杜洛克和二花脸猪耳软骨组织的差异比较分析

Comparative analysis of the differences in ear cartilage tissues between Duroc and Erhualian pigs

  • 摘要: 【目的】比较杜洛克和二花脸猪耳软骨组织的差异,为揭示猪外耳面积变异的分子机制提供参考。【方法】采集100日龄全同胞雄性杜洛克(n=5)和二花脸(n=5)猪外耳软骨组织进行石蜡切片及HE染色和Masson三色染色,对比两者结构上的区别;体外分离培养耳软骨细胞,使用CCK8法检测其增殖能力;联合转录组测序,比较二花脸(n=3)和杜洛克(n=3)耳软骨组织中基因表达的差异,筛选影响软骨生长相关的基因并使用实时荧光定量PCR(qRT-PCR)进行验证。【结果】 HE染色和Masson三色染色结果显示,与杜洛克相比,二花脸耳软骨细胞形成更多的由2~8个细胞组成的同源细胞群,软骨组织中含有更多的弹性纤维。细胞增殖检测试验表明,二花脸耳软骨细胞在体外培养36、48和60 h后增殖速率显著大于杜洛克(P<0.05)。转录组测序共筛选到251个差异表达基因(DEGs),其中杜洛克较二花脸上调基因165个,下调基因86个; GO功能注释显示上调基因主要富集于骨骼系统发育和细胞外基质等通路,下调基因主要富集于细胞对维甲酸应答和Wnt信号等通路。基因网络和功能分析表明,杜洛克耳软骨中高量表达的MMP3、BGLAPGDF5基因等促进软骨组织细胞外基质降解和矿化过程;二花脸耳软骨中Wnt信号通路抑制基因DKK1高量表达,Wnt信号强度受到负调控;高量表达的HMGA2、WNT10BAXIN2基因促进了二花脸外耳软骨细胞的增殖。qRTPCR验证了上述结果。【结论】二花脸耳软骨细胞较杜洛克具有更加旺盛的增殖分裂能力。杜洛克耳软骨通过MMP3、BGLAPGDF5等基因诱导细胞外基质降解和成骨分化过程;二花脸耳软骨组织中Wnt信号通路受到负调控,通过HMGA2、WNT10BAXIN2等基因促进软骨细胞的增殖及耳软骨的生长。

     

    Abstract: 【Objective】The comparison of the differences in ear cartilage tissues between Duroc and Erhualian pigs was conducted to provide insights into the molecular mechanisms underlying the variation in outer ear area in pigs.【Method】 Outer ear cartilage tissues from male sibling pigs of Duroc(n=5)and Erhualian(n=5),aged 100 days,were collected for paraffin sectioning. Hematoxylin-eosin(HE)staining and Masson trichrome staining were performed to observe and compare the structural differences between these two groups. Duroc and Erhualian ear chondrocytes were then isolated and cultured in vitro to detect cell proliferation ability using CCK8. Finally,transcriptome sequencing was combined with a comparison of gene expression differences in ear cartilage tissues of Erhualian(n=3)and Duroc(n=3). Genes related to cartilage growth were screened and verified by real-time fluorescence quantitative PCR(qRT-PCR).【Result】 HE and Masson trichrome stainings revealed that compared to Duroc,Erhualian ear chondrocytes formed more isogenous groups consisting of 2-8 cells,and the cartilage tissues contained more elastic fibers. The cell proliferation ability testing demonstrated that the proliferation rate of Erhualian ear chondrocytes was significantly higher than that of Duroc after 36,48, and 60 h of in vitro culture(P<0.05). A total of 251 differentially expressed genes(DEGs)were identified through transcriptome sequencing,including 165 up-regulated genes and 86 down-regulated genes of Duroc comparing with Erhualian. GO functional annotation revealed that the up-regulated genes were mainly enriched in pathways related to skeletal system development and extracellular matrix,while the down-regulated genes are mainly enriched in pathways associated with cellular response to retinoic acid and Wnt signaling. Gene network and functional analysis showed that MMP3, BGLAP and GDF5 genes with high expression in Duroc ear cartilage tissues promoted the degradation and mineralization of extracellular matrix in cartilage tissues. The Wnt signaling pathway inhibitory gene DKK1 was highly expressed in Erhualian ear cartilage,suggesting negative regulation of the Wnt signaling pathway. Highly-expressed HMGA2,WNT10B and AXIN2 genes promoted the proliferation of Erhualian outer ear chondrocytes. qRT-PCR verified above results.【Conclusion】The ear chondrocytes of Erhualian pigs exhibit a more robust proliferation and division ability compared to that of Duroc pigs. In Duroc ear cartilage,genes such as MMP3,BGLAP,and GDF5 induce extracellular matrix degradation and osteogenic differentiation processes. In Erhualian ear cartilage tissues,the Wnt signaling pathway is negatively regulated,and genes such as HMGA2,WNT10B and AXIN2 promote the proliferation of ear chondrocytes and the growth of ear cartilage.

     

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