小肠潘氏细胞防御素的研究进展

小肠潘氏细胞防御素是存在于哺乳动物小肠肠腺底部的潘氏细胞颗粒内的一类抗微生物肽。本文概述了小肠潘氏细胞防御素的一般特征、基因结构和抗微生物活性的国内外研究进展。     

小肠潘氏细胞的功能特性及与畜禽肠道疾病的关系研究

潘氏细胞位于小肠隐窝（又名小肠腺）基底部，是高度特化的分泌性上皮细胞，能够分泌α-防御素、溶菌酶、分泌性磷脂酶A2等抗菌物质。潘氏细胞在维持肠道稳态和调节小肠微生物群等方面具有重要作用。本文综述了潘氏细胞的发生与特性、α-防御素的重要作用，潘氏细胞与畜禽肠道炎症及功能障碍的关系。     

小肠潘氏细胞防御素在肠道免疫中作用的研究进展

防御素(defensins)是一类低分子质量抗菌肽,广泛地分布于动物和植物界。小肠潘氏细胞防御素是存在于哺乳动物小肠肠腺底部的潘氏细胞颗粒内的一类抗菌肽,是肠道天然屏障的重要组成部分。作者综述了小肠潘氏细胞防御素的分类、分布、分子特征和抗微生物活性及其在肠道免疫中的作用。     

肠道微生态改变对小鼠小肠潘氏细胞数的影响

为了观察小鼠小肠微生态的改变对其小肠潘氏细胞(Paneth cell,PC)的影响,试验利用饲喂抗生素、益生菌和肠道致病性大肠杆菌等改变肠道微生物菌群环境,通过苏木精-伊红(H.E.)染色、显微镜观察计数等方法进行研究。结果表明:连续饲喂抗生素导致小鼠小肠内的潘氏细胞数明显减少;致病性大肠杆菌导致小肠内的潘氏细胞数减少,而且随着感染天数的增加,潘氏细胞数逐渐降低;连续饲喂乳酸菌后,小鼠小肠潘氏细胞数呈先减少后增加的趋势。说明肠道微生态的改变对小鼠小肠潘氏细胞数有影响,且致病菌与益生菌具有相反的作用。     

便秘马肠道潘氏细胞结构改变与消长状况的研究

对12例无胃肠道疾患对照马和22例便秘马的肠道潘氏细胞进行了较详细的光镜和电镜观察。结果表明:①对照马肠道潘氏细胞位于小肠腺的潘氏细胞、干细胞部,占有小肠腺基底部的大部分面积,每个小肠腺所含的潘氏细胞数可多达数十个,细胞的内质网与高尔基复合体均较发达和粗大,具有一般分泌腺细胞的结构特征;潘氏细胞的出现率以十二指肠最少(66％),空肠次之(82％),回肠最多(98％)。根据潘氏细胞形态与结构的不同,可区分为成熟型、未成熟型和退化型三种,其中以成熟型最多(56％),未成熟型次之(36％),退化型最少(8％),大体上显示了马体生理状态下潘氏细胞分泌代谢活动的动态平衡。②便秘马肠道成熟型潘氏细胞的嗜酸颗粒减少—消失或减少—粘集,细胞体呈空壳状或葫芦状,主要细胞器发生退行性改变;未成熟型的嗜酸颗粒也减少,分化停滞,表明其分泌机能处于衰竭或停止状态。便秘马较对照马成熟型潘氏细胞数极显著地减少(P<0.01),退化型潘氏细胞数极显著地增多(P<0.01)。未成熟型潘氏细胞数增多不显著(P>0.05),但其分化停滞。便秘马肠道各型潘氏细胞形态结构与消长状况的明显改变,可作为病理组织学上判定肠道菌群失调症的参考指标。     

小肠氨基酸的代谢:动物营养的新视角

<正>(上接22期)6肠道氨基酸代谢的重要性近年来的研究发现,肠道氨基酸代谢具有重要的生理和营养功能。这些功能包括几个方面:①给小肠黏膜细胞提供能量;②为小肠黏膜细胞和肠腔内微生物合成蛋白质、氨基酸、谷胱甘肽、多胺、嘌呤和嘧啶核苷供给氮源和碳骨架;③维持小肠黏膜细胞的数量及其吸收、防御、细菌屏障和免疫功能;④调节肠腔内微生物种群的多样性和活性。     

海狸鼠消化管显微及亚显微结构

应用光镜、电镜和组织化学技术,对 １０ 只成年海狸鼠消化管的食管、胃底、小肠和大肠壁的显微和亚显微结构进行了研究。结果显示,在 Ｈ Ｅ 染色,海狸鼠食管粘膜上皮细胞角化程度较高,胃底腺壁细胞的数量几乎与主细胞的相等或稍多,小肠肠腺底部含有大量潘氏细胞; Ｓｃｈｉｆｆ 氏染色,胃粘膜的上皮细胞、胃底腺的颈粘液细胞、十二指肠腺的粘液细胞、肠腺的潘氏细胞和杯状细胞均为阳性; Ｍ ａｓｓｏｎｆｏｎｔａｎａ 氨银法染色,在胃底腺和肠腺内均见有银亲合细胞。扫描电镜下,胃粘膜表面见有较多胃小凹,小肠上皮细胞的微绒毛形成许多指状突起。透射电镜观察,胃底部壁细胞中线粒体、滑面内质网、细胞内小管发达,小肠上皮细胞的微绒毛密集排列,细胞间见有连接复合体结构,胃底腺和肠腺内见有散在分布的内分泌细胞。     

禽类防御素研究进展

生物有机体合成的内源性抗微生物多肽具有广谱、高效的抗微生物活性,是其天然免疫的重要组成成份。近二十年来已发现120多种抗微生物多肽。防御素(defensins)是其中的一个大家族。防御素是一类富含精氨酸的抗微生物肽,其分子内含有由6个保守半胱氨酸残基形成的三对二硫键,根据其分子内二硫键的连接位置不同可分为α-防御素和β-防御素。     

重组防御素的开发应用

<正>抗微生物多肽是一种古老而广泛存在的天然防御成分。在无脊椎动物中,由于没有完善的适应性免疫系统,抗微生物多肽可能是最主要的防御成分。     

防御素研究进展

防御素是广泛分布于动物和植物界的一类富含半胱氨酸的阳离子内源性抗微生物肽,是内源性抗微生物肽中的一个大家族。根据防御素分子内半胱氨酸的位置和连接方式、前体性质及表达位置的差异,可分为α-防御素、β-防御素、θ-防御素、昆虫防御素和植物防御素5种类型。防御素是由29个~54个氨基酸残基组成的小分子肽,具有广泛的生物学活性。防御素分子可以直接作用并杀死细菌、真菌和病毒等病原微生物,除此之外,防御素还具有细胞毒、免疫调节以及创伤和神经损伤的修复等多种生物学活性。文章概述了防御素的分子结构特征、分布、生物学活性及国内外研究概况。     

Fine Structural and Histochemical Study of Equine Paneth Cells

Ultrastructure. lysozyme and glycoconjugate activity in duodenal Paneth cells were observed concurrently in the horse. Paneth cells were seen to uniformly line the base of the equine intestinal glands. The round secretory granules have centrally located electron densities with peripherally located electron lucent halos. Histochemically, the peripheral halo layer was positively stained for carbohydrates by the periodic acid-thiocarbohydrazide-silver protein-physical development (PA-TCH-SP-PD) method and the entire granules reacted positively to the WGA. The central core area reacted with anti-lysozyme. We identified a young (Type I) and an old (Type II) cell population in the same crypt, but we suggest that the observed populations are variations of the same cell type with the varied appearance due to aging of the secretory granules.     

Methionine deficiency and its hydroxy analogue influence chicken intestinal 3-dimensional organoid development

Methionine and its hydroxy analogue(MHA)have been shown to benefit mouse intestinal regeneration.The intestinal organoid is a good model that directly reflects the impact of certain nutrients or chemicals on intestinal development.Here,we aimed to establish a chicken intestinal organoid culture method first and then use the model to explore the influence of methionine deficiency and MHA on intestinal organoid development.The results showed that 125-mm cell strainer exhibited the highest efficiency for chicken embryo crypt harvesting.We found that transforming growth factor-b inhibitor(A8301)supplementation promoted enterocyte differentiation at the expense of the proliferation of intestinal stem cells(ISC).The mitogen-activated protein kinase p38 inhibitor(SB202190)promoted intestinal organoid formation and enterocyte differentiation but suppressed the differentiation of enteroendocrine cells,goblet cells and Paneth cells.However,the suppression of enteroendocrine cell and Paneth cell differentiation by SB202190 was alleviated at the presence of A8301.The glycogen synthase kinase 3 inhibitor(CHIR99021),valproic acid(VPA)alone and their combination promoted chicken intestinal organoid formation and enterocyte differentiation at the expense of the expression of Paneth cells and goblet cells.Chicken serum significantly improved organoid formation,especially in the presence of A8301,SB202190,CHIR99021,and VPA,but inhibited the differentiation of Paneth cells and enteroendocrine cells.Chicken serum at a concentration of 0.25%meets the requirement of chicken intestinal organoid development,and the beneficial effect of chicken serum on chicken intestinal organoid culture could not be replaced by fetal bovine serum and insulin-like growth factor-1.Moreover,commercial mouse organoid culture medium supplemented with A8301,SB202190,CHIR99021,VPA,and chicken serum promotes chicken organoid budding.Based on the chicken intestinal organoid model,we found that methionine deficiency mimicked by cycloleucine suppressed organoid formation and organoid size,and this effect was reinforced with increased cycloleucine concentrations.Methionine hydroxy analogue promoted regeneration of ISC but decreased cell differentiation compared with the results obtained with L-methionine.In conclusion,our results provide a potentially excellent guideline for chicken intestinal organoid culture and insights into methionine function in crypt development.     

营养、大肠杆菌和氨基酸对猪小肠上皮细胞抗菌肽和信号通路蛋白表达的影响

为了研究应激和氨基酸对上皮抗菌肽表达的作用和分子机制,试验选用猪小肠上皮细胞系IPEC-J2作为研究对象,饥饿和大肠杆菌感染分别作为营养应激和细菌应激,丙氨酸和异亮氨酸作为氨基酸处理,收集细胞mRNA,利用荧光定量PCR测定β防御素和相关信号通路蛋白表达水平。结果表明:与对照组(DMEM/F12培养基)相比,饥饿显著降低了小肠上皮细胞猪防御素2(p BD-2)、猪防御素3(p BD-3)和猪防御素EP2c(p EP2c)及沉默信息调节因子2同源蛋白1(Sirt1)、叉头转录因子1(Fox O1)和叉头转录因子4(Fox O4)的表达(P0.05),大肠杆菌对β防御素表达无显著影响(P0.05)。与对照组(饥饿培养基)相比,丙氨酸处理未显著影响p BD-2和p BD-3的表达(P0.05),但显著提高了p EP2c表达水平(P0.05);异亮氨酸处理使p BD-2、p BD-3和p EP2c表达水平显著升高(P0.05)。与对照组相比,丙氨酸和异亮氨酸处理不同程度影响信号通路蛋白转录,丙氨酸处理显著提高了Sirt1和Fox O4的表达水平(P0.05),异亮氨酸处理显著促进了Sirt1、Fox O1和Fox O4的表达(P0.05)。由此得出,营养应激降低猪小肠上皮细胞中β防御素的表达,而氨基酸的补充可促进其表达,该调控作用可能与Sirt1和叉头转录因子(Fox O)信号通路蛋白有关。     

家畜防御素体内组织表达及体外表达研究进展

防御素是广泛分布于动物、植物、昆虫体内的一类富含半胱氨酸的阳离子内源性抗菌肽,在动物免疫调节及植物自身防御方面扮演着重要的角色。对防御素的概念、类型、结构特征、作用机理进行了综述,总结了防御素在牛、马、羊、猪、鹿、骆驼等家畜体内组织表达及体外表达研究进展,并对防御素在未来生物制药和畜牧饲料生产方面的应用前景进行了展望。     

Immunohistochemical and histoplanimetrical study on the spatial relationship between the settlement of indigenous bacteria and the secretion of bactericidal peptides in rat alimentary tract

To clarify the regulatory mechanism by bactericidal peptides secretion, the secretion of bactericidal peptides was immunohistochemically and histoplanimetrically compared with the degree of Gram-positive/negative bacterial colonization throughout the rat alimentary tract. In the associated exocrine glands from the oral cavity to the stomach, no comparable differences were observed under the changes of development of indigenous bacterial colonies. In the small intestine, immunopositive granules for lysozyme and secretory phospholipase A2 (sPLA2) were markedly decreased, whereas immunopositive vacuoles in the Paneth cells were more increased at sites with hyper-development of indigenous bacterial colonies in the intervillous spaces than at sites with no or less development. No changes in exocrine glands were observed in the large intestine because of the constant existence of large quantities of bacteria. Gram-positive bacterial colonies on the mucosal surfaces were dominant from the oral cavity to the stomach. Gram-negative bacteria were dominant in the large intestine, and the distributions of both Gram-positive and negative bacteria were intermediate in the small intestine. These findings suggest that lysozyme and sPLA2 secreted from the Paneth cells contribute to the regulation of the proliferation of indigenous bacteria in the intervillous spaces of the small intestine, and that the inversion of distributions of Gram-positive and -negative bacteria in the alimentary tract might be caused by the secretion of lysozyme and sPLA2 in the small intestine.     

The polymorphism in the β4-defensin gene and its association with production and somatic cell count in Holstein-Friesian cows

The aim of this study was to find a polymorphism of the bovine β4‐defensin gene and search for its association with milk yield and composition and with the somatic cell count in milk. The data were from the years 1999 to 2004 on 212 Holstein‐Friesian (HF) dairy cows, descended from 70 sires. Based on the sequence of the bovine β4‐defensin gene (GenBank no. AF008307 ) the primers were designed for the amplification of the 924‐bp or 393‐bp long fragments. The 924‐bp long fragment was sequenced and the sequence was compared with that available in the GenBank. Ten putative nucleotide sequence polymorphisms were found in the intron of the bovine β4‐defensin gene. One of them, a C→T transition at position 2239, that creates a new NlaIII (Hin1II) restriction site, was genotyped with polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) in a cohort of 212 HF cows. The CC genotype was the most common (72%). The heterozygous CT genotype was found in 26% of the genotyped cows and four cows (2%) were TT homozygotes. In order to determine the relationship between the polymorphism of the β4‐defensin gene and milk production traits a multi‐trait repeatability test‐day animal model was used. The Derivative‐free Multivariate analysis program was used for computation. The differences between estimates for genotypes were checked using Student's t‐test. The model included the animal genotype, year‐season of calving and parity as fixed effects and the animal additive genetic effect and permanent environmental effect of individual cows as well as dates of the tests as random effects. Significant associations were found between the RFLP‐NlaIII and milk fat, protein and lactose contents. Also, a significant effect was shown of the defensin genotype on the somatic cell count in the milk.     

Characterization of metastatic intestinal adenocarcinoma with differentiation into multiple morphologic cell types in a Virginia opossum.

A captively maintained mature male opossum (Didelphis virginiana) utilized in a research protocol was presented with clinical signs of chronic diarrhea and severe muscle wasting. At necropsy, there was multifocal mural gastric, intestinal, and urinary bladder thickening, concurrent bilateral hydroureter and hydronephrosis, and extensive fibrous abdominal adhesions. Histologic evaluation revealed intestinal adenocarcinoma with coelomic metastasis to the stomach and urinary bladder. The adenocarcinoma was evaluated using histochemistry and electron microscopy. Paneth, enteroendocrine, and goblet cell differentiation was documented in primary and metastatic sites. This unique presentation of intestinal adenocarcinoma has not previously been reported in the opossum or any other animals. Intestinal neoplasia with Paneth cell differentiation is extremely rare and has been reported in humans with familial adenomatous polyposis.     

A cell wall preparation of Enterococcus faecalis strain EC-12 stimulates β-defensin expression in newly hatched broiler chicks

In our previous study, a cell wall preparation of Enterococcus faecalis strain EC‐12 (EC‐12) prevented the colonization of vancomycin‐resistant enterococci (VRE) in newly hatched broiler chicks. Relatively early prevention against VRE colonization by EC‐12 administration may be related to the enhanced innate immune system. In this study we examined the effect of EC‐12 on the kinetics of immunoglobulin A (IgA) concentration in the cecal digesta of chicks from 1 day to 14 days old to evaluate humoral immunity. We also examined the effect of EC‐12 on β‐defensin mRNA expression and lysozyme production. The IgA concentration in the cecal digesta showed a decline in chicks from 2 to 8 days old and then a recovery up to when they were 14 days old. The concentration tended to be higher in EC‐12‐administered chicks than in control chicks at 14 days old. EC‐12 administration did not stimulate the lysozyme activities in the alimentary tract. However, β‐defensin mRNA expression in the tongue and the bursa of Fabricius was higher in chicks administered EC‐12 than in the control chicks at 5 days old. These results indicate that β‐defensin may be one of the major defense mechanisms inhibiting VRE colonization in young chicks.     

猪防御素基因pBD-2在大肠杆菌中的重组和融合表达

哺乳动物防御素是动物机体在抵御病原微生物的防御反应中产生的一类重要的抗菌肽物质,具有十分广泛的抗菌谱,在先天免疫上起重要作用。本研究根据已报道的pBD-2基因cDNA序列,合成了3条基因片段(1、2、3)。片段1、2和片段2、3各有15个碱基互补配对,PCR扩增延伸得到pBD-2基因,将pBD-2基因克隆至原核表达载体pGEX-KG,成功地在大肠杆菌BL21(DE3)中表达出pBD-2融合蛋白。pBD-2基因的成功表达为进一步研究其抗菌活性打下了基础。