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本试验旨在检测乌金猪和具有乌金猪血缘的约大乌猪不同组织间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表达的差异,并探讨东亚飞蝗抗菌活性物质(LAAS)对猪胎儿皮肤成纤维细胞(PFSF)免疫应激参数和防御素(pBDs)基因mRNA表达的调节作用。采用实时荧光定量PCR方法检测2月龄乌金猪与约大乌猪心脏、肝脏、肺脏、肾脏、空肠、回肠、十二指肠、皮肤、肌肉、胰腺、睾丸或卵巢这12种组织中pBD-1、pBD-2和pBD-3基因mRNA的表达差异;采用培养至第4~5代的PFSF,通过脂多糖(LPS)诱导建立免疫应激模型,在DMEM/F12培养液中添加不同浓度(0、75、150、300、600和1 200μg/mL)的LAAS,考察LAAS对PFSF免疫应激参数和pBD-1、pBD-2、pBD-3基因mRNA表达的影响。结果表明:1)乌金猪血清溶菌酶(LSZ)活性、免疫球蛋白G(IgG)含量显著高于约大乌猪(P<0.05),血清一氧化氮(NO)含量极显著低于约大乌猪(P<0.01)。2)约大乌猪大多数组织中pBD-1、pBD-2和pBD-3基因mRNA的表达量高于乌金猪;2个品种猪的pBD-1和pBD-3基因mRNA均在皮肤和卵巢中表达量较高,pBD-2基因mRNA在肝脏和肾脏中表达量较高。3)300μg/mL LAAS极显著降低了培养液中乳酸脱氢酶(LDH)活性(P<0.01);LAAS极显著增加了培养液中NO含量(除75μg/mL组正常细胞外)(P<0.01),正常细胞和应激细胞均在LAAS浓度为600μg/mL时达到最高;LAAS浓度为0、75和150μg/mL时应激细胞一氧化氮合酶(NOS)活性显著或极显著高于正常细胞(P<0.05或P<0.01);添加1 200μg/mL LAAS可极显著降低培养液中的LSZ活性(P<0.01),LAAS浓度为150、300、600和1 200μg/mL时应激细胞与正常细胞中LSZ活性差异显著或极显著(P<0.05或P<0.01)。4)应激细胞pBD-1、pBD-2和pBD-3基因mRNA的表达量均高于正常细胞;当LAAS浓度为150μg/mL时,pBD-1、pBD-2和pBD-3基因mRNA的表达量最高。结果提示,乌金猪和约大乌猪防御素基因的表达存在明显的品种差异性和组织特异性,适宜浓度的LAAS能够降低PFSF中LDH活性,提高NO含量及NOS、LSZ活性,上调应激细胞与正常细胞防御素的表达量。
The purpose of this study was to detect the expression of β defensin-1 (pBD-1), β defensin-2 (pBD-2) and β defensin- 3) mRNA expression and to explore the role of LAAS in modulating immune stress parameters and expression of pBDs mRNA in porcine fetal skin fibroblast (PFSF). The real-time PCR method was used to detect the expression of pBD-1 in 12 tissues of 2-month-old Wujin pigs and about 6-day-old Uv pigs, including heart, liver, lung, kidney, jejunum, ileum, duodenum, skin, muscle, pancreas, 1, pBD-2 and pBD-3 gene mRNA expression; using cultured to the 4th to 5th generations of PFSF, induced by lipopolysaccharide (LPS) to establish an immune model, DMEM / F12 medium was added at different concentrations ( 0,75,150,300,600 and 1 200μg / mL) LAAS. The effects of LAAS on PFSF immunoreactive parameters and mRNA expressions of pBD-1, pBD-2 and pBD-3 were investigated. The results showed that: 1) The serum lysozyme (LSZ) activity and the immunoglobulin G (IgG) content in Wujin pigs were significantly higher than that in Dabuwu (P <0.05), and the content of serum nitric oxide (NO) Pigs (P <0.01). 2) The mRNA expression of pBD-1, pBD-2 and pBD-3 in most tissues of Jupava was significantly higher than that of Jigui pigs; the mRNAs of pBD-1 and pBD-3 in the two breeds were both in the skin and ovary The expression of pBD-2 mRNA was higher in liver and kidney. LAAS significantly decreased lactate dehydrogenase (LDH) activity in culture medium (P <0.01); LAAS significantly increased the content of NO in culture medium (except for 75μg / mL normal cells) (P < 0.01). The normal cells and stressed cells reached the highest at the LAAS concentration of 600μg / mL. At LAA concentrations of 0,75 and 150μg / mL, the activity of NOS in stressed cells was significantly or extremely significantly higher than that of LAAS Normal cells (P <0.05 or P <0.01). The addition of 1 200 μg / mL LAAS significantly reduced the activity of LSZ in culture medium (P <0.01). When LAAS concentration was 150, 300, 600 and 1 200 μg / mL, There was a significant or significant difference in LSZ activity between cells and normal cells (P <0.05 or P <0.01). 4) The mRNA expressions of pBD-1, pBD-2 and pBD-3 in stress cells were higher than those in normal cells. When LAAS concentration was 150μg / mL, the expression of pBD-1, pBD- The highest expression. The results suggest that there are obvious variety differences and tissue specificities in the expression of defensin genes in Wujin and Jinduwu pigs. Appropriate concentration of LAAS can reduce the activity of LDH, increase the content of NO and the activity of NOS and LSZ in PFSF, Normal cellular defensin expression levels.