcium ion binding, cytokine activity, etc. KEGG showed that the differentially expressed genes were mainly enriched in neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction and other pathways. （4）The common gene THBS1 was screened out. （5）PCR and Western Blot experiments verified that THBS1 was down-regulated in the model group and up-regulated after the treatment （P < 0.05）. TGF -βexpression levels in the three groups detected with the use of Western Blot decreased indicated a decrease in the model group but an increase after the 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 um）
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 um ）
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 in 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 in 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 in 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（um） 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；组间比较，※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；组间比较，※※※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归一值 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；组间比较，※※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。组间比较，※P < 0.05。
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