【作者】 杨慧；

【导师】 潘康成； 陈正礼；

【作者基本信息】 四川农业大学 ， 基础兽医学， 2007， 硕士

【摘要】 本研究为探讨芽孢杆菌制剂对小鼠肠粘膜结构及粘膜免疫的影响进行了研究。将96只雄性健康小鼠随机分为芽孢杆菌PAb04组、芽孢杆菌PAS38组、抗生素组、对照组4组。对照组饲喂基础日粮，芽孢杆菌PAb04组、PAS38组和抗生素组分别在基础日粮中添加10~6cfu／g的PAb04、PAS38和20u／g土霉素，分别于饲喂20d、40d、60d后各宰杀8只，采取十二指肠、空肠，测量了小肠粘膜厚度和绒毛长度，并计算了上皮内淋巴细胞(IEL)的数量，研究了小肠粘膜中生长抑素(SS)阳性细胞、IgA阳性细胞、CD4+、CD8+T淋巴细胞及肥大细胞的分布及其数量的变化。1．采用H．E染色方法测量了小肠粘膜厚度和绒毛长度。结果显示，芽孢杆菌制剂可显著增加小肠粘膜厚度和绒毛长度，而抗生素组十二指肠和空肠粘膜厚度和绒毛长度则无显著变化，推测芽孢杆菌制剂对小鼠小肠的消化吸收功能具有促进作用，并且优于抗生素。2．采用H．E染色计算了IEL的数量。结果表明，芽孢杆菌制剂可以增加小鼠肠道内IEL的数量。在饲喂的整个过程中，各组小鼠肠道内IEL的数量均有不同程度的升高。饲喂40d后，芽孢杆菌PAS38组十二指肠和空肠中IEL数量均显著高于抗生素组和对照组(P＜0.05)；饲喂60d后，芽孢杆菌PAS38组十二指肠和空肠中IEL数量比对照组显著增加(P＜0.05)，表明芽孢杆菌PAS38可以增加小鼠肠道内IEL的数量。3．采用免疫组化技术(SABC法)分别研究了小鼠小肠中IgA阳性细胞、CD4+、CD8+T淋巴细胞数量的变化。结果显示：在饲喂的整个过程中，芽孢杆菌PAb04组十二指肠和空肠中IgA、CD4+、CD8+T淋巴细胞表达量比对照组均显著增加(P＜0.05)；而芽孢杆菌PAS38组十二指肠和空肠中IgA、CD4+、CD8+T淋巴细胞表达量则比对照组极显著增加(P＜0.01)；而芽孢杆菌PAS38组与抗生素组相比也呈现显著性。结果表明，芽孢杆菌制剂尤其是芽孢杆菌PAS38可有效增加小肠中IgA、CD4+T、CD8+T淋巴细胞的水平，提高机体的粘膜免疫力，且效果优于抗生素。4．应用甲苯胺蓝染色技术研究了小鼠小肠中肥大细胞数量的变化。结果显示：饲喂20d后，芽孢杆菌PAb04组空肠中肥大细胞数量比对照组显著增加(P＜0.05)，芽孢杆菌PAS38组则极显著增加(P＜0.01)，芽孢杆菌PAb04组、PAS38组均比抗生素组显著增加(P＜0.05)；饲喂40d后，芽孢杆菌PAS38组十二指肠中肥大细胞数量比对照组显著增加(P＜0.05)；饲喂60d后，芽孢杆菌PAb04组十二指肠中肥大细胞数量比对照组显著增加(P＜0.05)，而芽孢杆菌PAS38组则比对照组极显著增加(P＜0.01)；芽孢杆菌PAS38组、Pab04组空肠中肥大细胞数量均比对照组极显著增加(P＜0.01)。结果表明，肥大细胞的数量可能反映了消化道粘膜的免疫水平。5．采用免疫组化技术(SABC法)研究了小鼠小肠中SS数量的变化。结果表明：饲喂20d后，芽孢杆菌PAS38组十二指肠中SS比对照组显著减少(P＜0.05)，芽孢杆菌PAb04组、PAS38组比抗生素组则极显著减少(P＜0.01)；芽孢杆菌PAb04组、PAS38组空肠中SS均比对照组、抗生素组极显著减少(P＜0.01)，并且芽孢杆菌PAS38组比PAb04组显著减少(P＜0.05)；饲喂40d后，芽孢杆菌PAb04组、PAS38组十二指肠中SS均比抗生素组显著减少(P＜0.05)，芽孢杆菌PAb04组比对照组显著减少(P＜0.05)，芽孢杆菌PAS38组则比对照组极显著减少(P＜0.01)；芽孢杆菌PAS38组空肠中SS比对照组极显著减少(P＜0.01)；饲喂60d后，各组间无明显差异。结果表明，芽孢杆菌制剂尤其是芽孢杆菌PAS38对小鼠肠粘膜结构及粘膜免疫具有明显的增强作用。更多还原

【Abstract】 Effects of Bacillus sp. as feed additive on the epithelial structures intestinal mucosal immunity of mice was studied. Nity-six healthy weaning mice were divided into 4 groups. Control group mice were fed the basic diet; PAb04 group mice were fed the diet with 10~6cfu/g of B. subilits PAb04; PAS38 group mice were fed the diet with 10~6cfu/g of B. cerecus PAS38; Antibiotic group mice were fed the diet with 20u/g of Oxyteracycline. Eight mice in each group were killed per time-point on day 20, 40, 60-day-old. The distribution and change of somatostatin(SS)-positive cells, IgA-positive cells, IEL, CD4+ and CD8+ T cells and mast cells induodenum and jejunum of mice were measured.1. H.E staining method was adopted to study the change of mucosa thickness and villus length in mouse small intestinal. The results showed that Bacillus sp. as additive of mice could increase mucosa thickness and villus length in mouse small intestinal.2. H.E staining method was used to study the change of IEL in mouse small intestinal. The results showed that Bacillus sp. as praperation of mice to increase the number of IEL in mouse small intestinal increased, the number of IEL in duodenum and jejunum of PAS38 group increased significantly (P＜0.05) comparing to control group and antibiotic group at the 40th day; Comparing to control group the number of IEL in duodenum and jejunum of PAS38 group increased significantly (P＜0.05) at the 60th day. The results suggest that Bacillus sp. PAS38 as additive of mice can increase the number of IEL in mouse small intestinal.3. The changes of the number of IgA-positive cells, CD4+ and CD8+ T cells in mouse small intestinal were studied by SABC immunohistochemistry. The results showed that in the all sixty days the number of IgA-positive cells, CD4+ and CD8+ T cells in duodenum and jejunum of PAB04 group increased significantly (P＜0.05) comparing to control group; Comparing to control group the number of IgA-positive cells, CD4+ and CD8+ T cells in duodenum and jejunum of PAS38 group increased significantly (P＜0.01); the number of IgA-positive cells, CD4+ and CD8+ T cells in duodenum and jejunum of PAS38 group increased significantly compared to antibiotic group. The results suggest that Bacillus sp. PAS38 as additive of mice can increase the number of IgA-positive cells, CD4+ and CD8+ T cells, thus mucosal immunization is promoted.4. Toluidine blue staining method was used to study the change of mast cells numble in mouse small intestinal. The results showed that the number of mast cells in jejunum of PAb04 group increased significantly (P＜0.05) and the number of mast cells in jejunum of PAS38 group increased significantly (P＜0.01) compared to control group at the 20th day; Comparing to antibiotic group the number of mast cells in jejunum of PAb04 group and PAS38 group increased significantly (P＜0.01) at the 20th day; the number of mast cells in duodenum of PAS38 group increased significantly (P＜0.05) compared to control group at the 40th day; the number of mast cells in duodenum of PAb04 group increased significantly (P＜0.05) compared to control group at the 60th day; the number of mast cells in duodenum of PAS38 group increased significantly (P＜0.01) compared to control group at the 60th day; Comparing to control group the number of mast cells in jejunum PAb04 group and PAS38 group increased significantly (P＜0.01).Our results suggest that the number of mast cells might reflect the digestive mucosal immunization.5.The changes of the number of SS-positive cells in mouse small intestinal were studied by SABC immunohistochemistry.The results showed that the number of SS-positive cells in duodenum of PAS38 group increased significantly (P＜0.05) compared to control group at the 20th day,Comparing to antibiotic group the number of SS-positive cells in duodenum of PAb04 group and PAS38 group increased significantly (P＜0.01)at the 20th day; the number of SS-positive cells in jejunum of PAb04 group and PAS38 group increased significantly (P＜0.01) compared to control group and antibiotic group at the 20th day, Comparing to PAb04 group the number of SS-positive cells in jejunum of PAS38 group increased significantly (P＜0.05) at the 20th day; the number of SS-positive cells in duodenum of PAb04 group and PAS38 group increased significantly (P＜0.05) compared to antibiotic group at the 40th day; Comparing to control group the number of SS-positive cells in duodenum of PAb04 group increased significantly (P＜0.05)and the number of SS- positive cells in duodenum of PAS38 group increased significantly (P＜0.01) at the 40th day; the number of SS- positive cells in jejunum of PAS38 group increased significantly (P＜0.01) compared to control group at the 40th day.The results suggest that Bacillus sp. PAS38 as feed of mice to increase mucosal immunity promoted.更多还原