F4ac型产肠毒素大肠杆菌菌液上清对仔猪小肠上皮细胞的免疫刺激作用

The Immunostimulation of F4ac ETEC-Culture Supernatant to Porcine Small Intestinal Epithelial Cells

【目的】猪源产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)是一类世界范围内流行的致仔猪腹泻的病原菌。依据养猪业生产实践中对致仔猪腹泻病原菌血清学鉴定结果可知,F4ac型ETEC的流行性最为广泛。到目前为止,对ETEC导致仔猪腹泻的机理已经有了比较透彻的阐释。但对ETEC培养液的免疫原性尚未有研究和描述。论文以F4ac型ETEC为研究对象,探索其菌液上清(即培养液)对仔猪小肠上皮细胞(IPEC-J2)的免疫刺激作用。【方法】首先收集F4ac型ETEC菌株200培养12h后的培养液,后经4℃,4 000 r/min,离心15 min收集培养液上清,将该上清液经0.22μm滤器过滤,并与DMEM/F12培养基以1﹕1的比例混合,以此混合液共孵育IPEC-J2细胞3 h,重复此处理3次获得处理组细胞;同时设新鲜的LB培养基与DMEM/F12培养基同样以1﹕1的比例共孵育3 h的IPEC-J2细胞为对照组细胞,对照处理亦重复3次。然后用TRIZOL试剂按照说明书提取对照组和攻毒组IPEC-J2细胞的总RNA,并用TaKaRa公司的PrimeScriptTM RT reagent Kit with gDNA Eraser(反转录试剂盒)按照说明书将提取的总RNA反转录成cDNA。应用文献报道或自行设计的跨内含子的引物通过实时荧光定量PCR方法,以猪的持家基因β-actin作为内参,检测IL8、TNF-α、CXCL2、IL6、IL1A、TLR4、SLPI、PLAU和MUC13共9个免疫相关基因和黏蛋白基因在攻毒组和对照组中的mRNA表达差异性。【结果】较之对照组,在攻毒组中,3个重要的促炎细胞因子基因IL8、TNF-α和CXCL2的mRNA均出现了显著性地过表达。其中,在攻毒组中,IL8的表达量为对照组的3.24倍(P<0.05);TNF-α的表达量亦为对照组的3.24倍(P<0.01);CXCL2的表达量为对照组的1.65倍(P<0.001)。在攻毒组和对照组之间,其他6个基因(IL6、IL1A、TLR4、SLPI、PLAU和MUC13)的表达差异未达到统计学上的显著水平。【结论】研究采用F4ac型ETEC菌株200的培养液上清对IPEC-J2细胞系进行3 h攻毒处理,并通过荧光定量PCR方法检测到了3个重要的促炎因子基因IL8、TNF-α和CXCL2在攻毒组较之非攻毒组中的过表达。这表明猪F4ac型ETEC培养液上清对仔猪小肠上皮细胞确实具有一定的免疫刺激作用。研究为更明确地了解猪F4ac型ETEC释放的肠毒素及黏附素等抗原物质对仔猪小肠上皮细胞的免疫刺激作用提供了一定的实验证据。

[ Objective ] Porcine enterotoxigenic Escherichia coli （ETEC） is a worldwide cause of bacteria induced diarrhoea in piglets. In the veterinary practices of pig production, serological identification of ETEC shows that F4ac is the most common serological type expressed in ETEC strains isolated from diarrheic piglets. So far, the mechanism by which ETEC produces diarrhoea in piglets has been clearly elucidated. However, the immunostimulation of ETEC-culture to host target cells has not been studied or described. In the present study, the immunostimulation of the supernatant of F4ac ETEC-culture to IPEC-J2 cells was studied. [Method] The culture ofF4ac ETEC strain 200 was collected and centrifuged （4~C, 4 000 r/min for 15 min） after 12 hours in culture, and the supernatant was sterilized by passing it through a 0.22 μm filter. The solution combined the sterilized supernatant with equivalent DMEM/F12 medium was used to challenge IPEC-J2 cells for 3 hours. The IPEC-J2 cells co-cultured with fresh LB medium and equivalent DMEM/F 12 medium were as the control group. For both treatments, each experiment was repeated three times. Total RNAs of the stimulated and control IPEC-J2 cells were extracted using TRIZOL Reagent following the manufacturer＇s instructions. According to the manufacturer＇s instructions, complementary DNA （eDNA） was synthesized from RNA using the Prime Script~ RT reagent Kit with gDNA Eraser （Perfect Real Time）. The eDNA samples were then analyzed with real time RT-PCR using a LightCycler~ 480 Real-Time PCR System. The real time RT-PCR reactions were performed in a final volume of 20 p^L with the Roche SYBR Green PCR Kit according to the manufacturer＇s instructions. The pig house-keeping gene fl-actin was used as the internal standards to correct the input of eDNA. Triplicate qRT-PCRs were performed on each cDNA and the average Ct was used for further analysis. The relative quantification values were calculated using the 2＂6/＇Ct. Differential mRNA expression profiles oflL8, TNF-a, CXCL2, 1L6, 1LIA, TLR4, SLPI, PLAU and MUC13 between the stimulated and control IPEC-J2 cell groups were tested by amplification using documented and self-designed primers in the presence of SYBR Green. [Result] Compared with the control group, the mRNA expression of three important cytokines IL8, TNF-a and CXCL2 were up-regulated in the stimulated treatment group with the fold-change oflL8 being 3.24 （P〈0.05）, the fold-change of TNF-a also being 3.24 （P〈0.01） and that of CXCL2 being 1.65 （P〈0.001）. For the rest six genes （1L6, ILIA, TLR4, SLP1, PLAU and MUC13）, no statistically significant difference was observed in the mRNA expression levels between the two groups. [Conclusion] In this study, the IPEC-J2 cells were challenged with the superuatant of F4ac ETEC-culture for 3 hours, and it was found that this stimulation increased the mRNA expression levels of three key pro-inflammatory genes IL8, TNF-a and CXCL2, which proved that the supernatant of F4ac ETEC-culture showed immunostimulation to the IPEC-J2 cells. The discovery in this study provided insights into the immunostimulation of enterotoxin and/or adhesion of F4ac ETEC to porcine intestinal epithelial cells.