表达Ⅰ型耐热肠毒素的重组大肠杆菌诱发仔猪小肠炎症的分子机制研究
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摘要
本试验旨在研究表达Ⅰ型耐热肠毒素(STa)的重组大肠杆菌诱发7日龄仔猪小肠炎症反应。选取24头7日龄健康仔猪,随机分为4个处理组:对照组(人工乳)、STa组(人工乳+2×10~9 CFU重组菌LMG194-pBAD-STa)、LMG194组(人工乳+2×10~9 CFU大肠杆菌LMG194)和K88组(人工乳+2×10~9 CFU大肠杆菌K88),每组6头。试验第5天进行攻毒,第7天屠宰取样,观察空肠组织形态学变化,并检测血清中炎性细胞因子含量及空肠免疫相关基因的相对表达量。结果显示,与对照组相比,STa与K88组仔猪空肠绒毛明显萎缩变短,有明显损伤甚至脱落;STa、LMG194及K88组血清中IL-10、IL-8和IL-6浓度均显著升高(P<0.05),LMG194和K88组血液和肠道iFABP浓度均显著降低(P<0.05),STa和K88组血清中TNF-α浓度显著降低、NF-κB浓度显著升高,LMG194组NF-κB浓度显著降低、TNF-α浓度显著升高(P<0.05);STa和LMG194组CCL2和CXCL9基因表达水平显著上调(P<0.05),VNN1基因表达水平显著下调(P<0.05),STa和LMG194组IFN-γ组基因水平分别显著上升和下降(P<0.05);K88组CXCL9和IFN-γ基因表达水平显著下调(P<0.05),VNN1基因表达水平显著上调(P>0.05),CCL2基因表达水平无明显差异(P>0.05)。以上结果表明,表达Ⅰ型耐热肠毒素STa的重组大肠杆菌几乎具有与K88一样的毒性,可导致7日龄仔猪小肠黏膜受损,诱发严重的炎症反应,导致仔猪腹泻,可作为仔猪腹泻模型的候选菌株。
This experiment was conducted to investigate the effects of recombinant E.coli expressing heat-stable enterotoxin(STa) on jejunum inflammation of 7 days old piglets.Twenty-four 7 days old piglets were allotted to four treatments:Control group(artificial milk),STa group(artificial milk+2×10~9 CFU E.coli LMG194-pBAD-STa),LMG194 group(artificial milk+2×10~9 CFU E.coli LMG194),and K88 group(artificial milk+2×10~9 CFU E.coli K88),six piglets each group.The pigs were treated with E.coli on the 5 th day and slaughtered on the 7 th day.The results showed that compared with control group,the jejunum villi of STa and K88 groups were significantly atrophied and shortened with obvious damage or even falling off.Compared with control group,the concentrations of IL-10,IL-8 and IL-6 in the serum of STa,LMG194 and K88 groups were significantly increased(P<0.05),and the concentrations of iFABP in the blood and intestinal tract of LMG194 and K88 groups were significantly decreased(P<0.05);The concentrations of TNF-α in serum of STa and K88 groups were significantly decreased,the concentration of NF-κB was significantly increased(P<0.05);The concentration of NF-κB in LMG194 group was significantly decreased,and the concentration of TNF-α was significantly increased(P<0.05).Compared with control group,the expression levels of CCL2 and CXCL9 genes in STa and LMG194 groups were significantly up-regulated(P<0.05),and the expression level of VNN1 gene was significantly down-regulated(P<0.05),the expression level of IFN-γ in STa and LMG194 groups was significantly increased and decreased,respectively(P<0.05);The expression of CXCL9 and IFN-γ genes was significantly down-regulated in K88 group(P<0.05),and the expression level of VNN1 gene was significantly up-regulated(P>0.05).There was no significant difference in CCL2 gene expression level(P>0.05).These results suggested that recombinant E.coli expressing type Ⅰ heat-stable enterotoxin STa was almost as toxic as E.coli K88,which could cause damage to intestinal mucosa of 7-day-old piglets,induce serious inflammatory reaction,lead to diarrhea in piglets,and could be used as a candidate strain for piglet diarrhea model.
This experiment was conducted to investigate the effects of recombinant E.coli expressing heat-stable enterotoxin(STa) on jejunum inflammation of 7 days old piglets.Twenty-four 7 days old piglets were allotted to four treatments:Control group(artificial milk),STa group(artificial milk+2×10~9 CFU E.coli LMG194-pBAD-STa),LMG194 group(artificial milk+2×10~9 CFU E.coli LMG194),and K88 group(artificial milk+2×10~9 CFU E.coli K88),six piglets each group.The pigs were treated with E.coli on the 5 th day and slaughtered on the 7 th day.The results showed that compared with control group,the jejunum villi of STa and K88 groups were significantly atrophied and shortened with obvious damage or even falling off.Compared with control group,the concentrations of IL-10,IL-8 and IL-6 in the serum of STa,LMG194 and K88 groups were significantly increased(P<0.05),and the concentrations of iFABP in the blood and intestinal tract of LMG194 and K88 groups were significantly decreased(P<0.05);The concentrations of TNF-α in serum of STa and K88 groups were significantly decreased,the concentration of NF-κB was significantly increased(P<0.05);The concentration of NF-κB in LMG194 group was significantly decreased,and the concentration of TNF-α was significantly increased(P<0.05).Compared with control group,the expression levels of CCL2 and CXCL9 genes in STa and LMG194 groups were significantly up-regulated(P<0.05),and the expression level of VNN1 gene was significantly down-regulated(P<0.05),the expression level of IFN-γ in STa and LMG194 groups was significantly increased and decreased,respectively(P<0.05);The expression of CXCL9 and IFN-γ genes was significantly down-regulated in K88 group(P<0.05),and the expression level of VNN1 gene was significantly up-regulated(P>0.05).There was no significant difference in CCL2 gene expression level(P>0.05).These results suggested that recombinant E.coli expressing type Ⅰ heat-stable enterotoxin STa was almost as toxic as E.coli K88,which could cause damage to intestinal mucosa of 7-day-old piglets,induce serious inflammatory reaction,lead to diarrhea in piglets,and could be used as a candidate strain for piglet diarrhea model.
引文
[1] 卢伟.黄芩与鼠李糖乳杆菌发酵产物对仔猪大肠杆菌腹泻治疗效果研究[D].长春:吉林农业大学,2016. LU W.Study on therapy effect of ferment products of radix scutellatiae and Lactobacillus rhamnosus for Escherichia coli-induced diarrhea in piglets[D].Jilin Agricultural University,2016.(in Chinese)
[2] NAGY B,FEKETE P Z.Enterotoxigenic Escherichia coli (ETEC) in farm animals[J].Veterinary Research,1999,30(2-3):259-284.
[3] SVENNERHOLM A M,WIKSTRAM M,LINDBLAD M,et al.Monoclonal antibodies against Escherichia coli heat-stable toxin (STa) and their use in a diagnostic ST ganglioside GM1-enzyme-linked immunosorbent assay[J].Journal of Clinical Microbiology,1986,24(4):585-590.
[4] QADRI F,SVENNERHOLM A M,FARUQUE A S G,et al.Enterotoxigenic Escherichia coli in developing countries:Epidemiology,microbiology,clinical features,treatment,and prevention[J].Clinical Microbiology Reviews,2005,18(3):465-483.
[5] ABE A,KOMASE K,BANGTRAKULNONTH A,et al.Trivalent heat-labile- and heat-stable-enterotoxin probe conjugated with horseradish peroxidase for detection of enterotoxigenic Escherichia coli by hybridization[J].Journal of Clinical Microbiology,1990,28(12):2616-2620.
[6] 王磊,周佳,吕阳,等.表达单一肠毒素STa、STb和LT的重组大肠杆菌的构建及毒力比较[A].中国畜牧兽医学会动物营养学分会第十二次动物营养学术研讨会论文集[C].2016. WANG L,ZHOU J,LYU Y,et al.Construction and virulence comparison of recombinant Escherichia coli expressing single enterotoxin STa,STb and LT[A].Proceedings of the 12th Animal Nutrition Symposium of the Animal Nutrition Society of China Animal Husbandry and Veterinary Society[C].2016.(in Chinese)
[7] 吕阳,张林,李雪妮,等.表达耐热肠毒素的重组大肠杆菌对7日龄仔猪肠道形态结构及抗氧化功能的影响[J].中国畜牧兽医,2017,44(9):2816-2821. LYU Y,ZHANG L,LI X N,et al.Effects of recombinant E.coli expressing heat-stable enterotoxin on intestinal morphology and antioxidant capacity in seven days old piglets[J].China Animal Husbandry & Veterinary Medicine,2017,44(9):2816-2821.(in Chinese)
[8] 吕阳,张林,李雪妮,等.表达Ⅰ型耐热肠毒素的重组大肠杆菌对7日龄仔猪肠道免疫、屏障及抗氧化功能的影响[J].畜牧兽医学报,2017,48(9):1761-1768. LYU Y,ZHANG L,LI X N,et al.Effects of recombinant Escherichia coli expressing heat-stable enterotoxin on intestinal immune and barrier function and antioxidant capacity of seven days old piglets[J].Acta Veterinaria et Zootechnica Sinica,2017,48(9):1761-1768.(in Chinese)
[9] 梅慧敏,周颖,张越,等.大豆抗原蛋白对仔猪肠道转运通道的影响[J].饲料工业,2016,37(23):46-50. MEI H M,ZHOU Y,ZHANG Y,et al.Effect of soybean allergenic protein on intestinal function of piglets[J].Feed Industry,2016,37(23):46-50.(in Chinese)
[10] 刘涛.谷氨酰胺对早期断奶仔猪肠道营养与免疫功能影响机理的研究[D].武汉:华中农业大学,2002. LIU T.Effects of glutamine supplementation on small intestinal nutrition and immune function in early-weaned piglets and study on the mechanism involved[D].Wuhan:Huazhong Agricultural University,2002.(in Chinese)
[11] TENG Y T.The role of acquired immunity and periodontal disease progression[J].Critical Reviews in Oral Biology & Medicine,2003,14(4):237-252.
[12] 田中秋,邓立普.TNF-α、IL-6在全身炎症反应综合征表达的研究进展[J].蛇志,2008,20(4):275-278. TIAN Z Q,DENG L P.Investigated progress of expression of tumor necrosis factor-α (TNFα),interleukin-6 (IL-6) in systemic inflammatory response syndrome (SIRS)[J].Journal of Snake,2008,20(4):275-278.(in Chinese)
[13] LOSITO A,KALIDAS K,SANTONI S,et al.Association of interleukin-6 -174G/C promoter polymorphism with hypertension and left ventricular hypertrophy in dialysis patients[J].Kidney International,2003,64(2):616-622.
[14] 王娜娜,刘庆民.TNF-α、IL-6、NF-κB及其抑制剂与急性胰腺炎研究进展[J].中华消化病与影像杂志:(电子版),2015,5:39-41. WANG N N,LIU Q M.Research progress of TNF-α,IL-6,NF-κB and their inhibitors and acute pancrea-titis[J].Chinese Journal of Digestion and Medical Imageology (Electronic Edition),2015,5:39-41.(in Chinese)
[15] 李瑶,吕德官,陈临溪.IL-8 及其受体药物与疾病的研究进展[J].中国药理学通报,2014,30(3):310-314. LI Y,LYU D G,CHEN L X.Research progress of IL-8 and its receptor drugs in diseases[J].Chinese Pharmacological Bulletin,2014,30(3):310-314.(in Chinese)
[16] 李冠兰,刘先哲.TNF-α、IL-10、基质金属蛋白酶与MODS的研究进展[J].现代生物医学进展,2006,6(11):115-116. LI G L,LIU X Z.Relationship between TNF-α,IL-10,matrix metalloproteinases and multiple organ dysfunction syndrome[J].Progress in Modern Biomedicine,2006,6(11):115-116.(in Chinese)
[17] 王辉,孟庆华.白细胞介素-10研究进展[J].继续医学教育,2007,21(32):58-62. WANG H,MENG Q H.Research progress of IL-10[J].Continuing Medical Education,2007,21(32):58-62.(in Chinese)
[18] 吴炜景.核因子κB对肿瘤坏死因子-α刺激的肺泡Ⅱ型上皮细胞NADPH oxidase 1基因调控机制的探讨[D].广州:南方医科大学,2012. WU W J.Nuclear factor-κB regulates the transcription of NADPH oxidase 1 in tumor necrosis factor alpha-incluced type Ⅱ alveolar epithelial cells[D].Guangzhou:Southern Medical University,2012.(in Chinese)
[19] 徐云鹏,王文栋,郝敏,等.高脂饮食对幼年大鼠小肠组织FABP2 mRNA表达的影响[J].卫生研究,2014,43(5):749-753. XU Y P,WANG W D,HAO M,et al.Effects of high-fat diet on FABP2 mRNA expression in small intestinal tissue of young rats[J].Journal of Hygiene Research,2014,43(5):749-753.(in Chinese)
[20] 常晓彤,侯丽娟,王振辉.FABP2基因及其多态性与脂代谢关系的研究[J].军事医学,2008,32(2):192-195. CHANG X T,HOU L J,WANG Z H.Association of FABP2 gene and its polymorphism with lipid metabolism[J].Bulletin of the Academy of Military Medical Sciences,2008,32(2):192-195.(in Chinese)
[21] QIAN B,DENG Y,IM J H,et al.A distinct macrophage population mediates metastatic breast cancer cell extravasation,establishment and growth[J].PLoS One,2009,4(8):e6562.
[22] 路慧丽,俞眉,韩伟.趋化因子CXCL9/Mig的研究进展[J].中国生物工程杂志,2006,26(10):57-61. LU H L,YU M,HAN W.Progress in the study of chemokine CXCL9/Mig[J].China Biotechnology,2006,26(10):57-61.(in Chinese)
[23] GROOM J R,LUSTER A D.CXCR3 in T cell function[J].Experimental Cell Research,2011,317(5):620-631.
[24] 牛宇欣,李慧珠,张海燕,等.IFN-γ激活巨噬细胞产生一氧化氮杀伤疟原虫的作用[J].中国寄生虫学与寄生虫病杂志,1997,15(6):335-339. NIU Y X,LI H Z,ZHANG H Y et al.Ole of nitric oxide produced by IFN-γ activated macrophagecsin the killing of malaria parasite[J].Chinese Journal of Parasitology and Parasitic Diseases,1997,15(6):335-339.(in Chinese)
[2] NAGY B,FEKETE P Z.Enterotoxigenic Escherichia coli (ETEC) in farm animals[J].Veterinary Research,1999,30(2-3):259-284.
[3] SVENNERHOLM A M,WIKSTRAM M,LINDBLAD M,et al.Monoclonal antibodies against Escherichia coli heat-stable toxin (STa) and their use in a diagnostic ST ganglioside GM1-enzyme-linked immunosorbent assay[J].Journal of Clinical Microbiology,1986,24(4):585-590.
[4] QADRI F,SVENNERHOLM A M,FARUQUE A S G,et al.Enterotoxigenic Escherichia coli in developing countries:Epidemiology,microbiology,clinical features,treatment,and prevention[J].Clinical Microbiology Reviews,2005,18(3):465-483.
[5] ABE A,KOMASE K,BANGTRAKULNONTH A,et al.Trivalent heat-labile- and heat-stable-enterotoxin probe conjugated with horseradish peroxidase for detection of enterotoxigenic Escherichia coli by hybridization[J].Journal of Clinical Microbiology,1990,28(12):2616-2620.
[6] 王磊,周佳,吕阳,等.表达单一肠毒素STa、STb和LT的重组大肠杆菌的构建及毒力比较[A].中国畜牧兽医学会动物营养学分会第十二次动物营养学术研讨会论文集[C].2016. WANG L,ZHOU J,LYU Y,et al.Construction and virulence comparison of recombinant Escherichia coli expressing single enterotoxin STa,STb and LT[A].Proceedings of the 12th Animal Nutrition Symposium of the Animal Nutrition Society of China Animal Husbandry and Veterinary Society[C].2016.(in Chinese)
[7] 吕阳,张林,李雪妮,等.表达耐热肠毒素的重组大肠杆菌对7日龄仔猪肠道形态结构及抗氧化功能的影响[J].中国畜牧兽医,2017,44(9):2816-2821. LYU Y,ZHANG L,LI X N,et al.Effects of recombinant E.coli expressing heat-stable enterotoxin on intestinal morphology and antioxidant capacity in seven days old piglets[J].China Animal Husbandry & Veterinary Medicine,2017,44(9):2816-2821.(in Chinese)
[8] 吕阳,张林,李雪妮,等.表达Ⅰ型耐热肠毒素的重组大肠杆菌对7日龄仔猪肠道免疫、屏障及抗氧化功能的影响[J].畜牧兽医学报,2017,48(9):1761-1768. LYU Y,ZHANG L,LI X N,et al.Effects of recombinant Escherichia coli expressing heat-stable enterotoxin on intestinal immune and barrier function and antioxidant capacity of seven days old piglets[J].Acta Veterinaria et Zootechnica Sinica,2017,48(9):1761-1768.(in Chinese)
[9] 梅慧敏,周颖,张越,等.大豆抗原蛋白对仔猪肠道转运通道的影响[J].饲料工业,2016,37(23):46-50. MEI H M,ZHOU Y,ZHANG Y,et al.Effect of soybean allergenic protein on intestinal function of piglets[J].Feed Industry,2016,37(23):46-50.(in Chinese)
[10] 刘涛.谷氨酰胺对早期断奶仔猪肠道营养与免疫功能影响机理的研究[D].武汉:华中农业大学,2002. LIU T.Effects of glutamine supplementation on small intestinal nutrition and immune function in early-weaned piglets and study on the mechanism involved[D].Wuhan:Huazhong Agricultural University,2002.(in Chinese)
[11] TENG Y T.The role of acquired immunity and periodontal disease progression[J].Critical Reviews in Oral Biology & Medicine,2003,14(4):237-252.
[12] 田中秋,邓立普.TNF-α、IL-6在全身炎症反应综合征表达的研究进展[J].蛇志,2008,20(4):275-278. TIAN Z Q,DENG L P.Investigated progress of expression of tumor necrosis factor-α (TNFα),interleukin-6 (IL-6) in systemic inflammatory response syndrome (SIRS)[J].Journal of Snake,2008,20(4):275-278.(in Chinese)
[13] LOSITO A,KALIDAS K,SANTONI S,et al.Association of interleukin-6 -174G/C promoter polymorphism with hypertension and left ventricular hypertrophy in dialysis patients[J].Kidney International,2003,64(2):616-622.
[14] 王娜娜,刘庆民.TNF-α、IL-6、NF-κB及其抑制剂与急性胰腺炎研究进展[J].中华消化病与影像杂志:(电子版),2015,5:39-41. WANG N N,LIU Q M.Research progress of TNF-α,IL-6,NF-κB and their inhibitors and acute pancrea-titis[J].Chinese Journal of Digestion and Medical Imageology (Electronic Edition),2015,5:39-41.(in Chinese)
[15] 李瑶,吕德官,陈临溪.IL-8 及其受体药物与疾病的研究进展[J].中国药理学通报,2014,30(3):310-314. LI Y,LYU D G,CHEN L X.Research progress of IL-8 and its receptor drugs in diseases[J].Chinese Pharmacological Bulletin,2014,30(3):310-314.(in Chinese)
[16] 李冠兰,刘先哲.TNF-α、IL-10、基质金属蛋白酶与MODS的研究进展[J].现代生物医学进展,2006,6(11):115-116. LI G L,LIU X Z.Relationship between TNF-α,IL-10,matrix metalloproteinases and multiple organ dysfunction syndrome[J].Progress in Modern Biomedicine,2006,6(11):115-116.(in Chinese)
[17] 王辉,孟庆华.白细胞介素-10研究进展[J].继续医学教育,2007,21(32):58-62. WANG H,MENG Q H.Research progress of IL-10[J].Continuing Medical Education,2007,21(32):58-62.(in Chinese)
[18] 吴炜景.核因子κB对肿瘤坏死因子-α刺激的肺泡Ⅱ型上皮细胞NADPH oxidase 1基因调控机制的探讨[D].广州:南方医科大学,2012. WU W J.Nuclear factor-κB regulates the transcription of NADPH oxidase 1 in tumor necrosis factor alpha-incluced type Ⅱ alveolar epithelial cells[D].Guangzhou:Southern Medical University,2012.(in Chinese)
[19] 徐云鹏,王文栋,郝敏,等.高脂饮食对幼年大鼠小肠组织FABP2 mRNA表达的影响[J].卫生研究,2014,43(5):749-753. XU Y P,WANG W D,HAO M,et al.Effects of high-fat diet on FABP2 mRNA expression in small intestinal tissue of young rats[J].Journal of Hygiene Research,2014,43(5):749-753.(in Chinese)
[20] 常晓彤,侯丽娟,王振辉.FABP2基因及其多态性与脂代谢关系的研究[J].军事医学,2008,32(2):192-195. CHANG X T,HOU L J,WANG Z H.Association of FABP2 gene and its polymorphism with lipid metabolism[J].Bulletin of the Academy of Military Medical Sciences,2008,32(2):192-195.(in Chinese)
[21] QIAN B,DENG Y,IM J H,et al.A distinct macrophage population mediates metastatic breast cancer cell extravasation,establishment and growth[J].PLoS One,2009,4(8):e6562.
[22] 路慧丽,俞眉,韩伟.趋化因子CXCL9/Mig的研究进展[J].中国生物工程杂志,2006,26(10):57-61. LU H L,YU M,HAN W.Progress in the study of chemokine CXCL9/Mig[J].China Biotechnology,2006,26(10):57-61.(in Chinese)
[23] GROOM J R,LUSTER A D.CXCR3 in T cell function[J].Experimental Cell Research,2011,317(5):620-631.
[24] 牛宇欣,李慧珠,张海燕,等.IFN-γ激活巨噬细胞产生一氧化氮杀伤疟原虫的作用[J].中国寄生虫学与寄生虫病杂志,1997,15(6):335-339. NIU Y X,LI H Z,ZHANG H Y et al.Ole of nitric oxide produced by IFN-γ activated macrophagecsin the killing of malaria parasite[J].Chinese Journal of Parasitology and Parasitic Diseases,1997,15(6):335-339.(in Chinese)