猪传染性胃肠炎病毒诱导PK-15细胞凋亡相关基因mRNA转录水平变化的研究
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摘要
为探究PK-15细胞感染猪传染性胃肠炎病毒(TGEV)后相关凋亡基因Bcl-XL、MCL-1及PUMA mRNA转录水平差异以及TGEV增殖与细胞凋亡之间的关联,本研究在TGEV感染PK-15细胞前后,应用流式细胞仪检测细胞凋亡率,结果显示TGEV具有诱导PK-15细胞发生凋亡的能力;并利用建立的q PCR方法对凋亡相关基因Bcl-XL、MCL-1和PUMA mRNA的转录水平进行测定,结果显示,感染TGEV (实验组)后Bcl-XL、MCL-1和PUMA mRNA转录水平与未感染病毒组(对照组)有明显差异,进一步表明TGEV感染PK-15细胞诱导了细胞凋亡的进程。本研究为了解TGEV诱导PK-15细胞凋亡的机制提供了实验依据。
To investigate the differences in transcriptional levels of apoptosis related genes in PK-15 cells infected with transmissible gastroenteritis virus of swine(TGEV), and the association between TGEV proliferation and apoptosis, the quantitative PCR(qPCR) was developed, and the transcriptional levels of apoptosis-related genes of Bcl-XL, MCL-1 and PUMA were detected by the qPCR methods before and after TGEV infection of PK-15 cells. Then the transcriptional regularity curve and the proliferation pattern of TGEV in PK-15 cells were determined by the qPCR, and the apoptosis rate of PK-15 cells was detected by flow cytometry before and after TGEV infection. The results showed that TGEV was able to induce apoptosis of PK-15 cells.The m RNA levels of apoptosis-related genes of Bcl-XL, MCL-1 and PUMA were measured by the established qPCR method. The results showed that the transcriptional levels of the Bcl-XL, MCL-1 and PUMA in the TGEV infected group were significantly different from those in the control group. Through the detection of apoptosis-related genes, it was confirmed that TGEV infection affected the process of PK-15 cell apoptosis, but the mechanism of inducing apoptosis has not been fully studied. This study provides a basis for further understanding the mechanism of TGEV-induced apoptosis of PK-15 cells.
To investigate the differences in transcriptional levels of apoptosis related genes in PK-15 cells infected with transmissible gastroenteritis virus of swine(TGEV), and the association between TGEV proliferation and apoptosis, the quantitative PCR(qPCR) was developed, and the transcriptional levels of apoptosis-related genes of Bcl-XL, MCL-1 and PUMA were detected by the qPCR methods before and after TGEV infection of PK-15 cells. Then the transcriptional regularity curve and the proliferation pattern of TGEV in PK-15 cells were determined by the qPCR, and the apoptosis rate of PK-15 cells was detected by flow cytometry before and after TGEV infection. The results showed that TGEV was able to induce apoptosis of PK-15 cells.The m RNA levels of apoptosis-related genes of Bcl-XL, MCL-1 and PUMA were measured by the established qPCR method. The results showed that the transcriptional levels of the Bcl-XL, MCL-1 and PUMA in the TGEV infected group were significantly different from those in the control group. Through the detection of apoptosis-related genes, it was confirmed that TGEV infection affected the process of PK-15 cell apoptosis, but the mechanism of inducing apoptosis has not been fully studied. This study provides a basis for further understanding the mechanism of TGEV-induced apoptosis of PK-15 cells.
引文
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[14]Hao Hong-ying,Dong Yan-bin,Bowling M T,et al.E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation[J].Bmc Cancer,2007,7(1):1-12.
[2]Martin S.Deconstructing the cell cycle[J].Nat Rev Mol Cell Bio,2011,12(11):689.
[3]Huard S,Elder R T,Liang D,et al.Human immunodeficiency virus type 1 Vpr induces cell cycle G2 arrest through Srk1/MK2-mediated phosphorylation of Cdc25[J].J Virol,2008,82(6):2904-2917.
[4]Nagaleekar V K,Tiwari A K,Kataria R S,et al.Bluetongue virus induces apoptosis in cultured mammalian cells by both caspase-dependent extrinsic and intrinsic apoptotic pathways[J].Arch Virol,2007,152(9):1751-1756.
[5]Planelles V,Andersen J L,Zimmerman E.Cell cycle arrest and apoptosis[P].WO 2006046951 A2,2006.
[6]Davy C,Doorbar J.G2/M cell cycle arrest in the life cycle of viruses[J].Virology,2007,368(2):219.
[7]Cory S,Adams J M.The Bcl2 family:Regulators of the cellular life-or-death switch[J].Nat Rev Cancer,2002,2(9):647-656.
[8]丁利.TGEV诱导PK-15细胞凋亡信号转导通路研究[D].杨凌:西北农林科技大学,2012.
[9]Wang Chun-xin,Youle R J.The Role of Mitochondria in Apoptosis[J].Annu Rev Genet,2009,43(1):11-22.
[10]Ding Li,Zhao Xiao-min,Huang Yong,et al.Regulation of ROSin transmissible gastroenteritis virus-activated apoptotic signaling[J].Biochem Bioph Res Co,2013,442(1-2):33-37.
[11]Jr Bruno F,Chen Su-lin,Oyler G A,et al.Aven and Bcl-xL,enhance protection against apoptosis for mammalian cells exposed to various culture conditions[J].Biotechnol Bioeng,2004,85(6):589-600.
[12]何太平,唐菁燕,宋春雷,等.抗调亡基因bcl-xl过表达提高工程细胞IVCC及目的蛋白表达的研究[J].中国细胞生物学学报,2012,(7):674-679.
[13]Chetoui N,Sylla K,Gagnonhoude J V,et al.Down-regulation of MCL-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis[J].Mol Cancer,2008,6(1):42.
[14]Hao Hong-ying,Dong Yan-bin,Bowling M T,et al.E2F-1 induces melanoma cell apoptosis via PUMA up-regulation and Bax translocation[J].Bmc Cancer,2007,7(1):1-12.