复方苦芩对感染猪传染性胃肠炎病毒的PK-15细胞中Bcl-2/BAX mRNA转录的影响

doi:10.3864/j.issn.0578-1752.2014.11.018

摘要/Abstract

摘要： 【目的】探讨中药复方苦芩对感染猪传染性胃肠炎病毒 (transmissible gastroenteritis virus of swine, TGEV)的猪肾细胞（PK-15）中凋亡基因Bcl-2/BAX mRNA转录的影响。【方法】体外扩增TGEV，用TGEV感染PK-15细胞的模型进行体外试验研究，试验中设有复方苦芩组、空白组、模型组及黄芪多糖组，在测定了病毒半数感染细胞量（TCID50），复方苦芩及黄芪多糖药液对PK-15细胞的最大无毒浓度后，制作细胞爬片采用瑞士染色法染色观察PK-15细胞的形态变化;按试剂盒操作提取各试验组RNA并及时反转录为cDNA，采用实时荧光定量PCR法检测细胞中Bcl-2和BAX mRNA转录情况。【结果】与空白组相比，模型组的PK-15细胞发生了明显的细胞病变效应（cytopathic effect,CPE）,细胞变细长，细胞间成网状，细胞核着色较浅，出现核浓缩、固缩、碎裂等现象；与模型组比，复方苦芩组与黄芪多糖组细胞病变得到改善，而复方苦芩组效果更加明显，大部分细胞核结构清晰，染色质着色均一，少部分细胞核变大或碎裂；与空白组相比，模型组Bcl-2 mRNA转录量减少（P＜0.01），复方苦芩组Bcl-2基因转录量增加，为模型组的1.403倍（P＜0.01）、黄芪多糖组的1.078倍（P＞0.05）；与空白组相比，模型组BAX mRNA转录量明显升高，而其他3组BAX基因转录差异不明显（P＞0.05），模型组BAX基因转录量为空白组的1.440倍、复方苦芩组1.290倍、黄芪多糖组的1.490倍，差异极显著（P＜0.01），复方苦芩组、黄芪多糖组BAX基因转录量分别为空白组的1.116倍、0.966倍，差异不显著（P＞0.05）；与空白组相比，模型组Bcl-2/BAX mRNA转录比值降低，差异极显著（P＜0.01）；与模型组相比，复方苦芩组、黄芪多糖组Bcl-2/BAX mRNA转录比值均增加，分别为模型组1.809倍和1.940倍，差异极显著（P＜0.01），复方苦芩组Bcl-2/BAX mRNA转录比值分别为空白组的0.749倍（P＜0.01）、黄芪多糖组的0.933倍（P＞0.05）。【结论】复方苦芩可通过上调PK-15细胞Bcl-2 mRNA转录，下调BAX mRNA的转录，从而抑制TGEV引起的PK-15细胞凋亡。

关键词: 复方苦芩 , 猪传染性胃肠炎病毒 , PK-15 , Bcl-2 , BAX , 荧光定量PCR

Abstract: 【Objective】 The objective of this study is to investigate the effect of Kuqin compound on the transcription of Bcl-2/BAX mRNA in porcine kidney cells (PK-15) infected with transmissible gastroenteritis virus (TGEV) . 【Method】 To study the drug (Kuqin compound) test in vitro by TGEV infected PK-15 cell model, the test was divided into Kuqin compound group, blank control group, model group, and astragalus polysaccharide group, respectively. The morphology change was observed in PK-15 cells by the Swiss staining method and the transcription of Bcl-2 and BAX mRNA was tested by RT-PCR method after determination of tissue culture infective dose (TCID50) of TGEV and the maximum of no toxic concentration of Kuqin compound and astragalus polysaccharide on PK-15 cells. 【Result】The following results were obtained from the observation. Compared with blank control group, the PK-15 cells in model group had obvious cytopathic effects (CPE) including elongated cell shape, intercellular drawing into mesh, and lighter staining in nucleus. The nucleus enrichment, pyknosis and fragmentation were also observed. On the other hand, compared with the model group, CPE in Kuqin compound and astragalus polysaccharide group was improved and the Kuqin compound had better effect with most nucleus structure clear, uniform staining in chromatin and fewer nucleuses became larger or fragmentized. The transcription amounts were also considered as an effect of compound Kuqin on infected PK-15 cells. Compared with blank, the transcription amount of Bcl-2 mRNA in model group was less (P＜0.01). However, the transcription amount of Bcl-2 mRNA in compound Kuqin group was increased by 1.403 times compared with the model group (P＜0.01), by 1.078 times with the Astragalus polysaccharide group (P＞0.05). For the BAX mRNA transcription, the model group had an obvious increase (increased by 1.440 times, 1.290 times and 1.490 times compared with the blank group, compound Kuqing group and the astragalus polysaccharide group) while no obvious changes among the other three groups (compound Kuqin was 1.116 larger than blank and astragalus polysaccharide was 0.966 of the blank). Moreover, the transcription ratio of Bcl-2/BAX in model group had a huge decrease compared with the blank group (P＜0.01). This ratio in compound Kuqin and astragalus polysaccharide groups was much larger compared with the model group (P＜0.01) with the number of 1.809 and 1.904 times, respectively; this number was 0.749 (P＜0.01) and 0.933 times (P＜0.05) of blank, respectively.【Conclusion】Kunqin compound could inhibit apoptosis in PK-15 cells infected by TGEV by up-regulating transcription of Bcl-2 mRNA and down-regulating transcription of BAX mRNA.

Key words: Kuqin compound , TGEV , PK-15 , Bcl-2 , BAX , real-time PCR

邵秋红, 凌榕镔, 朱兆荣, 刘娟, 郭志兴, 王鹏. 复方苦芩对感染猪传染性胃肠炎病毒的PK-15细胞中Bcl-2/BAX mRNA转录的影响[J]. 中国农业科学, 2014, 47(11): 2242-2250.

SHAO Qiu-Hong, LING Rong-Bin, ZHU Zhao-Rong, LIU Juan, GUO Zhi-Xing, WANG Peng. Effect of Kuqin Compound on mRNA Transcription of Bcl-2/BAX in PK-15 Cells Infected by TGEV[J]. Scientia Agricultura Sinica, 2014, 47(11): 2242-2250.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.11.018

https://www.chinaagrisci.com/CN/Y2014/V47/I11/2242

参考文献

[1] 殷震, 刘景华. 动物病毒学. 第二版. 北京：科学出版社, 1997:681-688.

Yin Z, Liu J H. Animal Virology. 2nd Ed. Beijing: Science Press, 1997：681-688.(in Chinese)

[2] 刘邓, 袁秀芳, 徐丽华, 张存, 叶伟成, 牛登元, 王一成. 猪传染性胃肠炎诊断技术研究进展. 动物医学进展, 2008, 29(12):64-68.

Liu D, Yuan X F, Xu L H, Zhang C, Ye W C, Niu D Y, Wang Y C. Progress on the diagnostic methods of porcine transmissible gastroenteritis. Progress in Veterinary Medicine, 2008, 29(12):64-68. (in Chinese)

[3] Galan C, Sola I, Nogales A , Thomas B, Akoulitchev A , Enjuanes L , Almazan F. Host cell proteins interacting with the 3′ end of TGEV coronavirus genome influence virus replication. Virology, 2009, 391(2):304-314.

[4] Jeong H P, Jeong H H, Hyuk M K. Sequence analysis of the ORF 7 region of transmissible gastroenteritis viruses isolated in Korea. Virus Genes, 2008, 36:71-78.

[5] 丁利. TGEV诱导PK-15细胞凋亡信号转导通络研究[D]. 西安：西北农林科技大学，2012.

Ding L .Signal transduction pathways of TGEV-induced apoptosis in PK-15 cells[D]. Xi’an: Northwest Agriculture and Forestry University , 2012. (in Chinese)

[6] 仝钢, 张彦明, 唐青海, 王津津. 猪传染性胃肠炎病毒(TGEV)对猪小肠黏膜上皮细胞活性的影响. 西北农业学报, 2011(1):40-44.

Tong G, Zhang Y M, Tang Q H, Wang J J. Influence of the transmissible gastroenteritis virus on activities of swine tntertinal epithelial cells. Acta Agriculturae Boreali-Occidentalis Sinica, 2011(1): 40-44. (in Chinese)

[7] 朱兆荣, 刘娟, 聂晨睿. 复方苦芩注射液对人工感染犬细小病毒病的防治.中兽医医药杂志, 2010(6):34-37.

Zhu Z R, Liu J, Nie C R. Curative and preventive effects of compound Kuqin injection against canine parvovirus diease in dogs. Journal of Traditional Chinese Veterinary Medicine, 2010(6):34-37. (in Chinese)

[8] 朱兆荣, 刘娟, 杜林林, 诸葛证, 刘俊玮, 吕雪. 复方苦芩对小鼠免疫功能及犬IL-4mRNA和IFN-γ mRNA表达的影响. 中国兽医科学, 2012, 42(8):875-880.

Zhu Z R , Liu J, Du L L, Zhu G Z, Liu J W, Lü X. Effect of Kuqin compound on immune function of experimental animals and on expressions of canine IL-4 m RNA and IFN-γ m RNA. Chinese Veterinary Science, 2012, 42(8):875-880. (in Chinese)

[9] 李梁, 刘娟, 诸葛证, 冯杰. 复方苦芩对细小病毒病犬组织结构影响. 中国兽医杂志, 2010(4):49-51.

Li L, Liu J, Zhuge Z, Feng J, Effect of Kuqin compound on organizational structure of canine infected canine parvovirus. Chinese Journal of Veterinary Medicine, 2010(4):49-51. (in Chinese)

[10] 诸葛证, 朱兆荣, 刘娟. 复方苦芩对细小病毒病犬红细胞及血红蛋白变化的影响. 内江科技, 2009, 30(12):25.

Zhuge Z, Zhu Z R, Liu J. Effect of Kuqin compound on red blood cells and hemoglobin changes in dogs infected by canine parvovirus . Neijiang Science & Technology, 2009, 30(12):25. (in Chinese)

[11] 刘俊玮, 刘娟, 杜林林, 郭志兴, 凌榕镔. 复方苦芩对犬细小病毒致心肌组织Bcl-2和 Bax mRNA的影响. 畜牧兽医学报, 2013, 44(1): 122-128.

Liu J W, Liu J, Du L L, Guo Z X, Ling R B. Effect of Shenqin compound on m RNA expression of Bcl-2 and Bax genes in canine myocardium infected by canine parvovirus. Chinese Journal of Animal and Veterinary Sciences, 2013,44(1): 122-128. (in Chinese)

[12] 杜林林, 刘娟, 刘聪, 李鑫, 吕雪. 复方参芩对人工感染心肌炎型细小病毒病犬的疗效试验. 中国畜牧兽医,2012,39(2): 178-182.

Du L L, Liu J, Liu C, Li X, Lü X. Therapeutic experiment of compound shenqin on sickness dog of myocarditis parvovirus caused by artifical infection. China Animal Husbandry & Veterinary Medicine, 2012, 39(2): 178-182. (in Chinese)

[13] 杜林林, 李梁, 刘娟, 吕雪. 复方苦芩对犬细小病毒致肠粘膜细胞凋亡及相关基因表达的影响. 中国兽医学报, 2012, 32(10):1511-1515.

Du L L, Li L, Liu J, Lü X . Effect of Kuqin compound on intestinal mucosa apoptosis and related gene expression casused by canine parvovirus. Chinese Journal of Veterinary Science, 2012, 32(10): 1511-1515. (in Chinese)

[14] 封海波, 曾宪垠, 刘娟, 朱兆荣, 杜林林, 吕雪. 8种中药成分体外抗犬细小病毒的研究. 中国兽医学报, 2012, 32(6):884-888.

Feng H B, Zeng X Y, Liu J, Zhu Z R, Du L L, Lu X .Study on activity of antivirus of eight Chinese herbal medicine ingredients on canine parvovirus in vitro. Chinese Journal of Veterinary Science, 2012, 32(6): 884-888. (in Chinese)

[15] Seung W K, Ramakrishnan M A, Peter C R, Sagar M G. Effects of humidity and other factors on the generation and sampling of a coronavirus aerosol. Aerobiologia, 2007, 23:239-248.

[16] 姜成刚. 中草药抗猪传染性胃肠炎病毒活性及作用机理研究[D]. 黑龙江: 东北农业大学, 2003.

Jiang C G. Studys on activity of antivirus and mechanism of action of Chinese herbal medicine on transmissible gastroenteritis of swine[D]. Heilongjiang: Northeast Agricultural University, 2003. (in Chinese)

[17] 姜成刚, 张秀英, 龙忠臣. 中药复方制剂抗猪传染性胃肠炎病毒活性的研究. 畜牧兽医学报, 2004(5):587-589.

Jiang C G, Zhang X J, Long Z C. Study on activity of antivirus and mechanism of action of complex prescription of Chinese herbal medicine on transmissible gastroenteritis of swine. Chinese Journal of Animal and Veterinary Sciences, 2004(5):587-589. (in Chinese)

[18] 卫文强. 抗猪传染性胃肠炎病毒中草药的筛选及作用机制初步研究[D]. 甘肃：甘肃农业大学, 2009.

Wei W Q. Screen of herbs against TGEV and preliminary study on the mechanism of effect[D]. Gansu: Gansu Agricultural University, 2009. (in Chinese)

[19] 王明方. 中药在兽医临床抗病毒的作用机理. 养殖技术顾问, 2010(11):157.

Wang M F. The mechanism of Chinese medicine in veterinary clinical antiviral. Technical Advisor for Animal Husbandry, 2010(11):157. (in Chinese)

[20] 杜晋平, 肖立新, 罗静波. 黄芪多糖作用研究进展.畜牧与饲料科学, 2013, 34(4):39-41.

Du J P, Xiao L X, Luo J B. Advance on effects of astragalus polysaccharide. Animal Husbandry and Feed Science, 2013, 34(4): 39-41. (in Chinese)

[21] Gonzalez J M, Gomez-Puertas P, Cavanagh D, Gorbalenya A. E, Enjuanes L. A comparative sequence analysis to revise the current taxonomy of the family Coronaviridae. Archives of Virology, 2003, 148:2207-2235.

[22] Smith E C, Denison M R. Implications of altered replication fidelity on the evolution and pathogenesis of coronaviruses. Current Opinion in Virology , 2012, 2:519-524.

[23] Rathmell J C, Lindsten T, Zong W X, Ryan M C, Craig B T. Deficiency in Bak and BAX perturbs thymic selection and lymphoid homeostasis. Nature Immunology, 2002, 3(10): 932-939.

[24] Jerry E C, John C F, Christopher P D．Mechanism of apoptosis induction by inhibition of the anti-apoptotic Bcl-2 proteins. Proceedings of the National Academy of Sciences. USA, 2008, 105( 51) : 20327．

[25] Jerry M A, Suzanne C. The Bcl-2-regulated apoptosis switch: mechanism and therapeutic potential. Current Opinion in Immunology, 2007,19(5) : 488-496[26] Schabitz W R, Sommer C, Zoder W, Kiessling M, Schwaninger M, Schwab S. Intravenous brain-derived neurotrophic factor reduces infarct size and counterregulates BAX and Bcl-2 expression after temporary focal cerebral ischemia. Stroke, 2000, 31(9): 2212-2217.