动物营养学报 >

2013 , Vol. 25 >Issue 5: 1037 - 1044

DOI: https://doi.org/10.3969/j.issn.1006-267x.2013.05.019

分子营养 Molecular Nutrition

L-精氨酸对猪胎儿皮肤成纤维细胞免疫应激参数和β防御素基因mRNA表达的影响

  • 骆雪 ,
  • 张春勇 ,
  • 安清聪 ,
  • 李美荃 ,
  • 陈克嶙 ,
  • 郭荣富
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  • 云南农业大学,云南省动物营养与饲料重点实验室,昆明 650201

收稿日期: 2012-11-06

  网络出版日期: 2013-04-12

基金资助

云南省重大科技专项项目(2012ZA018);云南现代农业生猪产业技术体系(A3006688)

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L-Arginine Affects Immune Stressed Parameters and mRNA Expression of β-Defensins Genes in Porcine Fetal Skin Fibroblasts

  • LUO Xue ,
  • ZHANG Chunyong ,
  • AN Qingcong ,
  • LI Meiquan ,
  • CHEN Kelin ,
  • GUO Rongfu
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  • Key Laboratory of Animal Nutrition and Feed Science of Yunnan Province, Yunnan Agricultural University, Kunming 650201, China

Received date: 2012-11-06

  Online published: 2013-04-12

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摘要

本试验旨在研究L-精氨酸(L-Arg)对猪胎儿皮肤成纤维细胞(PFSF)免疫应激参数和β防御素基因mRNA表达的影响。采用培养至4～5代的PFSF,通过脂多糖诱导建立免疫应激模型,在DMEM/F12培养液中添加不同浓度(0、35、70、140、280和560 μg/mL)的L-Arg,培养24 h后,检测培养液一氧化氮(NO)含量及乳酸脱氢酶(LDH)、一氧化氮合酶(NOS)和溶菌酶(LSZ)活性;采用实时定量PCR法检测PFSF β防御素1(pBD-1)、β防御素2(pBD-2)和β防御素3(pBD-3)基因mRNA表达量。结果表明:1)L-Arg能够降低培养液LDH活性,正常细胞和应激细胞分别在L-Arg浓度为70和140 μg/mL时达到最低;2)L-Arg能够极显著提高培养液NO含量(P<0.01),正常细胞和应激细胞均在L-Arg浓度为140 μg/mL达到最高;3)L-Arg能够显著或极显著增加正常细胞培养液NOS活性(P<0.05或P<0.01),而对应激细胞无显著影响(P>0.05),L-Arg浓度为35和70 μg/mL时,应激细胞显著或极显著高于正常细胞(P<0.05或P<0.01);4)560 μg/mL L-Arg可显著提高培养液LSZ活性(P<0.05);5)70 μg/mL L-Arg可极显著提高正常细胞和应激细胞pBD-1和pBD-2基因mRNA表达量(P<0.01),140 μg/mL L-Arg可极显著提高正常细胞和应激细胞pBD-3基因mRNA表达量(P<0.01)。结果提示,适宜浓度的L-Arg能够降低培养液LDH活性,提高NO含量以及NOS和LSZ活性,上调应激细胞和正常细胞β防御素基因mRNA表达;本试验条件下,L-Arg的适宜添加浓度为70～140 μg/mL。

关键词： L-精氨酸; 脂多糖; 免疫应激; β防御素; 基因表达

本文引用格式

骆雪 , 张春勇 , 安清聪 , 李美荃 , 陈克嶙 , 郭荣富 . L-精氨酸对猪胎儿皮肤成纤维细胞免疫应激参数和β防御素基因mRNA表达的影响[J]. 动物营养学报, 2013 , 25(5) : 1037 -1044 . DOI: 10.3969/j.issn.1006-267x.2013.05.019

Abstract

This experiment was conducted to investigate the effects of L-arginine (L-Arg) on the immune stress parameters and mRNA expressions of β-defensins genes in porcine fetal skin fibroblasts (PFSF). The cultured 4 to 5 generations of PFSF were used, and immune stress model was induced by lipopolysaccharides (LPS). Adding different concentrations (0, 35, 70, 140, 280 and 560 μg/mL) of L-arginine in DMEM/F12 culture medium. After being cultured for 24 hours, the activities of lactate dehydrogenase (LDH), nitric oxide synthase (NOS) and lysozyme (LSZ) and nitric oxide (NO) content in culture medium were determined, and the mRNA expressions of β-defensin-1 (pBD-1), β-defensin-2 (pBD-2) and β-defensin-3 (pBD-3) genes were detected by the method of real-time qPCR. The results showed as follows: 1) L-arginine could reduce LDH activity in culture medium, and that in normal cells and stressed cells reached the lowest when L-arginine concentrations were 70 and 140 μg/mL, respectively; 2) L-arginine could significantly increase NO content in culture medium (P<0.01), and that in normal cells and stressed cells both reached the highest when L-arginine concentration was 140 μg/mL; 3) L-arginine could significantly increase NOS activity in normal cells (P<0.05 or P<0.01), but had no significant effects on stressed cells (P>0.05), when L-arginine concentrations were 35 and 70 μg/mL, NOS activity in stressed cells was significantly higher than that in normal cells; 4) the supplementation of 560 μg/mL L-arginine can significantly increase LSZ activity in culture medium (P>0.01); 5) the supplementation of 70 μg/mL L-arginine significantly increased the mRNA expressions of pBD-1 and pBD-2 genes, and the supplementation of 140 μg/mL L-arginine significantly increased the mRNA expression of pBD-3 gene. The results indicate that the proper concentration of L-arginine can decrease LDH activity, but increase NO content and the acitivities of NOS and LSZ in PFSF, and up-regulate the mRNA expressions of β-defensins genes; the optimal concentration of L-arginine is 70 to 140 μg/mL in the present study.

Key words： L-arginine; lipopolysaccharide; immnue stress; β-defensins; gene expression

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