新生仔猪小肠上皮细胞的分离培养和鉴定

为更快捷地进行猪肠道病毒性、细菌性疾病及细胞永生化的研究提供便利,本研究使用多种酶消化法及其他培养法尝试分离培养肠上皮细胞.结果,利用组织块培养法能成功建立新生仔猪小肠上皮细胞株,获得的上皮细胞具有较强的增殖能力,22 h内贴壁并发生细胞分裂,6～7 d明显增殖,10～12d汇合成单层.连续传代培养至12代,细胞基本失去上皮样特征.倒置显微镜下观察,10代前培养细胞为多角状或卵圆形,单层生长不重叠,呈铺路石状排列,11～12代细胞发生变形,间隙增大.细胞角蛋白18鉴定二者均为阳性.电镜下,纹状缘整齐,紧密连接结构清晰可见.结果表明利用组织块培养法可以获得能够连续传代、活力较好的猪小肠上皮细胞.     

Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells

ABSTRACT: This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a+CD11R1high and CD4+ cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.     

新生山羊小肠上皮细胞株的建立和鉴定

进行肠上皮细胞的分离培养是研究小肠功能、营养物质吸收机制及其调控的主要手段之一。本试验利用组织块培养法成功的建立了山羊小肠上皮细胞株。获得的上皮细胞具有较强的增殖能力,24h内贴壁并发生分裂。纯化后按照0.8～0.9×104个/cm2的密度种植时,3～4d连成一片。倒置显微镜下观察培养细胞为多角状或卵圆形,单层生长不重叠,呈铺路石状排列,细胞角蛋白鉴定为阳性。采用组织块培养法可以获得具有增殖能力的山羊小肠上皮细胞,为研究IEC的生理病理等提供有用的实验模型。     

Role of intestinal epithelial cells in the innate immune defence of the pig intestine

The intestinal epithelium serves as a dynamic barrier, which in the course of its normal function, maintains regulated uptake of nutrients and water while excluding potential pathogens. Over the past decade many studies have also revealed the immunological importance of intestinal epithelial cells (IEC). IEC have developed a variety of mechanisms to reduce the risk of infection by invasive pathogens or damage by toxic compounds. The effective maintenance of a physical barrier function is dependent on the establishment of well-organised intercellular junctions and a constant state of regeneration/renewal of the epithelium. IEC also participate in the innate immune responsiveness of the intestine by their ability to secrete mucus and antimicrobial peptides. IEC are also able to secrete cytokines and to respond to exogenous chemokines. This review summarises the current knowledge of the innate immune mechanisms developed by porcine IEC.     

Innate immune activation of swine intestinal epithelial cell lines (IPEC-J2 and IPI-2I) in response to LPS from Salmonella typhimurium

The innate immune system has the basic function of identifying and eradicating microbial invaders and alerting the adaptative immune system to their presence. In this study, the porcine intestinal innate immune response was evaluated by analysing the expression of TLRs, cytokines and chemokines in two porcine epithelial cell lines from different regions: IPEC-J2 (jejunum) and IPI-2I (ileum). Both cells lines were stimulated with 1microg of LPS from Salmonella typhimurium. RNA was collected at 30min, 1, 2, 3 and 4h after treatment. Expression of TLR-1, -2, -3, -4, -6, -8, -9, -10, TNF-alpha, IL-1beta, -8 and MCP-1 was quantified relative to the quantity of Cyclophilin-A mRNA using real-time quantitative PCR (RTQ-PCR). The results obtained show up differences in the gene expression between both cell lines IPEC-J2 and IPI-2I as response to LPS from S. typhimurium during the activation time, which may suggest an in vivo variability in the innate immune response against pathogens in different regions of the host's gut.     

丁酸钠对体外培养猪肠黏膜上皮细胞寡肽转运载体及钠氢交换载体mRNA表达的影响

为探讨丁酸钠对肠道寡肽转运载体(PepT1)及钠氢交换载体(NHE2、NHE3)mRNA表达丰度的调控,取刚出生未吮乳的仔猪小肠黏膜组织,建立猪小肠上皮细胞(IEC)分离及原代培养方法。分别用0,2,4,8 mmol/L的丁酸钠处理体外培养的猪小肠黏膜上皮细胞96 h后,提取细胞总RNA,以18S rRNA为内标基因,用实时荧光定量RT-PCR法(SYBR GreenⅠ试剂盒)检测PepT1、NHE2及NHE3 mRNA在不同浓度丁酸钠处理细胞中的表达丰度。结果表明,体外培养的猪小肠黏膜上皮细胞用丁酸钠处理96 h后,PepT1和NHE2 mRNA的表达丰度均显著增加(P0.05),且呈现剂量依赖效应;NHE3 mRNA表达丰度的剂量效应不明显,只有在高浓度丁酸钠(8 mmol/L)组时才显著高于对照组(P0.05)。     

Age-dependent resistance to transmissible gastroenteritis of swine. III. Effects of epithelial cell kinetics on coronavirus production and on atrophy of intestinal villi.

Coronavirus titers in small intestine, degree of villous atrophy and apparent rates of regeneration of intestinal villi were compared in newborn, 3-week-old and adult pigs for 1 week after they were exposed to the transmissible gastroenteritis virus of swine. The response within the newborn group was homogeneous, resulting in high virus titers, maximal villous atrophy and comparatively slow regeneration. In general, virus titers were lower, villous atrophy was less severe and regeneration more rapid in both older groups than in the newborn pigs. However, the response varied greatly in the older groups. The 3-week-old group was divided into two populations. The major population had low virus titers and developed partial villous atrophy, whereas the minor population had marked villous atrophy and virus titers comparable to those of the newborn pigs. These observations support the hyposthesis that the accelerated replacement of villous epithelium in the small intestine of pigs during the first 3 weeks contributes to the innate age-dependent resistance to transmissible gastroenteritis. The accelerated replacement of villous epithelial cells in older pigs contributes to resistance in two ways. The increased proliferative capacity of crypt epithelium results in a more rapid regeneration of atrophic villi; and the comparatively young villous absorptive cells resulting from accelerated replacement produce less virus per cell than the older ones of the newborn pig.     

“非洲猪瘟+饲料无抗”后对猪只营养体系的创新思考

“非洲猪瘟和饲料禁抗”后,猪场对生物安全、猪只免疫营养、肠道健康等的需求进一步加强。文章从猪场需求转变着手,着重从配方设计理念、添加剂筛选、发酵饲料的应用、饲料加工体系和猪场饲喂体系等方面系统阐述对行业变化的应对思考,以期为生猪高效养殖提供实践依据。     

肠道免疫相关的猪富含半胱氨酸肠蛋白2三维建模、分子特征及mRNA表达的组织分布

本研究利用广泛用于肠道感染机制研究的IPEC-J2细胞系探究poCRIP2在猪胃肠道炎症中的作用。在识别poCRIP2基因全长序列的基础上研究poCRIP2的有关特征,同时对其组织表达模式进行分析,并建立有效的poCRIP2三维结构模型以解释其功能,了解poCRIP2在细菌感染过程中的免疫作用及其与NF-κB通路的关系。采用逆转录聚合酶链反应（RT-PCR）技术从猪心获得poCRIP2的全长序列;利用Primer Premier5.0和NCBI Primer BLAST程序设计了特异性引物对,采用保家基因β-肌动蛋白（β-actin）对结果进行归一化处理,测试CRIP2基因在猪体内的组织分布;采用生物信息学方法对poCRIP2的特性进行分析,构建poCRIP2蛋白模型;利用ROBETTA服务器,以高度同源的小鼠LIM-homeodomain protein islet 1（Isl1）（PDB：4 JCJ）为模板,模拟poCRIP2的三维结构;用PROCHECK和ProSA对模型进行了分析;应用Procheck对蛋白质折叠进行立体化学和拓扑分析,并应用ProSA对蛋白质折叠进行评价;使用PyMOL程序对建模结果进行评估,分析poCRIP2的保守结构域;设计肠道感染试验,分析poCRIP2在肠道免疫中的功能。结果表明,从猪心获得的poCRIP2 cDNA全长序列为1 118 bp,poCRIP2蛋白与人和小鼠的同源性分别为94.23%和93.75%,它还含有两个保守区（LIM-TLP和LIM-CRP）。poCRIP2的组织表达模式分析表明,poCRIP2在所有组织中均有表达,其中在小肠、肺、胃、脾和肌肉等中表达较低。poCRIP2在革兰阴性菌作用下的表达水平比对照组高1~2倍,但NF-κB通路在同一作用下被激活,无抑制作用,poCRIP2可能不与NF-κB通路相互作用,而与其它通路共同作用于肠道免疫。本研究成功获得了猪CRIP2基因的全长序列,poCRIP2的组织表达模式表明,poCRIP2与人CRIP2相似,建立了可靠的三维结构模型,基于该模型推测poCRIP2中包含的功能主要由β折叠和α-螺旋结构实现,革兰阴性菌感染肠道后poCRIP2能被显著上调,poCRIP2应不依赖于NF-κB通路,在肠道免疫中起其他作用。     

Porcine IPEC-J2 intestinal epithelial cells in microbiological investigations

IPEC-J2 cells are porcine intestinal columnar epithelial cells that were isolated from neonatal piglet mid-jejunum. This cell line forms polarized monolayers with high transepithelial electrical resistance when cultured on 0.4 μm pore-size filters. The cell line is unique in that it is derived from small intestinal tissue (compared to the common human colon-derived lines HT-29, T84, and Caco-2) and is not transformed (compared to the porcine small intestinal line, IPI-2I). Porcine intestinal epithelial cells more closely mimic human physiology than analogous rodent cell lines (e.g. IEC-6 or IEC-18), which is important in studies of zoonotic infections; in addition, they provide specificity to study porcine-derived infections. IPEC-J2 cells are increasingly being used in microbiological studies to examine the interactions of various animal and human pathogens, including Salmonella enterica and pathogenic Escherichia coli, with intestinal epithelial cells. The IPEC-J2 cell line has also been employed in some probiotic studies, in which the cells have been used as an initial screening tool for adhesiveness and anti-inflammatory properties of the potential probiotic microorganisms. The validity of these studies is not clear as follow-up studies to assess the efficacy of the probiotics in vivo have not been published to date. The aims of this review are to provide a comprehensive overview of the microbiological studies that have been conducted with IPEC-J2 cells and a reference guide of key cellular and immune markers that have been identified in this cell line that may prove to be useful in future studies.     

体外培养猪小肠上皮细胞大豆凝集素结合位点的标记

本研究旨在获得体外培养状态下的猪小肠上皮细胞,研究猪小肠上皮细胞与大豆凝集素的结合情况.选取新生未哺乳健康仔猪,取小肠组织对小肠上皮细胞进行分离培养,并进行纯化.纯化后的细胞以角蛋白抗体-8为—抗进行细胞免疫化学试验鉴定,鉴定后的猪小肠上皮细胞以FITC-SBA为探针进行细胞凝集素荧光标记化学试验,对猪小肠上皮细胞大豆凝集素结合位点进行标记.结果表明:试验分离纯化的细胞经角蛋白抗体-8鉴定为阳性,所分离纯化的细胞为猪小肠上皮细胞,经检测其纯度达90％以上,猪小肠上皮细胞FITC-SBA标记结果为阳性.体外培养的猪小肠上皮细胞表面存在大量的大豆凝集素结合位点,进一步明确了猪小肠上皮细胞是大豆凝集素结合的主要细胞类型之一,为单胃动物大豆凝集索抗营养机制研究提供理论依据.     

Defects in small intestinal epithelial barrier function and morphology associated with peri-weaning failure to thrive syndrome (PFTS) in swine

The objective of this study was to investigate intestinal function and morphology associated with peri-weaning failure to thrive syndrome (PFTS) in swine. Jejunum and distal ileum from control and pigs exhibiting PFTS was harvested at weaning, 4 and 11 days post-weaning (PW) for intestinal barrier function studies and histological analyses (n=6 pigs per group). Marked disturbances in intestinal barrier function was observed in PFTS pigs, compared with controls, indicated by lower (p<0.05) TER and increased (p<0.01) permeability to FITC dextran (4 kDa). Intestines from weaned pigs, subjected to a 4-day fast, exhibited minor disturbances in intestinal barrier function. Villus atrophy and crypt hyperplasia were observed in the PFTS intestine compared with control and fasted pigs. These data demonstrate that PFTS is associated with profound disturbances in intestinal epithelial barrier function and alterations in mucosal and epithelial morphology in which anorexia is not the sole factor.     

小肠上皮细胞的培养及在动物营养上的研究进展

小肠是动物消化吸收的主要场所,体外分离培养小肠黏膜上皮细胞(intestinal epithelial-cells,IEC)为研究小肠消化吸收、物质转运及肠道免疫等生物学功能提供了重要手段,文章综述了体外培养小肠上皮细胞的有关方法及鉴定,简要介绍了小肠上皮细胞培养在动物研究中的应用概况。     

低聚糖对断奶羔羊小肠黏膜免疫相关细胞的影响

选用陶赛特F2代断奶公羔40只,随机分成4组,研究低聚糖对断奶羔羊小肠黏膜免疫相关细胞的影响。结果表明:日粮中添加低聚果糖FOS(0.3%)、低聚异麦芽糖IMO(0.3%)和复合低聚糖(0.1%FOS+0.2%IMO)均可提高断奶羔羊胸腺指数和脾脏指数(P>0.05)及小肠黏膜上皮内sIEL数量(P<0.05);FOS可提高小肠黏膜上皮GC的数量I,MO和复合低聚糖可降低GC数量(P>0.05)I;MO和复合低聚糖可提高断奶羔羊小肠黏膜中MC细胞的数量(P>0.05)。提示断奶羔羊日粮中添加低聚糖可增加其小肠黏膜免疫相关细胞的数量,增强断奶羔羊小肠黏膜免疫功能,提高其免疫力。     

Changes in intestinal villi, cell area and intracellular autophagic vacuoles related to intestinal function in chickens

1. Morphological changes in the intestinal villi, cell area and cell mitosis number in the duodenal epithelial cells were compared in cockerels fasted for 1, 2 and 3 d, and also when refed for 1 and 2 d after 3 d of fasting, to demonstrate whether these morphological changes are related to intestinal function. Alterations in the fine structure of vacuoles in epithelial cells were also examined in each group to investigate whether the vacuolar changes are associated with these morphological changes, and to obtain an index for judging the nutritional condition of the chicken intestine. 2. Fasting induced decreases in villus height, cell area and cell mitosis number, which recovered rapidly after refeeding, suggesting that these parameters are related to changes in intestinal function and may be useful for assessing intestinal function. 3. At 1 d of fasting, small electron-dense bodies appeared in the absorptive epithelial cells, some of them fusing with each other. As the fasting period increased, these small bodies developed to moderate-sized nascent autophagic vacuoles containing various kinds of electron-dense contents and finally became large autophagic vacuoles with electron-lucent contents. Some vacuoles showed positive acid phosphatase reactions, which indicated that they were lysosomal autophagic vacuoles containing hydrolytic enzymes. 4. After 1 d of refeeding the large autophagic vacuoles seen after 3 d fasting rapidly decreased to the small electron-dense bodies seen after 1 d of fasting. 5. These findings suggest that intestinal epithelial cells have the ability to digest their own cell components to supply nutrients during fasting by means of lysosomal active autophagic transport mechanisms: after refeeding, the epithelial cells return to the absorption of nutrients. 6.The present results demonstrate that the autophagic vacuolar changes are correlated with changes in intestinal villus height, cell area and cell mitosis number induced by fasting and refeeding; this indicates that autophagic vacuoles are a useful index of the nutritional condition of chicken intestine. The greater the number of electron-lucent vacuoles there are in the duodenal absorptive cells, the lower the nutritional condition of the chicken intestine.     

Fatty acids,inflammation and intestinal health in pigs

The intestine is not only critical for nutrient digestion and absorption, but also is the largest immune organ in the body.However, in pig production, inflammation induced by numerous factors, such as pathogen infection and stresses(e.g.,weaning), results in intestinal mucosal injury and dysfunction, and consequently results in poor growth of pigs. Dietary fatty acids not only play critical roles in energy homeostasis and cel ular membrane composition, but also exert potent effects on intestinal development, immune function, and inflammatory response. Recent studies support potential therapeutic roles for specific fatty acids(short chain and medium chain fatty acids and long chain polyunsaturated fatty acids) in intestinal inflammation of pigs. Results of these new lines of work indicate trophic and cytoprotective effects of fatty acids on intestinal integrity in pigs. In this article, we review the effect of inflammation on intestinal structure and function, and the role of specific fatty acids on intestinal health of pigs, especial y under inflammatory conditions.     

Intestinal microbiota and its interaction to intestinal health in nursery pigs

The intestinal microbiota has gained increased attention from researchers within the swine industry due to its role in promoting intestinal maturation,immune system modulation,and consequently the enhancement of the health and growth performance of the host.This review aimed to provide updated scientific information on the interaction among intestinal microbiota,dietary components,and intestinal health of pigs.The small intestine is a key site to evaluate the interaction of the microbiota,diet,and host because it is the main site for digestion and absorption of nutrients and plays an important role within the immune system.The diet and its associated components such as feed additives are the main factors affecting the microbial composition and is central in stimulating a beneficial population of microbiota.The microbiotaehost interaction modulates the immune system,and,concurrently,the immune system helps to modulate the microbiota composition.The direct interaction between the microbiota and the host is an indication that the mucosa-associated microbiota can be more effective in evaluating its effect on health parameters.It was demonstrated that the mucosa-associated microbiota should be evaluated when analyzing the interaction among diets,microbiota,and health.In addition,supplementation of feed additives aimed to promote the intestinal health of pigs should consider their roles in the modulation of mucosa-associated microbiota as biomarkers to predict the response of growth performance to dietary interventions.