干扰SERBP1表达对山羊鼻甲骨原代细胞增殖、周期及凋亡的作用

doi:10.11843/j.issn.0366-6964.2022.08.028

摘要/Abstract

摘要： 旨在克隆山羊SERBP1基因，揭示干扰SERBP1基因后对山羊鼻甲骨原代细胞增殖、细胞周期和细胞凋亡的作用，为进一步研究羊口疮病毒诱导细胞凋亡的分子机制奠定基础。本研究以简州大耳羊为试验动物，利用RT-PCR技术克隆SERBP1基因cDNA序列并进行生物信息学分析；实时荧光定量PCR （RT-qPCR）检测SERBP1基因在不同组织的表达水平；并筛选有效干扰序列；MTT法检测siRNA干扰SERBP1对山羊鼻甲骨原代细胞增殖的影响，流式细胞仪检测siRNA干扰SERBP1对细胞周期和凋亡的影响，RT-qPCR检测对凋亡相关基因P53、Caspase7、Caspase3等的影响。成功扩增山羊SERBP1基因1 293 bp，包含5'UTR 44 bp，CDS区1 179 bp，3'UTR 70 bp，编码392个氨基酸残基，不存在信号肽，主要定位于细胞核；预测到SERBP1可能与RPL31、RPS3等10个蛋白存在相互作用。SERBP1基因在山羊胰腺中表达水平最高；筛选到干扰效率为70%的有效干扰序列；siRNA干扰SERBP1基因后抑制了山羊鼻甲骨原代细胞增殖和凋亡，且细胞被阻滞于G₀/G₁期；同时可下调Caspase3、Caspase7、BCL2L11和Bax基因mRNA的表达，而对Bcl-2、P53、PARP1基因mRNA的表达无显著影响。干扰山羊SERBP1基因抑制了山羊鼻甲骨原代细胞增殖和凋亡，并诱导细胞周期停滞。研究结果为进一步深入研究SERBP1基因功能，揭示ORFV125抑制细胞凋亡的分子机制提供重要的试验数据。

关键词: 山羊, SERBP1基因, 干扰, 细胞周期, 细胞凋亡

Abstract: The aim of this study was to clone goat SERBP1, and to reveal the role of interfering with SERBP1 gene on the proliferation, cell cycle and apoptosis of goat turbinate bone primary cells. These data may provide important experimental data for further mechanism study underlying cell apoptosis induced by Orf virus (ORFV). With the Jianzhou goats as the experimental animal, the SERBP1 gene sequence was cloned by RT-PCR and analyzed by bioinformatics. Real-time fluorescent quantitative PCR(RT-qPCR) was performed to determine the expression of SERBP1 in different tissues, and screening the most effective interference sequence. The MTT method was used to detect the effects of siRNA interference SERBP1 on cell proliferation of goat turbinate bone cells, and the effects of siRNA interference with SERBP1 on the cell cycle and apoptosis were detected by flow cytometry, and the effects on apoptosis-related genes P53, Caspase7 and Caspase3 were detected by RT-qPCR. A total length of 1 293 bp SERBP1 gene sequence was cloned successfully, including 44 bp of 5'UTR, 1 179 bp of CDS region, and 70 bp of 3'UTR,encoding 392 amino acids, with no signal peptide and mainly located in the nucleus. It was predicted that SERBP1 might interact with 10 proteins including RPL31 and RPS3. The expression mRNA of SERBP1 gene was the highest in goat pancreas, and an effective interference sequence with an interference efficiency of 70% was selected. siRNA interference of SERBP1 gene inhibited the proliferation and apoptosis of goat turbinate primary cells, and the cells were arrested in G₀/G₁ phase. The mRNA expression of Caspase3, Caspase7, BCL2L11 and Bax gene was down-regulated but no significant difference was determined in the mRNA expression of Bcl-2, P53, and PARP1 gene. Interference with goat SERBP1 gene could inhibit the proliferation and apoptosis of goat turbinate bone cells and induce cell cycle arrest. These data may lay a foundation for further study of SERBP1 gene function, and also facilitate the mechanism research underlying cell apoptosis inhibited by ORFV125 gene.

Key words: goat, SERBP1 gene, interference, cell cycle, cell apoptosis

中图分类号:

S826.2

徐颂为, 向华, 张焕容, 杨璐, 王宪军, 撒朗文朱, 朱江江. 干扰SERBP1表达对山羊鼻甲骨原代细胞增殖、周期及凋亡的作用[J]. 畜牧兽医学报, 2022, 53(8): 2708-2720.

XU Songwei, XIANG Hua, ZHANG Huanrong, YANG Lu, WANG Xianjun, SALANG Wenzhu, ZHU Jiangjiang. Effects of Interfering SERBP1 on the Cell Proliferation, Cell Cycle and Apoptosis in Goat Turbinate Bone Primary Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(8): 2708-2720.

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