CN109952996A - 一种高效调控内源抗菌肽表达的方法及应用 - Google Patents
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Abstract
本发明公开了一种高效调控内源抗菌肽表达的方法及应用。将适宜浓度的丁酸钠添加至饲料或细胞的培养基中实现体内或体外对猪内源抗菌肽表达的调控。该方法的用途是:上调仔猪回肠和结肠中抗菌肽pBD2和pBD3表达,减轻肠道炎症反应,改善肠道绒毛形态;增强巨噬细胞的功能,抑制细菌,清除细菌;增强肠道上皮屏障功能,增强上皮细胞抵抗E coil O157:H7的能力,增强仔猪的抗病能力,减少养殖业中抗生素的使用。
Description
技术领域
本发明属于生物医学技术领域,特别是涉及一种高效调控内源抗菌肽表达的方法
及应用。
背景技术
养殖过程中大量抗生素被用于促进动物生长或治疗其疾病,导致耐药菌株产生、畜产品残留、生态污染等问题,并且禁用、减用抗生素已成为现今畜牧人的共识,而提高动物自身机体免疫则是减少抗生素使用的一个重要手段。
抗菌肽,也称为宿主防御多肽,具有广谱抗菌活性,还可以抗真菌、寄生虫、肿瘤等。抗菌肽具有抗炎症反应,招募免疫细胞,促进上皮损伤修复,在肠道屏障中直接抑菌杀菌等功能,保护机体免受感染,若能高效调控抗菌肽的表达将有效减抗、禁抗。
猪的内源抗菌肽有PG1-5、PMAP-23、PMAP-36、PMAP-37、PR39、Prophenin-1和Prophenin-2、fensins、protegrins等。关于调控内源抗菌肽表达的方法已有一些报道:在日粮中添加高浓度氧化锌可以显著提高PR39基因的表达水平,但随粪便排出的高浓度氧化锌会造成环境污染,使畜牧养殖业无法绿色发展;乳铁蛋白可以使断奶仔猪骨髓中PR39和protegrin-1表达量分别显著提高143%和217%,虽然差异已经显著,但内源抗菌肽增加的比例并不高,同样,谷氨酰胺也不够高效,在日粮中添加1%谷氨酰胺可以使PR39在骨髓和空肠黏膜中的表达量分别提高50.5%和24.0%;视黄酸处理的猪骨髓细胞在6或12小时后PR39表达显著增加,但是起效时间长,需要6小时以上,不够快速;分别在0日龄、7日龄、11日龄、26日龄时,给新生仔猪灌胃Lactobacilli salivarius B1制剂,可以使仔猪在14日龄和28日龄时肠道中pBD2显著提高,这种方法操作繁琐并且出现的效果不稳定,并不能通过微生物制剂的作用有效地调控内源抗菌肽的表达。所以当前养殖业仍急需一种可以稳定、高效、快速、便捷调控猪内源抗菌肽表达的方法。
发明内容
为了克服现有技术的不足,本发明的目的是提供一种高效调控内源抗菌肽表达的方法及应用。
一种高效调控内源抗菌肽表达的方法,在基础日粮中加入质量百分比为0.2%的丁酸钠,饲喂后,抗菌肽pBD2和pBD3在仔猪回肠和结肠中的表达水平提高。
一种丁酸钠体外调控猪内源抗菌肽表达的方法,以终浓度为4 mmol/L溶于培养基中处理猪巨噬细胞或者以终浓度为8 mmol/L溶于培养基中处理猪肠道上皮细胞后,提高上述细胞中内源抗菌肽的表达。
一种高效调控仔猪内源抗菌肽表达的饲料,在基础日粮中加入质量百分比为0.2%的丁酸钠。
一种提高E coli 0157:H7抵抗能力的药剂,含有丁酸钠,通过添加入猪饲料中进入猪的体内,用于提高E coli 0157:H7抵抗能力。
本发明的有益效果:
1、提高pBD2和pBD3在仔猪回肠和结肠中的表达水平,减轻由E coil O157:H7引起的肠道炎症反应,改善肠道绒毛形态;
2、增强巨噬细胞的功能,抑制细菌,清除细菌;
3、增强肠道上皮屏障功能,增强肠道上皮细胞抵抗E coil O157:H7的能力,增强仔猪的抗病能力,减少抗生素在养殖业中的使用。
附图说明
图1是丁酸钠对仔猪回肠中抗菌肽表达的调控,以上结果为6次独立重复实验平均值。
图2是丁酸钠对仔猪结肠中抗菌肽表达的调控,以上结果为6次独立重复实验平均值。
图3是不同浓度的丁酸钠对猪巨噬细胞中抗菌肽表达的调控,以上结果为3次独立重复实验平均值。
图4是不同处理时间下4 mmol/L丁酸钠对猪巨噬细胞中抗菌肽表达的调控,以上结果为3次独立重复实验平均值。
图5是丁酸钠对猪小肠上皮细胞中抗菌肽表达的调控,以上结果为3次独立重复实验平均值。
图6是丁酸钠对由E coil O157:H7引起的绒毛高度降低的缓解,以上结果为6次独立重复实验平均值。
图7是丁酸钠对由E coil O157:H7引起的隐窝深度增加的缓解,以上结果为6次独立重复实验平均值。
图8是丁酸钠对由E coil O157:H7引起的绒毛隐窝比值降低的缓解,以上结果为6次独立重复实验平均值。
图9是丁酸钠对由E coil O157:H7引起的肠道跨膜电阻TER值降低的缓解,以上结果为6次独立重复实验平均值。
图10是丁酸钠对猪巨噬细胞的抑制细菌功能的调控,以上结果为3次独立重复实验平均值。
具体实施方式
下面结合附图和实施例对本发明进行进一步的阐述。
实施例1 丁酸钠对仔猪回肠中抗菌肽表达的调控
使用购自西格玛奥德里奇(上海)贸易有限公司的丁酸钠(纯度 > 98%),以质量百分比为0.2%的比例添加至基础日粮中,饲喂28日龄的杜×长×大仔猪,连续饲喂10天,取仔猪回肠检测抗菌肽基因RNA表达水平。抗菌肽pBD2和pBD3在仔猪回肠中的表达量分别显著提高4.78倍和4.15倍,而对抗菌肽pBDl的表达量仅提高了1.05倍,没有显著影响(见图1)。
实施例2 丁酸钠对仔猪结肠中抗菌肽表达的调控
使用购自西格玛奥德里奇(上海)贸易有限公司的丁酸钠(纯度 > 98%),以质量百分比为0.2%的比例添加至基础日粮中,饲喂28日龄的杜×长×大仔猪,连续饲喂10天,取仔猪结肠检测抗菌肽基因RNA表达水平,抗菌肽pBD2和pBD3在仔猪结肠中的表达量分别显著提高5.86倍和4.53倍,而对抗菌肽pBDl的表达量仅提高了1.12倍,没有显著影响,(见图2)。
实施例3 不同浓度的丁酸钠在体外对猪巨噬细胞抗菌肽表达的调控
使用购自西格玛奥德里奇(上海)贸易有限公司的丁酸钠(纯度 > 98%),分别以0mmol/L、1 mmol/L、2 mmol/L、4 mmol/L的浓度添加至猪巨噬细胞的培养基中,培养24 h后,与对照组(0 mmol/L)相比,培养基中添加4 mmol/L丁酸钠可使猪巨噬细胞中抗菌肽pBD2、pBD3、PG1-5、PMAP37和PR39的表达量分别提高7.21倍、3.45倍、6.83倍、2.86倍和6.32倍,而培养基中添加4 mmol/L丁酸钠使抗菌肽pBDl表达量仅提高了1.11倍,对其表达没有显著影响(见图3)。
实施例4 不同处理时间下4 mmol/L丁酸钠在体外对猪巨噬细胞中抗菌肽表达的调控
使用购自西格玛奥德里奇(上海)贸易有限公司的丁酸钠(纯度 > 98%),以4 mmol/L的浓度添加至猪巨噬细胞的培养基中,分别培养0 h、3 h、6 h、12 h、24 h后,与对照组(0 h)相比,丁酸钠可在3 h内迅速提高猪巨噬细胞抗菌肽的表达,分别使pBD2、pBD3、PG1-5、PMAP37和PR39的表达量提高4.06倍、5.56倍、2.75倍、5.56倍和2.93倍,然而,4 mmol/L丁酸钠培养3h使抗菌肽pBDl表达量降低了6.25%,没有显著影响(见图4)。
实施例5 不同浓度的丁酸钠在体外对猪小肠上皮细胞抗菌肽表达的调控
使用购自西格玛奥德里奇(上海)贸易有限公司的丁酸钠(纯度 > 98%),分别以0mmol/L、1 mmol/L、2 mmol/L、4 mmol/L的浓度添加至猪小肠上皮细胞的培养基中,培养24h后,与对照组(0 mmol/L)相比,培养基中添加2 mmol/L的丁酸钠可使猪小肠上皮细胞中抗菌肽pBD2和pBD3的表达量分别提高1.68倍和1.78倍,培养基中添加4 mmol/L的丁酸钠可使猪小肠上皮细胞中抗菌肽pBD2和pBD3的表达量分别提高2.12倍和2.03倍,然而,对抗菌肽pBDl的表达没有显著影响,添加2 mmol/L、4 mmol/L的丁酸钠仅分别使pBDl的表达量提高了1.12倍、1.21倍(见图5)。
实施例6 丁酸钠对由E coil O157:H7引起的肠道炎症反应的缓解
丁酸钠可以提高肠道抗菌肽的表达水平,从而可减轻由E coil O157:H7引起的肠道炎症反应。将28日龄的杜×长×大仔猪分为四组,每组6头,均饲养10天,对照组仅饲喂基础日粮,攻毒组饲喂基础日粮并在饲养的第2、3、4天用E coil O157:H7攻毒,丁酸钠组饲喂含有质量百分比为0.2%丁酸钠的基础日粮,丁酸钠攻毒组饲喂含有质量百分比为0.2%丁酸钠的基础日粮并在饲养的第2、3、4天用E coil O157:H7攻毒。由E coil O157:H7引起的肠道炎症反应表现为绒毛高度显著降低、隐窝深度显著增加、绒毛隐窝比值显著降低,以及肠道跨膜电阻TER值显著降低,而含有质量百分比为0.2%丁酸钠的饲料可以减轻这种肠道炎症反应(见图6、图7、图8、图9)。
实施例7 丁酸钠对猪巨噬细胞抑菌功能的调控
丁酸钠提高猪巨噬细胞抗菌肽的表达水平,从而调控猪巨噬细胞的功能,抑制细菌,清除细菌。使用购自西格玛奥德里奇(上海)贸易有限公司的丁酸钠(纯度 > 98%),分别以0mmol/L、4 mmol/L、8 mmol/L的浓度添加至猪巨噬细胞的培养基中,培养24 h后,清洗并裂解猪巨噬细胞,取细胞裂解物的上清液与E coli 0157:H7孵育后,发现4 mmol/L和8 mmol/L丁酸钠处理后的细胞裂解物上清液可以使E coli 0157:H7增殖分别极显著降低46.15%、47.05%(如图10)。这说明丁酸钠可以通过上调猪巨噬细胞中抗菌肽的表达来增强猪巨噬细胞的功能,抑制细菌,清除细菌。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形和等同替换。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形和等同替换,均在本发明的保护范围之内。
Claims (4)
1.一种高效调控内源抗菌肽表达的方法,其特征在于,在基础日粮中加入质量百分比为0.2%的丁酸钠,饲喂后,抗菌肽pBD2和pBD3在仔猪回肠和结肠中的表达水平提高。
2.一种丁酸钠体外调控猪内源抗菌肽表达的方法,其特征在于,以终浓度为4 mmol/L溶于培养基中处理猪巨噬细胞或者以终浓度为8 mmol/L溶于培养基中处理猪肠道上皮细胞后,提高上述细胞中内源抗菌肽的表达。
3.一种高效调控仔猪内源抗菌肽表达的饲料,其特征在于,在基础日粮中加入质量百分比为0.2%的丁酸钠。
4.一种提高E coli 0157:H7抵抗能力的药剂,其特征在于,含有丁酸钠,通过添加入猪饲料中进入猪的体内,用于提高E coli 0157:H7抵抗能力。
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