黄姑鱼肠道上皮细胞体外分离培养及鉴定

本研究旨在建立黄姑鱼(Nibea albiflora)肠道上皮细胞的体外分离培养及鉴定方法,为海水鱼类肠道功能及其发病机制相关研究提供细胞模型.以黄姑鱼肠道组织为研究对象,采用胰蛋白酶(0.25％)、胶原蛋白酶(1 mg/mL)和透明质酸酶(0.16 mg/mL)联合消化法对肠道组织进行不同时间(0、20、40、60和...     

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.     

紫锥菊多糖对LPS损伤后IEC-6细胞分泌IL-1ɑmRNA的影响简

为了研究紫锥菊多糖对LPS损伤后IEC一6细胞分泌IL-1αmRNA的影响，以体外培养大鼠小肠上皮细胞为模型，进行细胞分组及收集，并给予LPS刺激造成内毒素损伤，根据GenBank中发布的IL-1α基因序列设计引物，以基因组R-NA为模板进行RT-PCR扩增，发现紫锥菊多糖对LPS刺激小肠上皮细胞产生的IL-1αmRNA的表达减少。     

Immunohistochemical study on the delayed progression of epithelial apoptosis in follicle-associated epithelium of rat Peyer's patch

It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.     

精氨酸对热应激处理的奶牛原代小肠上皮细胞增殖和凋亡的影响

本试验旨在研究精氨酸(L-Arginine,L-Arg)对热应激奶牛原代小肠上皮细胞增殖和凋亡的影响,探究精氨酸对热应激奶牛小肠上皮细胞损伤的修复作用.首先以体外培养的奶牛原代小肠上皮细胞为模型,分为对照组(Con组,37℃)、试验组[42℃培养6h后,更换成无精氨酸培养基(HS组)或者不同浓度(2、4、6、8、10 ...     

鸡小肠上皮细胞的分离培养与鉴定

选择组织块法分离培养鸡小肠上皮细胞(intestinal epithelial cells,IEC),采用机械刮除法和相差消化－相差贴壁法纯化细胞,0.05%的Trypsin-EDTA对获得的IEC进行消化传代,免疫细胞化学法鉴定IEC。结果表明,组织块培养法可分离出活性较强的IEC,并获得纯化的上皮细胞;形态学和免疫细胞化学法检测显示获得的细胞表面抗原呈阳性,鉴定为IEC;纯化的IEC可在体外稳定传代。     

Effect of dipeptide on intestinal peptide transporter 1 gene expression: An evaluation using primary cultured chicken intestinal epithelial cells

Peptide transporter 1 (PepT1) is a transporter responsible for absorbing dipeptide and tripeptide in enterocytes and is upregulated by dipeptide in mammals. It has not been certain whether intestinal PepT1 expression is responsive to dipeptides in chickens because of the lack of in vitro study using the cultured enterocytes. This study established a primary culture model of chicken intestinal epithelial cells (IECs) in two-dimensional monolayer culture using collagen gel by which the response of chicken PepT1 gene expression to dipeptide stimuli was evaluated. The cultured chicken IECs showed the epithelial-like morphology attached in a patch-manner and exhibited positive expression of cytokeratin and epithelial cadherin, specific marker proteins of epithelial cells. Moreover, the chicken IECs exhibited the gene expression of intestinal cell type-specific marker, villin1, mucin 2, and chromogranin A, suggesting that the cultured IECs were composed of enterocytes as well as goblet and enteroendocrine cells. PepT1 gene expression was significantly upregulated by synthetic dipeptide, glycyl-l-glutamine, in the cultured IECs. From the results, we herein suggested that dipeptide is a factor upregulating PepT1 gene expression in chicken IECs.     

Culture of Piglet Intestinal Epithelial Cells in vitro and the Infectivity of Porcine Epidemic Diarrhea Virus on the Cells

The study was aimed to establish a simple in vitro culture system for piglet small intestinal epithelial cells,and to provide materials for researches on porcine epidemic diarrhea virus (PEDV).In this study,newborn piglets that did not eat colostrum were used as the initial donors,and the primary cells were separated in vitro by scraping the intestinal mucosa from the intestinal lumen and mechanical separation and dispersion.The piglet intestinal epithelial cells were purified using 0.1% trypsin differential digestion method.Compared the effects of newborn weak piglets and normal piglets as donors on the activity of primary intestinal epithelial cells.MTT method was used to compare the proliferation activity of primary cells of different generations.Immunofluorescence and Real-time RT-PCR were used to detect the infection and proliferation of PEDV strain CV777 in primary intestinal epithelial cells.The results showed that the isolation and culture method in vitro used in this study could obtain primary intestinal epithelial cells with good proliferation activity,with obvious S-type cell proliferation curves.The cells with high purity and single morphology could be obtained by differential digestion,and had good proliferation activity after five consecutive passages.The proliferative activity of intestinal epithelial cells isolated from weak piglets and normal piglets had no obvious difference,and provided new way for reducing the cost of primary cell culture.The results of immunofluorescence and Real-time RT-PCR showed that PEDV could infect the primary intestinal epithelial cells,and replicated and proliferated in them.In this study,we established a simple,practical and low-cost method for in vitro culture of piglet primary small intestinal epithelial cells.The primary cells cultured by this method could be used as basic materials for the isolation and culture of PEDV and related research.     

Cellular kinetics of villous epithelial cells and m cells in rabbit small intestine

The cellular kinetics of villous columnar epithelial cells and M cells in the rabbit small intestine were determined by the use of 5-bromo-2'-deoxyuridine (BrdU) as a tracer. To identify M cells, vimentin antibody was used. The BrdU-labeled nuclei of columnar epithelial cells reached the base of intestinal villi in all portions at 1 day after BrdU administration. Thereafter, BrdU-labeled cells migrated toward the villous tip, but they did not move at a uniform speed. The epithelial cells which existed in intestinal villi on circular folds moved faster than those on mucosa other than circular folds. At 7 days after BrdU administration, the leading edge of BrdU-labeled epithelial cells already disappeared from the villous tip in all portions of the small intestine. In the ileal Peyer's patch, the BrdU-labeled nuclei of microvillous epithelial cells and vimentin-positive M cells appeared near the intestinal crypt orifice at 1 day after BrdU administration, and then migrated toward the luminal surface of the follicle-associated epithelium (FAE). As they moved toward the upper portion of FAE, the number of BrdU-labeled M cells on the side of the dome decreased simultaneously. The leading edge of BrdU-labeled epithelial cells disappeared from the top of the FAE within 7 days. These results suggest that M cells may differentiate from the undifferentiated cells in intestinal crypts within 1 day and disappear from the top of the FAE after the change of their form from M cells into microvillous epithelial cells.     

仔猪小肠上皮细胞的体外培养及猪流行性腹泻病毒对其感染性研究

本研究旨在建立一种操作简单的仔猪小肠上皮细胞体外培养体系，为猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）的相关研究提供材料。研究以未吃初乳的新生仔猪作为肠道供体，采用肠腔面刮取肠黏膜和机械分离分散的方式进行原代细胞的体外分离。采用0.1%胰蛋白酶差速消化法进行仔猪小肠上皮细胞的纯化；比较新生弱仔猪和正常仔猪作为供体对原代小肠上皮细胞活性的影响；MTT法比较不同代次原代细胞的增殖活性；免疫荧光和实时荧光定量RT-PCR方法检测PEDV毒株CV777在原代小肠上皮细胞感染和增殖情况。结果显示，本研究建立的体外分离培养方法能获得增殖活性良好的原代小肠上皮细胞，具有明显的"S"型细胞增殖曲线。通过胰酶差速消化可得到纯度高、形态单一的小肠上皮细胞，同时细胞连续传代5次仍保持良好的增殖活性。弱仔猪和正常仔猪分离培养的小肠上皮细胞的增殖活性比较显示，两者并没有明显区别，这为降低原代细胞培养的成本提供新的思路。免疫荧光和实时荧光定量RT-PCR结果显示，PEDV可感染本方法分离培养的仔猪原代小肠上皮细胞，并在其中进行复制增殖。本研究建立了一种操作简单、实用性强、成本较低的仔猪原代小肠上皮细胞体外培养方法，该方法培养的原代细胞可作为PEDV分离培养和相关研究的基础材料。     

动物消化道乳酸菌粘附机制及影响因素研究进展

消化道益生乳酸菌群对动物健康起着非常重要的作用。乳酸菌通过粘附作用与宿主消化道上皮细胞紧密结合,形成生理性菌膜。乳酸菌黏附上皮细胞的机理有多种学说,主要包括乳酸菌粘附的特异性与非特异性、乳酸菌粘附的表面分子与肠道粘膜上皮细胞粘附受体学说等。影响乳酸菌与肠道上皮细胞粘附和定植的因素主要包括PH值、Ca2 浓度和温度。     

槲皮素对猪肠上皮细胞利用蛋白质的作用机制

本试验旨在研究槲皮素促进猪肠上皮细胞利用蛋白质的作用及机制。猪肠上皮细胞孵育48 h后试验组分别用含0.1、0.2、0.4、0.8和1.6 mg/L槲皮素的二甲基亚砜(DMSO)溶液处理72 h,对照组采用0.2%DMSO处理。采用二喹啉甲酸(BCA)测定受试细胞中蛋白质的含量;采用实时荧光定量PCR(RT-qPCR)法测定氨基酸和小肽转运载体以及哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关基因的mRNA相对表达量;采用Western blot法测定mTOR信号通路相关基因的蛋白表达。结果表明:与对照组相比,1)0.4和0.8 mg/L槲皮素均极显著增加猪肠上皮细胞中蛋白质的含量(P<0.01)。2)1.6 mg/L槲皮素极显著提高猪肠上皮细胞中兴奋性氨基酸转运载体1(EAAC1)、谷氨酰胺载体2(ASCT2)、氨基酸转运载体A2(ATA2)、L型氨基酸转运载体2(LAT2)、阳离子氨基酸转运载体1(CAT1)、b 0,+系统氨基酸转运载体(rBAT)、y+L系统氨基酸转运载体1(y+LAT1)、y+L系统氨基酸转运载体2(y+LAT2)和寡肽转运载体1(PepT1)mRNA相对表达量(P<0.01)。3)0.4 mg/L槲皮素极显著降低猪肠上皮细胞中结节性硬化复合物1(TSC1)mRNA相对表达量(P<0.01);0.8 mg/L槲皮素极显著增加mTOR和核糖体蛋白S6(RPS6)mRNA相对表达量并极显著降低TSC1 mRNA相对表达量(P<0.01);1.6 mg/L槲皮素极显著增加mTOR、真核起始因子4E结合蛋白1(4E-BP1)、真核细胞翻译起始因子4E(eIF4E)、真核细胞翻译起始因子4B(eIF4B)、真核细胞翻译起始因子4A(eIF4A)和RPS6 mRNA相对表达量(P<0.01)。4)0.1和1.6 mg/L槲皮素极显著提高猪肠上皮细胞中mTOR、eIF4E和eIF4A蛋白表达量并极显著降低4E-BP1蛋白表达量(P<0.01)。由此可见,槲皮素可通过调控氨基酸转运载体、小肽转运载体及mTOR信号通路相关基因的表达来促进猪肠上皮细胞对蛋白质的利用。     

Special sugar expression on apoptotic epithelial cells of Peyer's patches and intestinal villi in rat small intestine

Our previous study clarified that the apical regions of both the follicle-associated epithelium (FAE) of Peyer's patches and the intestinal villi are the only adhesion sites of indigenous bacteria in rat jejuno-ileum. To survey the ligands against bacterial lectins, sugar expression patterns on epithelial cells were lectin-histochemically investigated using 21 lectins in the jejuno-ileal Peyer's patches of rats. As a result, (D-glcNAc)(2-4), detected by Solanum tuberosum (STL) and by Lycopersicon esculentum (LEL), and beta-D-gal(1-3)-D-galNAc detected by Peanut agglutinin (PNA), were strongly expressed on the brush borders of the apical regions of the FAE and the intestinal villi. On the other hand, neither sugar was expressed on the brush borders of the basal regions of both FAE and intestinal villi. The positive intensities for the lectins correlated with the progression of epithelial apoptosis in the FAE and in the intestinal villi. Moreover, the double staining with lectin histochemical method and the in situ nick end-labeling method could simultaneously detect the strong expression of both sugars and nuclear DNA fragmentation in epithelial cells at the late apoptotic stage. Other sugar expression patterns in the intestinal villi were similar with those in the FAE. There were no lectins specific for M cells in the FAE. From these findings, the possible sugars of ligands against some indigenous bacterial lectins, expressing specially on the apoptotic epithelial cells, might be narrowed down in rat jejuno-ileum.     

Effects of dietary butyrate supplementation on intestinal integrity of heat‐stressed cockerels

Butyrate modulates intestinal epithelial cell structure and function. Three hundred and sixty Lohmann LSL‐Classic layer cockerels were used to investigate the effect of butyrate on heat stress‐induced intestinal injury and intestinal integrity. The experiment was conducted from day 85 to 105 of age. The birds were divided randomly into three treatments: control, heat stress (HS), and heat stress provided with butyrate (HSB) at a level of 0.35 g/kg of diet. The control was reared at 21 ± 1 °C throughout the experiment. The HS and HSB treatments were exposed to a cyclic HS (35 ± 1 °C from 09:00 to 13:00 and 21 ± 1 °C from 13:00 to 09:00). Intestinal and mucosal weights, villus height, villus surface area (VSA), absorptive epithelial cell area and intestinal beneficial bacteria were lower in the HS treatment than in the other two treatments (p < 0.05). Heat‐stressed cockerels exhibited the highest (p < 0.05) villi injury scores and serum endotoxin levels compared with the other treatments. Dietary inclusion of butyrate increased (p < 0.05) intestinal and mucosal weights, villus height, VSA, absorptive epithelial cell area and intestinal beneficial bacteria counts and reduced (p < 0.05) HS‐induced injury in intestinal epithelia as well as intestinal permeability to endotoxin. In conclusion, dietary butyrate exerted protective effects against intestinal damage induced by HS and improved intestinal health and integrity.     

Pathology of infectious diarrhea of infant rats (IDIR) induced by an antigenically distinct rotavirus

Suckling rats were inoculated with a group B rotavirus to determine the progression of the morphologic changes induced in the intestine by this virus. Several changes were observed by light microscopy 1 day after viral inoculation: shortening of small intestinal villi, villous epithelial necrosis, and villous epithelial syncytia. The lesions were most often present in the distal small intestine, although other small intestinal segments were affected to a lesser degree. By day 3 post-inoculation, epithelial necrosis, and syncytia were no longer present; however, the villous epithelium was disorganized and irregularly vacuolated, and intestinal crypt epithelium was hyperplastic. Alterations in villous height to crypt depth ratios were present in portions of the small intestine for the remainder of the 12-day study period. Epithelial syncytia appeared to form by the breakdown of the lateral interdigitating membranes of the absorptive villous epithelium. Viral particles, abundant in the syncytia, appeared to form from amorphous or reticular arrays of viral precursor material. Group B rotaviral antigens, as detected by indirect immunofluorescence, were present in large amounts in the small intestinal villous epithelium only on the first day after viral inoculation. These studies show that two important diagnostic features of group B rotaviral infections of rats, epithelial syncytia and viral antigen as determined by immunofluorescence, are present only on the first day of disease. These findings should be taken into consideration when attempting to diagnose disease induced by this agent.     

益生菌大肠杆菌Nissle 1917抗逆性能、猪肠上皮细胞黏附率及抑菌效果研究

本试验旨在研究益生菌大肠杆菌Nissle 1917(Ec N)抗逆性能、猪肠上皮细胞黏附率及抑菌效果。采用体外法对Ec N进行生长曲线绘制和耐酸、耐胆盐、耐热性能的测定;以猪肠上皮细胞IPEC-J2细胞为体外细胞模型,考察了Ec N对该细胞的黏附率以及对致病菌大肠杆菌K88的黏附抑制率;同时通过蛋白质印迹法检测了Ec N对IPEC-J2细胞β-防御素-2和Toll样受体4的水平的影响。结果表明:1)Ec N对高酸、高胆盐和高温环境具有一定耐受能力。2)Ec N对IPEC-J2细胞的黏附作用以对数期最佳,黏附率达33.96%,显著高于迟缓期、稳定期和衰亡期(P0.05)。3)Ec N对致病菌大肠杆菌K88具有良好的抑制效果,黏附抑制率达87.84%。4)Ec N还能上调IPEC-J2细胞β-防御素-2和Toll样受体4水平。结果提示,益生菌Ec N具有较好的抗逆性能,能够良好地黏附猪肠上皮细胞,对致病菌大肠杆菌K88具有良好的抑制作用。     

哺乳动物肠上皮细胞的原代培养

肠上皮细胞是由多功能干细胞分化成的一个高度组织化系统.从离体的肠组织中分离的肠隐窝单位或肠上皮细胞可在数小时内保持高度活力,但要长期(达到10 d)原代培养肠上皮细胞仍然很困难.文章对肠上皮细胞的分离、鉴定、原代培养所需维持培养基和生长培养基的设计及培养条件等方面进行了综述.     

4-Phenylbutyric acid accelerates rehabilitation of barrier function in IPEC-J2 cell monolayer model

As the first line of defence against pathogens and endotoxins crossing the intestine-blood barrier, the intestinal epithelial barrier plays a determinant role in pigs' health and growth. 4-Phenylbutyric acid (4-PBA), an aromatic fatty acid, was reported to benefit homeostasis of endoplasmic reticulum and protein synthesis. However, whether 4-PBA affects intestinal epithelial barrier function in pigs is unknown. This study aimed to explore the effects of 4-PBA on the intestinal barrier function, using in vitro models of well-differentiated intestinal porcine epithelial cell (IPEC-J2) monolayers in the transwell plates. Cell monolayers with or without 4-PBA (1.0 mmol/L) treatment were challenged with physical scratch, deoxynivalenol (DON, 2.0 μg/mL, 48 h), and lipopolysaccharide (LPS, 5.0 μg/mL, 48 h), respectively. Transepithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran (FD-4) permeability were measured to indicate barrier integrity and permeability. Real-time PCR and Western blot were conducted to determine relative gene and protein expressions of tight junction proteins. As expected, physical scratch, DON, and LPS challenges decreased TEER and increased FD-4 permeability. 4-PBA treatment accelerated cell mitigation and rehabilitation of the physical scratch-damaged intestinal epithelial barrier but did not alleviate DON or LPS induced barrier damage. However, once 48-h DON and LPS challenges were removed, rehabilitation of the epithelial barrier function of IPEC-J2 monolayer was accelerated by the 4-PBA treatment. Also, the relative gene and protein expressions of zonula occludens-1 (ZO-1), occludin, and claudin-1 were further upregulated by the 4-PBA treatment during the barrier rehabilitation. Taken together, 4-PBA accelerated the IPEC-J2 cell monolayer barrier recovering from physical scratch, DON-, and LPS-induced damage, via enhancing cell mitigation and expressions of tight junction proteins.     

The effects of mycotoxins, fungal food contaminants, on the intestinal epithelial cell-derived innate immune response

Mycotoxins are structurally diverse fungal metabolites that can contaminate a variety of dietary components consumed by animals and humans. It is considered that 25% of the world crop production is contaminated by mycotoxins. The clinical toxicological syndromes caused by ingestion of moderate to high amounts of mycotoxins and their effect on the immune system have been well characterized. However, no particular attention has been focused on the effects of mycotoxins on the local intestinal immune response. Because of their location, intestinal epithelial cells (IECs) could be exposed to high doses of mycotoxins. As a component of the innate local immune response, intestinal epithelial cells have developed a variety of mechanisms which act to reduce the risk of infection by microorganisms or intoxication by toxic compounds. This review summarises the innate immune response developed by intestinal epithelial cells and reports the literature concerning the effects of mycotoxins on them. Particularly, the effects of mycotoxins on the maintenance of a physical barrier by epithelial cells will be discussed together with their effect on extrinsic protective components of the innate intestinal immunity: mucus secretion, antimicrobial peptide generation, IgA and pro-inflammatory cytokine release.     

Experimental infection of specific-pathogen-free chickens with avian rotaviruses

One-to-70-day-old specific-pathogen-free chickens were infected with rotaviruses isolated from chickens, turkeys, and pheasants. Chicks infected at 1 or 7 days of age remained largely free of infection: virus could be isolated from a few chicks of these ages, but virus antigen could not be detected in the epithelial cells of the intestinal villi. Treating the virus with trypsin before inoculation did not markedly enhance virus infectivity. The same batches of virus successfully infected chickens at 56 and 70 days of age: virus could be isolated between 1 and 5 days postinfection, and virus antigen was detected in the epithelial cells of the intestinal villi. Rotaviruses isolated from pheasants were infectious for 49-day-old chickens. All infections in the older chickens remained subclinical.