猪β-防御素基因表达特点及维生素A的调节作用
摘要
防御素是抗菌肽家族成员之一,具有广泛的抗菌、抗病毒、抗寄生虫和免疫调节作用,是机体免疫系统的重要组成部分。维生素A作为一种免疫调节剂,对动物的抗感染能力、细胞因子以及相关基因表达水平的影响十分明显。然而,关于猪防御素基因表达规律及维生素A对其表达的调节作用目前尚未见报道。本研究旨在考察猪防御素基因表达的品种和组织差异及维生素A对其表达的影响,以揭示猪防御素基因的表达特点和维生素A的调节作用。本试验首先考察了不同抗病力的两种猪体内防御素-1、2、3基因表达的差异,并通过体外细胞培养试验比较了不同维生素对防御素-1、2、3基因表达的影响,重点研究了在动物试验中维生素A对猪防御素-1、2、3基因表达的影响,初步探讨了维生素A影响猪防御素-1、2、3基因表达的信号转导机制。本研究包括以下四个试验：
试验一DLY和梅山仔猪pBD基因表达差异的研究
通过观察防御素基因在DLY和梅山仔猪各个组织中的表达水平,比较了两个猪种之间的表达差异。本实验随机选择健康的7日龄梅山猪和DLY(杜×长×大)杂交猪各6头,公母各半。屠宰后立即取其小肠黏膜、心、肝、脾、肺、肾、脑、皮肤、肌肉、口腔黏膜、呼吸道上皮、舌头、胸腺、生殖道上皮、睾丸(公)或卵巢(母),提取总RNA,用Real-time PCR方法检测pBD-1、2、3基因mRNA表达水平。
结果表明：所有组织中均能检测出pBD基因mRNA的表达。其中梅山猪中,pBD-1、2、3在舌头和口腔黏膜的表达量最高；而DLY杂交猪中,pBD-1、3在舌头和口腔黏膜的表达量最高,pBD-2在肾脏和肝脏的表达量最高。绝大部分组织的pBD基因表达量均为梅山猪高于DLY杂交猪,其中小肠黏膜、舌、口腔黏膜和生殖道黏膜等与外界接触的组织中的pBD-1、2、3表达量均是以梅山猪为高。由此显示,猪pBD的表达存在明显的品种差异和组织特异性,梅山猪体内pBD-1、2、3基因在多个组织中的表达量要高于DLY三元杂交猪。
试验二VA、VD、VE对IPEC-J2细胞系pBD基因mRNA和蛋白质表达的影响
为了考察VA、VD、VE对pBD-1、2、3基因mRNA与蛋白质表达的影响,本实验选用猪小肠上皮细胞系IPEC-J2为研究对象,在无血清DMEM/F12培养基上分别添加不同浓度的维生素A、D、E培养,24小时后,提取总RNA和蛋白。用Real-timePCR法测定pBD-1、2、3基因mRNA表达水平,用ELISA方法测定pBD-1、2、3蛋白含量。分析不同浓度维生素对pBD基因与蛋白表达的影响。
结果表明：维生素A、D、E均能够不同程度的诱导猪小肠上皮细胞pBD基因的表达并能促进pBD蛋白的分泌。其中,0.5-20μmol/L维生素A可显著提高pBD-1、pBD-2和／或pBD-3基因的mRNA表达水平和蛋白质表达水平；5-50 nmol/L维生素D显著提高了pBD-1、pBD-2或pBD-3基因的mRNA表达水平或蛋白质表达水平；20和50μg/ml维生素E显著提高了pBD-1和pBD-3基因的mRNA表达水平或蛋白质表达水平。结果提示,在本实验浓度范围内,维生素A、D、E可以促进pBD-1、2、3基因mRNA与蛋白的表达,其中维生素A对pBD-1、2、3基因表达的促进作用效果较好。
试验三VA对DLY和荣昌仔猪pBD基因表达的影响
为了考察动物试验中维生素A对内源防御素表达的影响,本试验选用产期相近、胎次相似、相同环境饲养健康的21日龄断奶、体重约3.5-4 kg荣昌猪断奶仔猪24头,按体重随机分为4组,1、3组饲喂维生素A缺乏日粮,2、4组饲喂正常水平维生素A日粮。另外选择产期相近、胎次相似、相同环境饲养健康的21日龄断奶、体重6.5-7kgDLY断奶仔猪30头,按体重随机分为5组,5、7组饲喂维生素A缺乏日粮,6、8组饲喂正常水平维生素A日粮,9组饲喂高剂量维生素A(5倍NRC水平)日粮。每个处理组设有6个重复,每个重复1头猪,试验期包括预饲两周和正式试验四周,在正式试验第14天对3、4、7、8和9组实验动物接种PRRS弱毒苗。进行饲养试验和屠宰试验。
结果表明：
1.DLY猪的日增重和日采食量均显著高于荣昌猪；攻毒(接种PRRS弱毒苗)显著降低了两种猪的日增重和日采食量,显著升高了其料重比；攻毒对DLY猪生产性能的影响强于荣昌猪；
2.DLY猪体内血清视黄醇浓度和肝脏视黄醇浓度均显著高于荣昌猪；攻毒可降低猪血清视黄醇浓度,显著提高肝脏视黄醇浓度；维生素A缺乏显著降低了血清视黄醇浓度和肝脏视黄醇浓度；高剂量维生素A显著升高了肝脏视黄醇浓度,升高血清视黄醇浓度；
3.攻毒显著提高了猪血清中免疫球蛋白IgA、IgG、IgM的浓度；维生素A缺乏显著降低了猪血清中免疫球蛋白IgA、IgG、IgM的浓度；攻毒对DLY猪血清中IgA和IgG的影响强于荣昌猪；
4.攻毒显著提高细胞因子IL-1a、IL-1B、IL-10的浓度；维生素A水平缺乏显著提高了细胞因子IL-1a、IL-1β的浓度,降低了细胞因子IL-10的浓度；
5.接种PRRS弱毒苗对pBD-1、3基因表达的影响不大,能显著提高pBD-2在肺、脾、肠系膜淋巴结和腹股沟淋巴结中的表达；日粮中维生素A水平对pBD-2基因表达的影响不大,能显著影响pBD-1、3在舌、口腔黏膜、呼吸道黏膜和小肠黏膜中的表达。
结果显示,攻毒降低了试验动物日采食量、日增重和血清维生素A,提高了猪血清中免疫球蛋白IgA、IgG、IgM的浓度,提高了pBD-2基因在肺、脾、肠系膜淋巴结和腹股沟淋巴结中的表达,相比较而言,攻毒对DLY猪的影响要强于荣昌猪；维生素A缺乏降低了血清视黄醇的含量与体内部分组织中pBD-1、3基因的表达,攻毒可以加剧这种作用。试验四VA影响pBD基因表达信号转导机制的研究
为初步揭示维生素A诱导pBD-1、2、3基因表达的信号转导机制,本试验以IPEC-J2细胞系为研究模型,考察信号通路阻断剂对维生素A诱导pBD-1、2、3基因mRNA表达的影响。试验选用的信号通路阻断剂有NF-κB通路阻断剂MG-132和MEK-ERK通路阻断剂PD98059,采用单因子试验设计,共分对照组、维生素A组、MG-132组、PD98059组、维生素A+MG-132组和维生素A+PD98059组6个处理组。
结果表明,MG-132极显著的降低了维生素A对pBD-2基因的诱导作用,PD98059极显著的降低了维生素A对pBD-1和pBD-3基因的诱导作用。结果提示,NF-κB通路可能是维生素A影响pBD-2基因表达的信号转导通路之一；MEK-ERK通路可能是维生素A影响pBD-1、3基因的表达的信号通路之一。
综上所述,本研究结果表明：
1.抗病力不同的梅山猪和DLY猪体内pBD的表达水平也不相同,抗病力较高的地方猪种——梅山猪具有较高的防御素表达量。
2.维生素A、D、E均能够诱导猪小肠上皮细胞pBD-1、2、3基因的表达,并能促进其蛋白质的分泌。其中,维生素A对pBD-1、2、3基因表达的诱导作用最为显著。
3.在正常和攻毒两种状态下,日粮维生素A的含量对舌、口腔黏膜、呼吸道黏膜和小肠黏膜等组织中pBD-1、3基因的表达有显著影响,高水平维生素A日粮可以促进这些组织中的pBD基因的表达。
4.本试验初步证明,MEK-ERK通路是维生素A诱导pBD-1、3基因的表达的信号转导通路之一；NF-κB通路是维生素A诱导pBD-2基因表达的信号通路之一。
Defensin is one kind of antibiotic peptide, which has the extensive effects of antibiosis, anti-virus, anti-parasitic and immune regulation, and defensin is an important component of immune system. As a kind of immune modulator, vitamin A has significant effect on the ability of anti-infection, cytokines and related gene expression of animals. But, the gene expression regularity of porcineβ-defensin and the regulative action of vitamin A on porcineβ-defensin have not been reported yet. This research intended to study the difference of porcineβ-defensin expressed in species and tissues, and the effect of vitamin on the expression, to investigate the expression trait of porcineβ-defensin and the regulative effect of vitamin A. This research firstly studied the difference of porcineβ-defensin1,2,3 genes expression in two kinds of pig species with different disease resistance, and the effects of different vitamin supplementation on the expression ofβ-defensin1,2,3 genes through cell culture experiment in vitro, focused on the effect of vitamin A on porcineβ-defensin1,2,3 genes expression in animal test, initially investigated the mechanism of signal transduction of porcineβ-defensin1,2,3 genes expression which influenced by vitamin A. This study includes the following four experiments.
Experiment 1:Research on the difference of pBD-1,2,3 genes expression of DLY and Meishan pigs
The expression difference ofβ-defensin gene in two pig species was compared through the study onβ-defensin expression level in different tissues of DLY and Meishan pigs. Six Meishan pigs and six DLY (Duroc×Landrace x Large) crossbred pigs all at 7-day-old were selected randomly, half male and half female. The mucous membrane of small intestine, heart, liver, spleen, lung, kidney, brain, skin, muscle, oral mucous membrane, respiratory epithelium, tongue, thymus, reproductive tract epithelium, and testis (male) or ovaries (female) were all selected immediately after pigs were slaughtered, and saved in liquid nitrogen for the extraction of total RNA to detect the levels of pBD-1,2,3 genes expression by real-time quantitative PCR.
The results showed that, the pBD gene mRNA was detected in all tissues. The expression of pBD-1,2,3 genes in the tongue and oral mucosa were the highest for Meishan pigs. The expression of pBD-1,3 genes in the tongue and oral mucosa were the highest for DLY crossbred pigs, and also for the expression of pBD-2 genes in the kidney and liver. For the expression of pBD gene in most tissues of Meishan pigs was higher than that of the DLY crossbred pigs. The expression level of pBD-1,2,3 genes in the small intestinal mucosa, tongue, oral mucosa and genital mucosa which contacting with the outside world of Meishan pigs was higher than DLY crossbred pigs. So, it illustrated that the apparent species difference and tissue specificity existed for porcine pBD gene expression, the expression level of pBD-1,2,3 genes in most tissues of Meishan pigs were higher than DLY crossbred pigs.
Experiment 2:The effect of VA, VD and VE on the expression of pBD genes and protein in cell line IPEC-J2
To study the effect of VA, VD and VE on the expression of pBD-1,2,3 genes mRNA and protein, porcine small intestinal cell line IPEC-J2 was used and cultured in serum free DMEM/F12 medium with different levels of vitamin A, D, E. Total RNA and protein were extracted after 24h. The expression of pBD-1,2,3 gene mRNA were determined by Real-time PCR, and the protein content of pBD-1,2,3 were measured by ELISA, to compare the effect of different level of vitamin on the expression of pBD gene and protein.
Results showed that, Vitamin A, D, E could induce the expression of pBD gene in porcine small intestinal epithelium and the secretion of pBD protein. Vitamin A at 0.5-20μmol/L improved the mRNA and protein expression level of pBD-1,2 and/or pBD-3 significantly. Vitamin D at 5-50nmol/L improved the mRNA or protein expression level of pBD-1, pBD2 or pBD-3 significantly. Vitamin E at 20 and 50μg/ml improved the mRNA or protein expression level of pBD-1 and pBD-3 significantly. Results illustrated that vitamin A, D, E could improve the mRNA and protein expression of pBD1,2,3 genes in this present condition, and vitamin A would be better for improving the expression of pBD1,2,3 genes.
Experiment 3:VA influenced the expression of pBDs gene in DLY and Rongchang piglets
24 Rongchang piglets (initially 3.5-4kg BW and 21-day-weaned) were randomly allotted to 4 treatment groups:group1,2,3 and 4 by weight; 30 DLY piglets (initially 6.5-7kg BW and 21-day-weaned) were randomly allotted to 5 treatment groups:group 5,6, 7,8 and 9 by weight, each treatment had 6 replicates with 1 piglets per replicate. The trial was conducted for 28 days. Groups 1,3,5 and 7 were fed on vitamin A deficient diet, group 9 were fed on higher vitamin A diet (5*NRC) and other groups were fed on normal vitamin A diet. The trial period included 2 weeks preliminary experiment and 4 weeks formal experiment. In formal experiment day 14, the piglets of group 3、4、7、8 and 9 were injected PRRS modified live vaccine vaccination or PBS as a control.
Results showed that,
1. DLY piglets' ADG and ADFI were significantly higher than Rongchang piglets. After piglets vaccinated PRRS, the performance of both Rongchang and DLY piglets were declined. The effect of PRRS to DLY piglets performance was bigger than to Rongchang piglets.
2. The retinol level in serum and liver of DLY piglets were significantly higher than Rongchang piglet. PRRS vaccination decreased the serum retinol level but improved the liver retinol level. Vitamin A deficient treatment significantly decreased the retinol level in serum and liver. High vitamin A diet improved both the liver and serum retinol level.
3. IgA、IgG and IgM in serum increased significantly after PRRS vaccination. Vitamin A deficient treatment significantly decreased the serum IgA、IgG and IgM contents. The effect of PRRS to DLY piglets serum IgA and IgG was greater than to Rongchang piglets.
4. IL-1a、IL-1βand IL-10 in serum increased significantly after PRRS vaccination. Vitamin A deficient treatment significantly increased the serum IL-la、IL-1βand decreased the serum IL-10 contents.
5. PRRS vaccination showed little effect on the mRNA expression of pBD-1 and pBD-3, but improved significantly the mRNA expression of pBD-2 in lung, spleen, mesenteric nodes and inguinal lymph nodes. The level of vitamin A in diet had little effect on the mRNA expression of pBD-2 gene, and had significant effect on the mRNA expression of pBD-1 and pBD-3 in tongue, oral mucosa, respiratory mucosa and mucous membrane of small intestine.
Results indicated that experimental animal'ADG, ADFI and serum retinol level were declined after PRRS vaccination, but the serum IgA、IgG、IgM increased significantly, the mRNA expression of pBD-2 in lung, spleen, mesenteric nodes and inguinal lymph nodes also increased. In comparison, the effect of PRRS to DLY piglets was greater than to Rongchang piglets. Vitamin A deficient treatment significantly decreased the serum retinol level and mRNA expression of pBD-1 and pBD-3 in some tissues, and this effect can be aggravated by PRRS vaccination.
Experiment 4:The possible signal transduction channels of VA influenced the expression of pBDs gene
In order to preliminary reveal the signal transduction mechanisms of vitamin A promoting induction expression of pBD-1,2,3 gene, the experiment used IPEC-J2 cell line model for the study to investigate the effet of signaling pathway blockade on induction expression of VA in pBD-1,2,3 gene mRNA. We chose MG-132 and PD98059 for NF-κB and MEK-ERK signaling pathway inhibitor, respectively, and applied single factor design with six treatment groups, that was control group, the vitamin A group, MG-132 group, PD98059 group, vitamin A+MG-132 group and the vitamin A+PD98059 group.
The results show that, MG-132 very significantly reduced the induction expression of pBD-2 gene of vitamin A, PD98059 significantly reduced the pBD-1 and pBD-3 gene induction of vitamin A. The results suggest that, NF-κB pathway may be one of the signal transduction pathway of vitamin A affection the expression of pBD-2 gene; MEK-ERK pathway may be one of the signaling pathways of vitamin A affection pBD-1,3 expression.
In conclusion, data reported here demonstrated:
1. The distribution and expression of pBD in Meishan and DLY pigs with different immunity were different, the expression of pBD1,2 and 3 in mucous membrane of small intestine, tongue, oral mucosa and mucous membrane of reproductive tract in Meishan pigs with higher immunity were all higher than DLY pigs.
2. Vitamin A, D and E could all induce the expression of pBD-1,2 and 3 gene in small intestine epithelium, and motivate the secretion of its protein, this mode of action exhibited the dose dependent relation. The promotive effect of vitamin A was the most significant one.
3. Under the situation of normal and PRRS vaccination, the content of vitamin A in diet showed the significant effect on the expression of pBD-1 and 3 genes in tongue, oral mucosa, Respiratory Mucosa and mucous membrane of small intestine, diet with higher vitamin A improved the expression of pBD gene in these tissues.
4. This study demonstrated initially that MEK-ERK passageway was one of the signal transduction channels of vitamin A inducing the expression of pBD-1 and 3 genes, NF-κB passageway was one of the signal transduction channels of vitamin A inducing the expression of pBD-2 gene.
试验一DLY和梅山仔猪pBD基因表达差异的研究
通过观察防御素基因在DLY和梅山仔猪各个组织中的表达水平,比较了两个猪种之间的表达差异。本实验随机选择健康的7日龄梅山猪和DLY(杜×长×大)杂交猪各6头,公母各半。屠宰后立即取其小肠黏膜、心、肝、脾、肺、肾、脑、皮肤、肌肉、口腔黏膜、呼吸道上皮、舌头、胸腺、生殖道上皮、睾丸(公)或卵巢(母),提取总RNA,用Real-time PCR方法检测pBD-1、2、3基因mRNA表达水平。
结果表明：所有组织中均能检测出pBD基因mRNA的表达。其中梅山猪中,pBD-1、2、3在舌头和口腔黏膜的表达量最高；而DLY杂交猪中,pBD-1、3在舌头和口腔黏膜的表达量最高,pBD-2在肾脏和肝脏的表达量最高。绝大部分组织的pBD基因表达量均为梅山猪高于DLY杂交猪,其中小肠黏膜、舌、口腔黏膜和生殖道黏膜等与外界接触的组织中的pBD-1、2、3表达量均是以梅山猪为高。由此显示,猪pBD的表达存在明显的品种差异和组织特异性,梅山猪体内pBD-1、2、3基因在多个组织中的表达量要高于DLY三元杂交猪。
试验二VA、VD、VE对IPEC-J2细胞系pBD基因mRNA和蛋白质表达的影响
为了考察VA、VD、VE对pBD-1、2、3基因mRNA与蛋白质表达的影响,本实验选用猪小肠上皮细胞系IPEC-J2为研究对象,在无血清DMEM/F12培养基上分别添加不同浓度的维生素A、D、E培养,24小时后,提取总RNA和蛋白。用Real-timePCR法测定pBD-1、2、3基因mRNA表达水平,用ELISA方法测定pBD-1、2、3蛋白含量。分析不同浓度维生素对pBD基因与蛋白表达的影响。
结果表明：维生素A、D、E均能够不同程度的诱导猪小肠上皮细胞pBD基因的表达并能促进pBD蛋白的分泌。其中,0.5-20μmol/L维生素A可显著提高pBD-1、pBD-2和／或pBD-3基因的mRNA表达水平和蛋白质表达水平；5-50 nmol/L维生素D显著提高了pBD-1、pBD-2或pBD-3基因的mRNA表达水平或蛋白质表达水平；20和50μg/ml维生素E显著提高了pBD-1和pBD-3基因的mRNA表达水平或蛋白质表达水平。结果提示,在本实验浓度范围内,维生素A、D、E可以促进pBD-1、2、3基因mRNA与蛋白的表达,其中维生素A对pBD-1、2、3基因表达的促进作用效果较好。
试验三VA对DLY和荣昌仔猪pBD基因表达的影响
为了考察动物试验中维生素A对内源防御素表达的影响,本试验选用产期相近、胎次相似、相同环境饲养健康的21日龄断奶、体重约3.5-4 kg荣昌猪断奶仔猪24头,按体重随机分为4组,1、3组饲喂维生素A缺乏日粮,2、4组饲喂正常水平维生素A日粮。另外选择产期相近、胎次相似、相同环境饲养健康的21日龄断奶、体重6.5-7kgDLY断奶仔猪30头,按体重随机分为5组,5、7组饲喂维生素A缺乏日粮,6、8组饲喂正常水平维生素A日粮,9组饲喂高剂量维生素A(5倍NRC水平)日粮。每个处理组设有6个重复,每个重复1头猪,试验期包括预饲两周和正式试验四周,在正式试验第14天对3、4、7、8和9组实验动物接种PRRS弱毒苗。进行饲养试验和屠宰试验。
结果表明：
1.DLY猪的日增重和日采食量均显著高于荣昌猪；攻毒(接种PRRS弱毒苗)显著降低了两种猪的日增重和日采食量,显著升高了其料重比；攻毒对DLY猪生产性能的影响强于荣昌猪；
2.DLY猪体内血清视黄醇浓度和肝脏视黄醇浓度均显著高于荣昌猪；攻毒可降低猪血清视黄醇浓度,显著提高肝脏视黄醇浓度；维生素A缺乏显著降低了血清视黄醇浓度和肝脏视黄醇浓度；高剂量维生素A显著升高了肝脏视黄醇浓度,升高血清视黄醇浓度；
3.攻毒显著提高了猪血清中免疫球蛋白IgA、IgG、IgM的浓度；维生素A缺乏显著降低了猪血清中免疫球蛋白IgA、IgG、IgM的浓度；攻毒对DLY猪血清中IgA和IgG的影响强于荣昌猪；
4.攻毒显著提高细胞因子IL-1a、IL-1B、IL-10的浓度；维生素A水平缺乏显著提高了细胞因子IL-1a、IL-1β的浓度,降低了细胞因子IL-10的浓度；
5.接种PRRS弱毒苗对pBD-1、3基因表达的影响不大,能显著提高pBD-2在肺、脾、肠系膜淋巴结和腹股沟淋巴结中的表达；日粮中维生素A水平对pBD-2基因表达的影响不大,能显著影响pBD-1、3在舌、口腔黏膜、呼吸道黏膜和小肠黏膜中的表达。
结果显示,攻毒降低了试验动物日采食量、日增重和血清维生素A,提高了猪血清中免疫球蛋白IgA、IgG、IgM的浓度,提高了pBD-2基因在肺、脾、肠系膜淋巴结和腹股沟淋巴结中的表达,相比较而言,攻毒对DLY猪的影响要强于荣昌猪；维生素A缺乏降低了血清视黄醇的含量与体内部分组织中pBD-1、3基因的表达,攻毒可以加剧这种作用。试验四VA影响pBD基因表达信号转导机制的研究
为初步揭示维生素A诱导pBD-1、2、3基因表达的信号转导机制,本试验以IPEC-J2细胞系为研究模型,考察信号通路阻断剂对维生素A诱导pBD-1、2、3基因mRNA表达的影响。试验选用的信号通路阻断剂有NF-κB通路阻断剂MG-132和MEK-ERK通路阻断剂PD98059,采用单因子试验设计,共分对照组、维生素A组、MG-132组、PD98059组、维生素A+MG-132组和维生素A+PD98059组6个处理组。
结果表明,MG-132极显著的降低了维生素A对pBD-2基因的诱导作用,PD98059极显著的降低了维生素A对pBD-1和pBD-3基因的诱导作用。结果提示,NF-κB通路可能是维生素A影响pBD-2基因表达的信号转导通路之一；MEK-ERK通路可能是维生素A影响pBD-1、3基因的表达的信号通路之一。
综上所述,本研究结果表明：
1.抗病力不同的梅山猪和DLY猪体内pBD的表达水平也不相同,抗病力较高的地方猪种——梅山猪具有较高的防御素表达量。
2.维生素A、D、E均能够诱导猪小肠上皮细胞pBD-1、2、3基因的表达,并能促进其蛋白质的分泌。其中,维生素A对pBD-1、2、3基因表达的诱导作用最为显著。
3.在正常和攻毒两种状态下,日粮维生素A的含量对舌、口腔黏膜、呼吸道黏膜和小肠黏膜等组织中pBD-1、3基因的表达有显著影响,高水平维生素A日粮可以促进这些组织中的pBD基因的表达。
4.本试验初步证明,MEK-ERK通路是维生素A诱导pBD-1、3基因的表达的信号转导通路之一；NF-κB通路是维生素A诱导pBD-2基因表达的信号通路之一。
Defensin is one kind of antibiotic peptide, which has the extensive effects of antibiosis, anti-virus, anti-parasitic and immune regulation, and defensin is an important component of immune system. As a kind of immune modulator, vitamin A has significant effect on the ability of anti-infection, cytokines and related gene expression of animals. But, the gene expression regularity of porcineβ-defensin and the regulative action of vitamin A on porcineβ-defensin have not been reported yet. This research intended to study the difference of porcineβ-defensin expressed in species and tissues, and the effect of vitamin on the expression, to investigate the expression trait of porcineβ-defensin and the regulative effect of vitamin A. This research firstly studied the difference of porcineβ-defensin1,2,3 genes expression in two kinds of pig species with different disease resistance, and the effects of different vitamin supplementation on the expression ofβ-defensin1,2,3 genes through cell culture experiment in vitro, focused on the effect of vitamin A on porcineβ-defensin1,2,3 genes expression in animal test, initially investigated the mechanism of signal transduction of porcineβ-defensin1,2,3 genes expression which influenced by vitamin A. This study includes the following four experiments.
Experiment 1:Research on the difference of pBD-1,2,3 genes expression of DLY and Meishan pigs
The expression difference ofβ-defensin gene in two pig species was compared through the study onβ-defensin expression level in different tissues of DLY and Meishan pigs. Six Meishan pigs and six DLY (Duroc×Landrace x Large) crossbred pigs all at 7-day-old were selected randomly, half male and half female. The mucous membrane of small intestine, heart, liver, spleen, lung, kidney, brain, skin, muscle, oral mucous membrane, respiratory epithelium, tongue, thymus, reproductive tract epithelium, and testis (male) or ovaries (female) were all selected immediately after pigs were slaughtered, and saved in liquid nitrogen for the extraction of total RNA to detect the levels of pBD-1,2,3 genes expression by real-time quantitative PCR.
The results showed that, the pBD gene mRNA was detected in all tissues. The expression of pBD-1,2,3 genes in the tongue and oral mucosa were the highest for Meishan pigs. The expression of pBD-1,3 genes in the tongue and oral mucosa were the highest for DLY crossbred pigs, and also for the expression of pBD-2 genes in the kidney and liver. For the expression of pBD gene in most tissues of Meishan pigs was higher than that of the DLY crossbred pigs. The expression level of pBD-1,2,3 genes in the small intestinal mucosa, tongue, oral mucosa and genital mucosa which contacting with the outside world of Meishan pigs was higher than DLY crossbred pigs. So, it illustrated that the apparent species difference and tissue specificity existed for porcine pBD gene expression, the expression level of pBD-1,2,3 genes in most tissues of Meishan pigs were higher than DLY crossbred pigs.
Experiment 2:The effect of VA, VD and VE on the expression of pBD genes and protein in cell line IPEC-J2
To study the effect of VA, VD and VE on the expression of pBD-1,2,3 genes mRNA and protein, porcine small intestinal cell line IPEC-J2 was used and cultured in serum free DMEM/F12 medium with different levels of vitamin A, D, E. Total RNA and protein were extracted after 24h. The expression of pBD-1,2,3 gene mRNA were determined by Real-time PCR, and the protein content of pBD-1,2,3 were measured by ELISA, to compare the effect of different level of vitamin on the expression of pBD gene and protein.
Results showed that, Vitamin A, D, E could induce the expression of pBD gene in porcine small intestinal epithelium and the secretion of pBD protein. Vitamin A at 0.5-20μmol/L improved the mRNA and protein expression level of pBD-1,2 and/or pBD-3 significantly. Vitamin D at 5-50nmol/L improved the mRNA or protein expression level of pBD-1, pBD2 or pBD-3 significantly. Vitamin E at 20 and 50μg/ml improved the mRNA or protein expression level of pBD-1 and pBD-3 significantly. Results illustrated that vitamin A, D, E could improve the mRNA and protein expression of pBD1,2,3 genes in this present condition, and vitamin A would be better for improving the expression of pBD1,2,3 genes.
Experiment 3:VA influenced the expression of pBDs gene in DLY and Rongchang piglets
24 Rongchang piglets (initially 3.5-4kg BW and 21-day-weaned) were randomly allotted to 4 treatment groups:group1,2,3 and 4 by weight; 30 DLY piglets (initially 6.5-7kg BW and 21-day-weaned) were randomly allotted to 5 treatment groups:group 5,6, 7,8 and 9 by weight, each treatment had 6 replicates with 1 piglets per replicate. The trial was conducted for 28 days. Groups 1,3,5 and 7 were fed on vitamin A deficient diet, group 9 were fed on higher vitamin A diet (5*NRC) and other groups were fed on normal vitamin A diet. The trial period included 2 weeks preliminary experiment and 4 weeks formal experiment. In formal experiment day 14, the piglets of group 3、4、7、8 and 9 were injected PRRS modified live vaccine vaccination or PBS as a control.
Results showed that,
1. DLY piglets' ADG and ADFI were significantly higher than Rongchang piglets. After piglets vaccinated PRRS, the performance of both Rongchang and DLY piglets were declined. The effect of PRRS to DLY piglets performance was bigger than to Rongchang piglets.
2. The retinol level in serum and liver of DLY piglets were significantly higher than Rongchang piglet. PRRS vaccination decreased the serum retinol level but improved the liver retinol level. Vitamin A deficient treatment significantly decreased the retinol level in serum and liver. High vitamin A diet improved both the liver and serum retinol level.
3. IgA、IgG and IgM in serum increased significantly after PRRS vaccination. Vitamin A deficient treatment significantly decreased the serum IgA、IgG and IgM contents. The effect of PRRS to DLY piglets serum IgA and IgG was greater than to Rongchang piglets.
4. IL-1a、IL-1βand IL-10 in serum increased significantly after PRRS vaccination. Vitamin A deficient treatment significantly increased the serum IL-la、IL-1βand decreased the serum IL-10 contents.
5. PRRS vaccination showed little effect on the mRNA expression of pBD-1 and pBD-3, but improved significantly the mRNA expression of pBD-2 in lung, spleen, mesenteric nodes and inguinal lymph nodes. The level of vitamin A in diet had little effect on the mRNA expression of pBD-2 gene, and had significant effect on the mRNA expression of pBD-1 and pBD-3 in tongue, oral mucosa, respiratory mucosa and mucous membrane of small intestine.
Results indicated that experimental animal'ADG, ADFI and serum retinol level were declined after PRRS vaccination, but the serum IgA、IgG、IgM increased significantly, the mRNA expression of pBD-2 in lung, spleen, mesenteric nodes and inguinal lymph nodes also increased. In comparison, the effect of PRRS to DLY piglets was greater than to Rongchang piglets. Vitamin A deficient treatment significantly decreased the serum retinol level and mRNA expression of pBD-1 and pBD-3 in some tissues, and this effect can be aggravated by PRRS vaccination.
Experiment 4:The possible signal transduction channels of VA influenced the expression of pBDs gene
In order to preliminary reveal the signal transduction mechanisms of vitamin A promoting induction expression of pBD-1,2,3 gene, the experiment used IPEC-J2 cell line model for the study to investigate the effet of signaling pathway blockade on induction expression of VA in pBD-1,2,3 gene mRNA. We chose MG-132 and PD98059 for NF-κB and MEK-ERK signaling pathway inhibitor, respectively, and applied single factor design with six treatment groups, that was control group, the vitamin A group, MG-132 group, PD98059 group, vitamin A+MG-132 group and the vitamin A+PD98059 group.
The results show that, MG-132 very significantly reduced the induction expression of pBD-2 gene of vitamin A, PD98059 significantly reduced the pBD-1 and pBD-3 gene induction of vitamin A. The results suggest that, NF-κB pathway may be one of the signal transduction pathway of vitamin A affection the expression of pBD-2 gene; MEK-ERK pathway may be one of the signaling pathways of vitamin A affection pBD-1,3 expression.
In conclusion, data reported here demonstrated:
1. The distribution and expression of pBD in Meishan and DLY pigs with different immunity were different, the expression of pBD1,2 and 3 in mucous membrane of small intestine, tongue, oral mucosa and mucous membrane of reproductive tract in Meishan pigs with higher immunity were all higher than DLY pigs.
2. Vitamin A, D and E could all induce the expression of pBD-1,2 and 3 gene in small intestine epithelium, and motivate the secretion of its protein, this mode of action exhibited the dose dependent relation. The promotive effect of vitamin A was the most significant one.
3. Under the situation of normal and PRRS vaccination, the content of vitamin A in diet showed the significant effect on the expression of pBD-1 and 3 genes in tongue, oral mucosa, Respiratory Mucosa and mucous membrane of small intestine, diet with higher vitamin A improved the expression of pBD gene in these tissues.
4. This study demonstrated initially that MEK-ERK passageway was one of the signal transduction channels of vitamin A inducing the expression of pBD-1 and 3 genes, NF-κB passageway was one of the signal transduction channels of vitamin A inducing the expression of pBD-2 gene.
引文
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