大豆凝集素与肉鸡小肠黏膜细胞结合规律的研究

该试验以75日龄爱拔益加肉鸡为试验对象,饲喂含有20%生大豆日粮。试验期后,取十二指肠、空肠前段、空肠中段、空肠后段、回肠制作石蜡切片,采用免疫组织化学SP法染色,利用图像分析仪分析大豆凝集素与肠上皮细胞结合情况。结果表明,在鸡的不同肠段,大豆凝集素与十二指肠和空肠前段结合的平均光密度值显著高于空肠中、空肠后和回肠（P＆lt;0.05）;在相同肠段不同部位的细胞,肠绒毛的平均光密度值最高,且与隐窝和肠壁的差异显著（P＆lt;0.05）。这表明了肉鸡的不同肠段上皮细胞糖基化模式具有差异性。     

大豆凝集素在鸡肠道内残留规律的研究

将生大豆粉碎,过40目筛,硫酸铵粗提后经N-乙酰基-D-半乳糖胺-epoxy-sepharose6B亲和层析体系纯化出大豆凝集素,以其为抗原免疫家兔制备多克隆抗体,并建立大豆凝集素间接竞争抑制ELISA定量检测方法,其检测下限低于10ng/mL。以含质量分数为20%的生大豆和1%Cr2O3(外源指示剂)的日粮饲喂鸡(75日龄)2周,检测其十二指肠、空肠前、空肠中、空肠后、回肠、盲肠、结直肠内食糜中大豆凝集素的活性残留率。结果表明:大豆凝集素在鸡肠道内活性残留由十二指肠到结直肠总地呈下降趋势,其中由十二指肠到空肠前的残留率变化达到了显著水平(P<0.05),说明大豆凝集素在鸡肠道内吸收和吸附主要发生在十二指肠至空肠前段。     

大豆凝集素对兔小肠组织的氧化应激与炎症因子的影响

为研究大豆凝集素对兔小肠组织的氧化应激以及炎症因子的影响,试验选择体质量（725.45±20.10）g、健康的35日龄断奶新西兰白兔30只,随机分为3组,每组10只,分别按0、17.5、87.5 mg/kg的剂量灌胃大豆凝集素,试验期10 d,试验结束后,采集十二指肠、空肠和回肠组织。结果显示：与0 mg/kg大豆凝集素组相比,87.5 mg/kg大豆凝集素组兔十二指肠、空肠及回肠的总抗氧化能力（T-AOC）显著降低,分别降低了31.96%、49.27%、36.35%,丙二醛（MDA）含量显著升高,分别升高了217.84%、132.46%、74.29%(P <0.05),十二指肠、空肠的超氧化物歧化酶（SOD）活性显著降低,分别降低了22.05%、45.51%(P <0.05),空肠和回肠的谷胱甘肽过氧化物酶（GSH-Px）活性显著降低,分别降低了36.73%、29.69%(P <0.05)。与0 mg/kg大豆凝集素组相比,87.5 mg/kg大豆凝集素组兔空肠的白细胞介素-6(IL-6)含量显著升高了21.12%(P <0.05),17.5 mg/kg和87...     

仔猪小肠黏膜特异性结合大豆凝集素的免疫组化研究

试验旨在研究大豆凝集素(SBA)与猪小肠不同部位黏膜特异性结合的程度。21日龄断奶仔猪日粮中添加纯化的SBA,饲喂1周后,无痛屠宰,立即取十二指肠、空肠前部、空肠中部、空肠后部和回肠组织,用10%的甲醛固定,制作常规石蜡切片,SP免疫组织化学染色,图像分析系统定量检测小肠组织中的棕色阳性产物的光密度值。结果表明:整个小肠黏膜与SBA都有特异性结合,光密度值由十二指肠到空肠中部呈下降趋势(P<0.05),但从空肠后部到回肠又出现上升趋势(P<0.05)。SBA在小肠前部(十二指肠和空肠前部)主要与绒毛的上半部分结合(P<0.05),且集中于上皮细胞表面;而在小肠后部(空场后部和回肠)较均匀地分布于黏膜上皮、固有层和中央乳糜管中。研究表明,猪小肠黏膜上皮细胞膜糖基化过程中含有N-乙酰基D-半乳糖胺或D-半乳糖,猪小肠后部对SBA的内吞作用较强。     

大豆凝集素与大鼠和小鼠小肠黏膜上皮细胞结合规律的研究

试验分别选择怀孕的6只SD大鼠和ICR小鼠,单笼饲养。仔鼠于分娩后19日龄断奶,在仔鼠第1、7、14、21、28日龄和第35日龄时分别从6窝仔鼠中每窝随机取1只,每个日龄6只,屠宰后取十二指肠、空肠和回肠制作石蜡切片,免疫组化染色后用图像分析仪检测其阳性染色物的光密度值。结果表明:大鼠和小鼠呈现相似的结合规律,大豆凝集素在十二指肠、空肠和回肠的结合没有显著差异,但不同日龄之间大豆凝集素的结合程度差异极显著(P<0.01)。在大鼠和小鼠出生时结合程度很低,然后逐渐增加,28日龄时达到最高。     

肉犊牛小肠黏膜免疫相关细胞的数量变化研究

分别选择1、4、6月龄各4头利杂犊牛的十二指肠、空肠、回肠,利用组织化学法和图像分析法研究犊牛小肠的上皮内淋巴细胞、杯状细胞和肥大细胞的数量变化.结果显示在同一月龄犊牛的小肠的上皮内淋巴细胞数量由十二指肠向空肠、回肠逐渐减少,而杯状细胞的数量逐渐增加,肥大细胞的密度逐渐降低.在1、6月龄犊牛十二指肠、空肠、回肠的上皮内淋巴细胞数量之间差异极显著(P＜0.01);4月龄十二指肠的上皮内淋巴细胞数量最多(P＜0.01).在1月龄犊牛十二指肠、空肠、回肠杯状细胞的数量差异极显著(P＜0.01);4和6月龄犊牛十二指肠和空肠之间的杯状细胞的数量差异不显著(P＞0.05),但二者与犊牛回肠杯状细胞的数量差异极显著(P＜0.01).各年龄犊牛十二指肠、空肠、回肠的肥大细胞密度差异极显著(P＜0.01),4月龄时犊牛小肠的肥大细胞密度最低.结果提示犊牛的黏膜免疫水平可能与其机体的发育相一致.     

不同种属动物消化道内大豆凝集素免疫活性残留的比较研究

比较研究大豆凝集素（SBA）在反刍动物（羊）、肉食动物（狗）、啮齿动物（兔）、杂食动物（猪）和禽类（鸡）等不同种属动物消化道不同区段食糜中的免疫活性的残留规律,并探讨SBA对不同种属动物抗营养机制的差异。各种属动物均选取半性成熟雄性8只（头）。日粮中含20%的生大豆和质量分数1%的Cr2O3（外源指示剂）,试验期为2周,最后一次进食2 h后,麻醉状态下颈动脉放血,立即取十二指肠、空肠前、空肠中、空肠后、回肠、盲肠、结肠（鸡为结直肠）各肠段内的食糜。食糜和饲料中具有免疫活性的SBA采用竞争性间接抑制ELISA方法检测,食糜和饲料中的Cr采用原子吸收光谱法检测。结果表明,同一种动物不同肠段内的SBA残留率不同,总的趋势是由十二指肠到结肠呈下降趋势,其中由十二指肠到空肠前段的残留率变化均达到显著水平（P〈0.05）,各肠段内均能检测到SBA。在不同动物的同一区段肠道内,SBA的残留率随动物的不同而不同,各肠段中SBA的残留率在兔均最低;十二指肠到空肠SBA的残留率在羊和鸡较高、在猪和狗居中;空肠之后在猪、羊和鸡较高。SBA在不同种属动物及其消化道不同部位内的免疫活性残留规律不同,SBA对不同种属动物的抗营养机制和抗营养作用可能不尽相同。     

绵羊自然感染贝氏莫尼茨绦虫对小肠黏膜免疫相关细胞的数量变化

运用组织学和组织化学染色法,分别对自然感染贝氏莫尼茨绦虫的成年绵羊（感染组）与正常成年绵羊（对照组）的小肠各肠段的上皮内淋巴细胞、浆细胞、杯状细胞和嗜酸粒细胞的分布和数量变化进行了比较研究。结果显示：感染组小肠各段上皮内淋巴细胞、浆细胞、杯状细胞和嗜酸粒细胞数量均显著高于对照组,其中感染组十二指肠、空肠和回肠的上皮内淋巴细胞数量分别比对照组增加了169.11%、230.38%和233.42%（P〈0.01）;嗜酸粒细胞数量分别比对照组增加了116.78%、123.87%和164.51%（P〈0.01）;浆细胞数量分别比对照组增加了127.34%、72.97%和328.26%（P〈0.01）;杯状细胞数量分别比对照组增加了33.40%、41.42%和133.17%。对照组和感染组上皮内淋巴细胞和杯状细胞从十二指肠到回肠均逐渐减少,相反,对照组和感染组嗜酸粒细胞数量从十二指肠到回肠均逐渐增多,对照组小肠固有层浆细胞数量从十二指肠到空肠增加,空肠到回肠减少,感染组小肠固有层浆细胞数量从十二指肠到回肠均逐渐增多;对照组十二指肠、空肠、回肠的上皮内淋巴细胞、上皮内杯状细胞、浆细胞和嗜酸粒细胞均差异极显著（P〈0.01）;感染组十二指肠、空肠、回肠的上皮内杯状细胞、浆细胞和嗜酸粒细胞均差异极显著（P〈0.01）,感染组上皮内淋巴细胞空肠与回肠差异极显著（P〈0.01）,但十二指肠与空肠差异不显著（P〉0.05）。研究结果表明,贝氏莫尼茨绦虫感染成年绵羊后,成年绵羊通过特异性黏膜免疫细胞上皮内淋巴细胞增生加强细胞免疫水平,浆细胞增生加强体液免疫水平,同时还通过非特异性黏膜免疫细胞,嗜酸粒细胞和杯状细胞的增生进一步加强黏膜免疫水平以抵抗贝氏莫尼茨绦虫对绵羊的感染。可见绵羊可以通过黏膜免疫相关细胞增生加强局部免疫力以监视虫体免疫逃逸来抵抗寄生虫的感染。     

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.     

研究肠道糖基化修饰的荧光凝集素免疫组化方法建立

糖基化修饰是生命体中最重要的一种蛋白质翻译后修饰之一，对生命体起着非常重要的作用。糖蛋白上的糖基可以作为凝集素的结合位点，因此凝集素可以用来鉴别和分析糖链结构，作为糖基化分析方法如质谱法的重要补充。本试验旨在建立和优化研究猪肠道糖基化修饰的荧光凝集素免疫组化方法，并应用该方法研究猪不同肠段糖基化修饰的差异以及断奶对仔猪肠道糖基化修饰的影响。结果表明：①荧光凝集素免疫组化方法最适条件为：切片脱蜡后，封闭切片30 min，采用5 μg/mL FITC标记植物凝集素（FITC-Lectin）室温孵育1 h，以含有DAPI的封片剂进行封片。该方法具有灵敏度高、特异性好以及定量更为多元化的特点。②生长猪回肠和结肠中糖基化修饰模式不同，回肠主要以岩藻糖、N-乙酰葡萄糖胺为主，而结肠中主要以N-乙酰葡萄糖胺、岩藻糖和甘露糖为主。除甘露糖分布在非杯状细胞的肠上皮细胞和固有层外，岩藻糖和N-乙酰葡萄糖胺主要分布在肠绒毛表面和杯状细胞中。③22~28日龄仔猪回肠中N-乙酰葡萄糖胺的含量随着仔猪日龄的增加而增加（P<0.05），断奶显著降低了28日龄仔猪回肠中N-乙酰葡萄糖胺的含量（P<0.05）。综上所述，本研究成功建立猪肠道糖基化修饰的研究方法，并通过此方法发现仔猪回肠中N-乙酰葡萄糖胺可作为肠道屏障功能完善的标志之一。     

Anti-nutritional effects of a moderate dose of soybean agglutinin in the rat.

This study was conducted to investigate the effects of a moderate dose of purified soybean agglutinin on performance and nitrogen digestibility in rats as well as to determine its effects on the protein, DNA and RNA content of the small intestine and pancreas. Twenty-four Sprague-Dawley rats were randomly allotted into one of four groups for a 10-day nitrogen balance experiment. The four groups of rats were fed 7 g of a casein-cornstarch based diet or a similar diet supplemented with 0.1, 0.2 or 0.4 mg/g purified soybean agglutinin. All experimental diets were adjusted to an identical nutrient level. Dose of soybean agglutinin had no significant effect on rat performance. Incorporation of soybean agglutinin in the diet reduced apparent protein digestibility and the utilization of dietary protein by increasing nitrogen loss from the faeces and urine. Fresh pancreatic weight increased in rats fed soybean agglutinin at a level of 0.4 mg/g in the diet compared to the control, but the dry pancreatic weight and the protein content of the pancreas did not differ among the four groups. However the DNA and RNA content of the pancreas had a tendency to increase with a higher level of soybean agglutinin. The weight of the jejunum and its protein, DNA and RNA content were not significantly affected by soybean agglutinin, but the dry weight and the RNA of the jejunum tended to increase with higher levels of soybean agglutinin in the diet. In conclusion, purified soybean agglutinin, at moderate levels in the rats diet, had negative effects on digestive function, such as nitrogen digestibility, nitrogen retention and nitrogen balance. As the level of soybean agglutinin increased, the effects became more pronounced. Meanwhile, hypertrophy of the pancreas was observed with higher doses of soybean agglutinin incorporation in the diets.     

Duration of resistance to experimental footrot infection in Romney and Merino sheep vaccinated with Bacteroides nodosus oil adjuvant vaccine

Groups of 10-12 Romney and Merino wethers were challenged simultaneously with homologous experimental footrot infection after having received the second of 2 doses of Bacteroides nodosus (strain 198) vaccine 0, 4, 8, 12, 16 and 20 weeks previously. Inoculations were carried out 28 days apart and unvaccinated sheep of both breeds were challenged as controls. Most Romneys that had been vaccinated up to 16 weeks prior to challenge were resistant to footrot whereas 8 of 10 controls were susceptible. This resistance was lost by about 20 weeks after vaccination. By contrast, protection against challenge in vaccinated Merinos lasted only about 4-5 weeks, although residual benefits of vaccination were apparent after longer intervals from the reduced number and severity of foot lesions among vaccinates compared with controls. Agglutinin titres, which did not differ markedly after similar intervals between Romneys and Merinos, reached maximum levels between 4 and 8 weeks after the second vaccine dose and subsequently declined. Although peak titres were generally recorded at the time of maximum protection in Merinos, the relationship between agglutinin levels and resistance in the Romneys was ill-defined.     

Localization of the lectin reactive sites in adult and prepubertal horse testes

The testes of prepubertal and adult horses were investigated using 10 horseradish peroxidase conjugated lectins combined with sialidase digestion and potassium hydroxide treatment, to localise the oligosaccharide sequences of glycoconjugates during spermatid maturation. In adult animals, the lectins showed a variable affinity for spermatids and Sertoli cell apical extensions. Soybean agglutinin (SBA), peanut agglutinin (PNA), Ricinus communis agglutinin (RCA-I) and wheat germ agglutinin (WGA) bound to the acrosomal structures of spermatids, whereas Griffonia simplicifolia agglutinin (GSA-II) labelled these structures only during Golgi and cap phases. These results suggested that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides and that these carbohydrate chains undergo modifications during spermiogenesis. Sialic acid residues were not detected throughout the acrosomal development. The lectin binding pattern of Sertoli cells was very similar to that of acrosome of spermatids during the maturation phase. In sexually immature horses, only the degenerated germinal cells and the Leydig cells showed reactivity towards lectins. The first cells reacted with SBA and Dolichos biflorus agglutinin (DBA), the latter with SBA, PNA, WGA, GSA-II, Canavalia ensiformis agglutinin (ConA), Lens culinaris agglutinin (LCA) and also with DBA after sialidase digestion.     

Effects of soybean agglutinin on nitrogen metabolism and on characteristics of intestinal tissues and pancreas in rats.

Two experiments were conducted to investigate the effects of increasing concentrations of supplemental purified soybean agglutinin on performance, apparent nitrogen digestibility, plasma insulin and cholecystokinine (CCK) levels in rats as well as on the growth of the small intestine and pancreas. In Experiment 1, a 10-day nitrogen balance trial was conducted with 24 male Sprague-Dawley rats (mean BW 85 g) that were randomly allotted to one of four dietary treatments. Rats in each group were provided daily with 7 g of a casein-cornstarch based diet (control) or a diet supplemented with purified soybean agglutinin at 0.4, 0.6 or 0.8 mg/g. Urine and faeces were collected daily and stored at -20 degrees C until analysis. In Experiment 2, 30 male Sprague-Dawley rats (mean BW 75 g) were divided into five groups for a 20-day growth experiment. Each rat was fed daily 7 g of a casein-cornstarch based diet (control) or a diet supplemented with purified soybean agglutinin at 0.4, 0.8, 1.2 or 2.0 mg/g. All experimental diets were adjusted to contain a similar level of nutrients. Results from the two experiments showed that supplementation of soybean agglutinin below 2.0 mg/g diet had no significant effect on rat performance. However, rats receiving 2.0 mg soybean agglutinin per gram of diet showed a significant reduction in weight gain compared to the control group. Incorporation of soybean agglutinin in the diet reduced apparent nitrogen digestibility and the retention of dietary nitrogen by increasing nitrogen loss from the faeces and urine. In addition, plasma CCK level increased with increasing inclusion of soybean agglutinin in the diet. On the contrary, the plasma insulin level declined as soybean agglutinin level increased. Soybean agglutinin induced a polyamine-dependent hyperplastic and hypertrophic growth of the small intestine and pancreas by increasing the contents of protein, RNA and DNA, though the increase in weight of small intestine was not significant. Furthermore, 1.2 and 2.0 mg soybean agglutinin per gram of diet promoted proliferation of the jejunum mucosa, while the structure of the brush border epithelium of small intestinal had no damaging change and no diarrhoea was observed in any treatment group. Based on these results, supplementation of low doses of soybean agglutinin or soy protein to parenterally-fed animals affected by atrophic small intestine may promote small intestinal growth.     

Spontaneous eosinophilic granulated round cell tumors in rats

Morphologic and histochemical characteristics were noted for three spontaneous tumors with eosinophilic cytoplasmic granules that occurred in aged Fischer 344 rats. Macroscopic lesions were widely distributed in the body, mainly involving the intra-abdominal adipose tissue, pancreas, and mesenterium. These lesions were generally hard swellings with nodular and sclerosing areas. Bloody ascites was a concomitant finding. Histologically, the tumor cells were round, from 9 to 30 microm in diameter with one or two round to oval nuclei, and characterized by eosinophilic granules (0.5-2.0 microm) that stained definitely to weakly positive with the periodic acid-Schiff reaction and demonstrated no metachromasia with toluidine blue stain. Furthermore, the granules were characterized by a positive reaction with lectin histochemistry for concanavalin A (Con A), wheat germ agglutinin (WGA), phaseolus vulgaris agglutinin (PHA-E4), lens culinaris agglutinin (LCA), and recinus communis agglutinin (RCA-I) in all tumors and for ulex europaeus agglutinin (UEA-I), peanut agglutinin (PNA), and soybean agglutinin (SBA) in one tumor. Positive reactions for anti-rat mast cell protease II and CD8 were not demonstrated immunohistochemically. Abundant glycogen was noted in the large tumor cells from one rat. With electron microscopy, the cytoplasmic granules were identified as electron-dense homogenous bodies bounded by a single unit membrane. These characteristics are similar to those of granulated metrial gland cells, but further study is needed to clarify the cell of origin for these tumors.     

Lectin histochemistry of dog major and minor salivary glands

The distribution of different carbohydrates in dog major and minor salivary glands was investigated using a peroxidase-labelled avidin-biotin method to demonstrate binding of six lectins (Canavalia ensiformis agglutinin [Con A], Dolichos biflorus agglutinin [DBA], Arachis hypogaea (peanut) agglutinin [PNA], Glycine max agglutinin [SBA], Tetragonolobus purpurea agglutinin [TGP] and wheat germ agglutinin [WGA]). With PNA, there was only weak staining in serous acini of parotid glands. Other lectins bound, to different degrees, to different components of the salivary glands; differences could be detected between glands and between binding of different lectins to serous and mucous acinar cells and to the epithelial cell cytoplasm, luminal surface and contents of ducts. These results provide a basis for the comparison of possible changes in carbohydrates which may occur in salivary gland diseases.     

The Tissue Distribution of la- and IgM-Positive Cells in Adult and Newborn Miniature Pigs

Distribution of Ia- and IgM-positive cells was compared in tissues of adult and newborn miniature pigs, using indirect immunoperoxidase and immunofluorescent techniques with monoclonal antibodies. The different distribution patterns were found mainly in adult jejunum and kidney. Both IgM- and Ia-staining were ascertained in the lamina propria between the Lieberkühn crypts and in the lymphoid follicles of the jejunum. Moreover, IgM-positive cells were demonstrated in the crypts while Ia-reaction was seen in the lamina propria of the villi. In the kidney, IgM-staining was limited to the glomeruli. On the contrary, a distinct Ia-reaction was found mainly in intertubular structures. Small differences in IgM- and Ia-positive cell distribution were also noted in the mesenteric lymph nodes and in the spleen. Developmental changes of the adult distribution patterns (chiefly that of IgM-positive cells) were found in all tissues studied at 2 days of age.     

Effects of inflammation and aqueous tear film deficiency on conjunctival morphology and ocular mucus composition in cats

An experimental model of keratoconjunctivitis sicca (KCS) was produced by removing the lacrimal gland and the gland of the third eyelid from the left eye of 6 cats. The right eye of each cat was left intact and used as a control. After 2 weeks, cats were euthanatized and the central portion of the upper eyelid from both eyes of each cat was excised. Histologic sections were stained with either hematoxylin and eosin or with a battery of biotinylated lectins including concanavalin A (conA), soybean agglutinin (SBA), wheat germ agglutinin (WGA), succinylated wheat germ agglutinin (S-WGA), Ulex europaeus agglutinin I (UEA), Dolichos biflorus agglutinin (DBA), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA), and PNA pretreated with neuraminidase. Consistent differences in histologic features were not observed between conjunctivas with KCS and control conjunctivas. A variable degree of mononuclear cell infiltration of the substantia propria was observed in control conjunctivas and those with KCS. In both groups, conjunctival goblet cell density decreased and epithelial stratification increased as the degree of submucosal inflammatory cell infiltration increased. Lectin binding sites for DBA, WGA, S-WGA, UEA, PNA, and PNA pretreated with neuraminidase were detected on conjunctival goblet cells of conjunctivas with KCS and control conjunctivas. The mucus/glycocalyx layer of conjunctival epithelial cells in both groups of conjunctivas bound lectins RCA, WGA, UEA, and conA, but inconsistently bound S-WGA. In both groups, DBA principally bound to the mucus layer overlying normal epithelium, whereas PNA pretreated with neuraminidase consistently bound to the mucus layer of stratified epithelial surfaces free of goblet cells. Binding of SBA to goblet cells and the mucus/glycocalyx layer was variable.