Comparison Of The Ultrastructure And The Intracellular Digestive Function Of Lysosome In The Small Intestinal Epithelium And Its Correlation With Intraluminal Digestive Enzymes In Sucking Lambs

Lamb production is the basic link in intensive mutton sheep breeding.Studies on the growth and development of small intestine of lambs mainly focused on the changes of intestinal morphology and digestive enzyme activities.The morphology and function of lysosomes digested by intestinal epithelial cells in sucking lambs are not well understood,especially the dynamic changes of the lysosomes in different sucking days have not yet reported.In this study,16 newborn lambs(2.77±0.08 kg)were randomly divided into 4 groups,namely,30 day(EEW-30),45 day(EW-45),60 day(NW-60)and 75 day(SC-75)old groups with 4 lambs in each group.Using transmission electron microscopy,immunohistochemical staining method,Western Blot,fluorescent quantitative PCR and ELISA methods the ultruramicromorphology and the locating of lysosomes in small intestinal epithelium were observed.Determine the relative expression of(Lysosomal-associated membrane protein 1/2,LAMP1/2)LAMP1 and LAMP2 gene m RNA and their protein,and analyze the correlation between the strength of intracellular digestion and the activity of digestive enzymes in the small intestine of suckling lamb.The main findings are as follows:1.Transmission electron microscopy showed that most lysosomes located in the apical region of intestinal epithelium.Most of the dense small globular primary lysosomes located in the duodenum,and most of the large endocytic vesicles(secondary lysosomes)located in the epithelium of jejunum and ileum.Immunohistochemical staining showed that the expression of Lamp2 was higher in the epithelial cells at the top of villi.2.Compared with sucking lambs of different ages,LAMP1 m RNA expression in the same intestinal segment.Duodenum showed that EEW-30 group was significantly lower than that in SC-75 group(P<0.05);Jejunum showed that EEW-30 group was significantly lower than that in EW-45 group(P<0.01).The m RNA expression of LAMP1 gene in jejunum was significantly higher than that in duodenum and ileum in EEW-30and EW-45 groups(P<0.01).There was no significant difference in LAMP2 m RNA expression between sucking lambs of different ages and different intestinal segments(P>0.05).The relative expression of LAMP1 protein in the same intestinal segment of sucking lambs at different ages showed that the lambs in Jejunum showed that NW-60 group were significantly lower than those in SC-75 group(P<0.01).The expression of LAMP1 protein in jejunum was significantly higher than that in ileum and duodenum in SC-75 group(P<0.01).The expression level of LAMP2 protein in the same intestinal segment of sucking lambs at different ages,duodenum and jejunum showed that EW-45 group and NW-60 group were significantly higher than EW-45 group and SC-75 group(P<0.01);Ileum showed that NW-60 group was significantly higher than other groups(P<0.01).The relative protein expression of LAMP2 was significantly higher in duodenum than in ileum at 30,45 and 60 days of age(P>0.05).Jejunum was significantly higher than ileum in 45,60 and 75 day old groups(P<0.01).3.The relative expression of LAMP2 protein in different intestinal segments was positively correlated with the activities of invertase and lipase(R~2=0.506,P=0.0095 and R~2=0.463,P=0.015),and negatively correlated with the activities of amylase(R~2=0.583,P=0.004).At 45 days of age,there was a significant moderate negative correlation with amylase and protease activities(R~2=0.451,P=0.017 and R~2=0.338,P=0.048).At 60 days of age,there was a significant moderate positive correlation with lipase activity(R~2=0.496,P=0.011).At the age of 75 days,there was a significant medium negative correlation with amylase activity(R~2=0.73,P=0.0004)and protease activity(R~2=0.457,P=0.016).The relative expression of LAMP2 protein in the same intestinal segment at different ages was only negatively correlated with duodenal invertase activity(R~2=0.279,P=0.035).It is suggested that the small intestinal intracellular lysosome digestion reflected by LAMPs expression in sucking lambs changes descendingly from duodenum to ileum.The intracellular digestion in the same segment of small intestine increase along with the increase of suckling age.The expression of LAMP2 is positive correlated with the activities of the small intestinal sucrase and lipase,and negatively correlated with the activities of protease,amylase and protease,which are mainly related to the segment of different small intestine but have little effect on suckling age.