| Fish is the lowest vertebrate,fish possesses the innate and adaptive immunity.The non-specific immunity plays an important role in immune response against invasion of foreign pathogens.The skin,gills and intestinal tissues of fish are covered by a large amount of mucus.This layer of mucus is mainly composed of mucoprotein 2(MUC2)secreted by mucous cell in the epithelium,which is mixed with water and other products to form the mucus gel on the surface of mucosal epithelial cells.The amount change of MUC2 can reflect the level of immunity of mucous tissue in the body,so the quantitative detection of mucous cells and MUC2 is particularly important for the diagnosis and treatment of fish body related diseases.The main results of this experiment are as follows:(1)This study was carried out on Cyprinus carpio L.Gene cloning technology was used to clone the MUC2 gene sequence,and its structural characteristics and biological informatics were analyzed.In this study,the length of the MUC2 gene sequence was 2663 bp,the relative molecular weight was 242256.95,and the main structure domain was VWD-C8-VWD-C8-TIL-VWC-CT conserved structure domain.The main structure of the protein was the α-helix,β-sheets and irregular-curl.Among them,8.56% of the amino acids were the α-helices 33% is β-sheets;irregular-curl accounted for 58.45% of the total amino acids of the protein.The gene has no transmembrane structure and is secretory mucin.(2)Real-time quantitative PCR(q RT-PCR)was used to detect the expression distribution of MUC2 in all tissues of healthy carp and the m RNA expression of MUC2 in tissues and organs of carp after challenged by LPS,Poly I:C and Aeromonas veronii.The results showed that the expression levels of healthy carp mucosal tissues(skin,gill and intestinal)were relatively high.The expression levelof sequence was: M-intestine> F-intestine> H-intestine> gill> skin> heart> liver> head kidney> muscle> kidney> blood> brain> spleen.The m RNA expression level of MUC2 in mucosal tissues and organs was detected after challenge.It was found that in the skin,the expression levels of MUC2 began to increase at 3h after infection,then,reached the maximum value(increased 5.56 times,11.26 times,12.51 times after challenge by LPS,Poly I:C and Aeromonas veronii,respectively)at 12 h and downregulated at 24 h.In gills,the expression level of MUC2 reached the peak at 6h after infection(increased 4.07 times,9.59 times,14.41 times),and then gradually decreased to normal value after 72 h.In the intestinal tract,the expression of MUC2 in different intestinal segments increased first and then decreased after the challenge,and the foregut reached the peak at 6h(increased 7.98 times,6.41 times and 4.47 times),midgut(increased 10.17 times,11.63 times,10.52 times)and hindgut(increased 15.47 times,17.32 times,6.30 times)also increased to the maximum at 6h.It was noteworthy that the expression level of MUC2 in midgut almost reached the peak at 3h after challenge,and the expression level was 9.35 times,9.78 times and 8.83 times of that of the control group,respectively.Obviously,the expression response of MUC2 was the most sensitive after stress in midgut.(3)Tissue sections were stained by AB-PAS staining,and the number and morphological distribution of mucous cells in skin,gill and intestinal mucosa were observed under an optical microscope.Conclusion: the number of mucous cells per unit area in healthy carp skin was 6.87±1.61,most of which were type IV and some were type II.The number of mucous cells per unit area of gill tissue was 9.33±1.21,and the main types were type IV,type II and a few type III.The number of mucous cells on both sides of intestinal villi per unit length in different intestinal segments varied,with 9.53±1.48 in the foregut,12.00±2.16 in the midgut,and 10.33±2.08 in the hindgut.The types of mucous cells in each intestinal segment were mainly type II and type IV.After LPS,Poly I: C and Aeromonas veronii infection,the secretion of mucous cells increased and then decreased,and at 12 h,skin(11.0±1.58,9.00±1.87,9.60±2.96),gills(12.5±1.14,16.6±1.14,19.2±1.48),foregut(14.20±1.64,18.00±1.58,17.2±1.64),and posterior intestine(17.60±4.27,15.00±2.74),respectively.The increase in the number of mucus cells in 25.60±5.22 reached a peak,while that in the midgut(15.80±0.83,18.00±2.12,16.40±2.30)reached a maximum at 6h.(4)Tissue sections were prepared by paraffin embedding and HE staining.Pathological changes of skin,gill and intestinal mucosa were observed under an optical microscope.Pathological changes were analyzed and it was found that tissue damage mainly occurred from 24 h,and after 48 h,the tissue was destroyed rapidly and a large number of cells were lytic and necrotic.Immunohistochemistry was used to preliminarily explore the changes of MUC2 content secreted by mucous cells.The results of this study can provide a theoretical basis for understanding the immune function of anti-infection of carp mucosa and provide reference for the diagnosis and treatment of fish-related diseases. |
PDF Full Text Request