SOCS2对山羊鼻甲骨细胞增殖、周期及凋亡的影响

Effects of SOCS2 on Proliferation, Cycle and Apoptosis of Turbinate Bone Cells in Goats

细胞因子信号转导抑制因子2 (suppressor of cytokine signaling 2,SOCS2)在动物体内参与调节多种生理和病理过程,如细胞凋亡、细胞增殖、细胞周期、肿瘤和炎症反应等。本研究在首次克隆山羊SOCS2基因的基础上,研究过表达和干扰SOCS2表达对山羊鼻甲骨细胞增殖、周期及凋亡的影响。以1周岁的健康简州大耳羊(6只)的心、肝、脾、肺、肾、瘤胃、大肠、小肠和山羊鼻甲骨细胞共9个样本为模板,采用反转录PCR技术克隆山羊SOCS2基因CDS区序列,并进行序列分析。利用RT-qPCR技术检测SOCS2基因在不同组织中的表达。将克隆得到的SOCS2基因CDS区连接载体构建真核表达载体pcDNA3.1-SOCS2。根据山羊SOCS2基因序列设计合成并筛选有效siRNA。利用Lipofectamine^(TM) 3000试剂将pcDNA3.1-SOCS2和siRNA片段分别转染至鼻甲骨细胞,采用蛋白免疫印迹技术检测SOCS2蛋白表达,MTT法检测SOCS2对细胞增殖的影响,流式细胞术检测SOCS2对细胞周期和凋亡的影响,并利用RT-qPCR技术检测细胞周期相关基因p21、CDK2和凋亡相关基因Caspase3、Caspase7、PARP1、p53、Bax、BCL2L11和Bcl-2的转录水平。结果显示,成功扩增山羊SOCS2基因序列,总长为1 065 bp, CDS区长为597 bp,编码198个氨基酸残基。SOCS2基因在肝中表达水平最高;SOCS2可以抑制细胞增殖。成功构建真核表达载体pcDNA3.1-SOCS2,且该基因在鼻甲骨细胞内成功表达。SOCS2过表达可导致G2/M+S期细胞数量显著增加,且下调CDK2和p21基因的mRNA表达;促进细胞凋亡,且上调Caspase3、Caspase7、Bax、p53、BCL2L11、Bcl-2基因的mRNA表达,PARP1表达水平无显著变化。而干扰SOCS2表达后,G0/G1期细胞数量显著升高,且CDK2和p21基因的mRNA表达上调;细胞凋亡被抑制,且Caspase3、Bax、p53、PARP1基因的mRNA表达下调,Caspase7、BCL2L11、Bcl-2基因表达水平无显著变化。综上所述,SOCS2可将细胞周期阻滞在G2/M+S期,促进细胞凋亡,抑制细胞增殖。研究结果为全面揭示山羊SOCS2功能提供试验数据。

Suppressor of cytokine signaling 2(SOCS2)is involved in regulating various physiological and pathological processes in animals,such as cell apoptosis,proliferation,cell cycle,tumor,and inflammatory response.In this study,based on the first cloned SOCS 2 gene from goats,the effects of overexpression and interference SOCS2 expression on proliferation,cycle and apoptosis of goat turbinate bone cells were studied.A total of 9 samples of heart,liver,spleen,lung,kidney,rumen,large intestine,small intestine and goat turbinate bone cells of healthy Jianzhou goats(6 animals)were used as samples,and the sequence of the CDS region of the goat SOCS 2 gene was cloned by reverse transcription PCR and analyzed the sequence.RT-qPCR was used to detect the expression of SOCS 2 gene in different tissues.The cloned SOCS 2 gene CDS region ligation vector was constructed as a eukaryotic expression vector pcDNA3.1-SOCS 2.Design,synthesis and screen effective siRNAs based on goat SOCS 2 gene sequences.After transfecting pcDNA3.1-SOCS 2 and siRNAs to turbinate osteocytes with Lipofectamine TM 3000 reagent,SOCS2 protein expression was detected with Western blot mothed,the MTT method detects the effect of SOCS2 on cell proliferation,flow cytometry to detect the effect of SOCS2 on cell cycle and apoptosis and RT-qPCR was used to detect the transcription levels of cell cycle-related genes p21,CDK 2 and apoptosis-related genes Caspase3,Caspase7,PARP1,p53,Bax,BCL2L 11 and Bcl-2.The results showed that the total length of goat SOCS 2 gene was 1065 bp,and the CDS zone length was 597 bp,encoding 198 amino acid residues.SOCS 2 gene is expressed at the highest levels in the liver tissue;SOCS2 can inhibit cell proliferation.The eukaryotic expression vector pcDNA3.1-SOCS 2 was successfully constructed,and the gene was successfully expressed in turbinate bone cells.SOCS2 overexpression can lead to a significant increase in the number of cells in the G2/M+S phase and down-regulation of mRNA expression in CDK 2 and p 21 genes.It promoted apoptosis and upregulated the mRNA expression of Caspase3,Caspase7,Bax,p53,BCL2L 11,and Bcl-2 genes,but there was no significant change in PARP 1 expression level.However,after interference the expression of SOCS2,the number of cells in G0/G1 stage increased significantly,and the mRNA expression of CDK 2 and p 21 genes was upregulated.The apoptosis was inhibited and the mRNA expression of Caspase3,Bax,p 53 and PARP 1 genes was downregulated,while the expression levels of Caspase7,BCL2L 11 and Bcl-2 did not change significantly.In summary,SOCS2 can block cells in the G2/M+S phase,promote apoptosis,and inhibit cell proliferation.The results of this study accumulate experimental data for a comprehensive reveal of the function of goat SOCS2.