Col X基因表达下调对鹿茸干细胞向软骨细胞分化的影响

发布时间：2018-06-29 10:24

  本文选题：X型胶原蛋白(Col + X)　； 参考：《农业生物技术学报》2017年05期

【摘要】：X型胶原蛋白(collagen type X,Col X)是影响软骨内成骨的关键因子之一。为研究Col X在鹿茸软骨内成骨中的作用,本研究采用RNA技术(RNA interface,RNAi)下调鹿茸干细胞中Col X的表达并研究离体情况下Col X下调后鹿茸软骨内成骨的变化。首先,本研究采集了生茸区骨膜组织(antlerogenic periosteum,AP)并进行原代培养,同时利用间充质干细胞表面标记物(Stro-1)对AP细胞进行定性分析。其次,针对东北梅花鹿(Cervus nippon)Col X mRNA,设计了4对特异性的sh RNA(S1~S4),并通过酶切连接法将其构建至慢病毒载体pLVTHM中。利用293T细胞包装慢病毒、收集浓缩病毒后利用悬液法感染AP细胞,同时利用微粒培养对其进行成软骨诱导。成软骨诱导培养21 d后,利用qRT-PCR和Western blot技术验证Col X的表达变化,最后通过组织切片结合HE染色和阿辛蓝染色鉴定成软骨过程变化。结果表明,本研究成功构建了包含sh RNA(S1~S4)的pLVTHM载体,慢病毒包装成功且病毒滴度达到1×108TU/m L。qRT-PCR和Western blot结果表明,4组sh RNA均能显著下调微粒体中Col X的表达(P0.05)。阿辛蓝染色发现,与阴性对照相比Col X下调后微粒体基质中阿辛蓝着色显著减少,说明基质中酸性粘多糖比例下降。同时,HE染色表明在粘多糖下降的同时伴随着部分软骨细胞的死亡表现为基质中无细胞陷窝空泡的增加。因此,综合判断Col X下调显著抑制或减缓微粒体成软骨过程,本研究为进一步研究Col X在哺乳动物软骨内成骨过程的作用提供理论依据。
[Abstract]:Type X collagen (collagen type XCol X) is one of the key factors affecting endochondral osteogenesis. In order to study the role of Col X in the osteogenesis of antler cartilage, RNA interface RNAi (RNAi) was used to down-regulate the expression of Col X in pilose antler stem cells and to study the changes of osteogenesis in antler cartilage after down-regulation of Col X in vitro. Firstly, antlerogenic peristeal AP was collected and cultured in primary culture, and the AP cells were qualitatively analyzed using Stro-1 as a surface marker of mesenchymal stem cells. Secondly, four pairs of specific sh RNAs (S1nS4) were designed for Cervus nippon Col X mRNAs and constructed into lentivirus vector pLVTHM by restriction endonuclease ligation. The lentivirus was packaged with 293T cells, then concentrated virus was collected and infected with AP cells by suspension method. Meanwhile, cartilage formation was induced by microparticle culture. After 21 days of chondrogenic induction and culture, the expression of ColX was confirmed by qRT-PCR and Western blot. Finally, the changes of cartilage formation were identified by tissue sections combined with HE staining and acinyl blue staining. The results showed that pLVTHM vector containing sh RNA (S1hS4) was successfully constructed. The results of lentivirus packaging and viral titer up to 1 脳 108TU / m L.qRT-PCR and Western blot showed that the expression of Col X in microsomes was significantly down-regulated in the four groups (P0.05). The results of acinyl blue staining showed that the percentage of acid mucopolysaccharide in microsomal matrix decreased significantly after down-regulation of Col X compared with negative control. At the same time, HE staining showed that the decrease of mucopolysaccharide was accompanied by the death of some chondrocytes. Therefore, the down-regulation of Col X could significantly inhibit or slow down the process of microsomal chondrogenesis. This study provides a theoretical basis for further study of the role of Col X in the process of endochondral osteogenesis in mammals.
【作者单位】： 中国农业科学院特产研究所/特种经济动物分子生物学国家重点实验室/吉林省鹿茸工程研究中心;吉林农业大学中药材学院;
【基金】：吉林省自然科学基金(No.20170101032JC) 中国农业科学院科技创新工程项目(No.CAAS-ASTIP-2014-ISAPS-04) 吉林省科技发展计划项目(No.20140204010YY)
【分类号】：S825
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