Study On The Secretion Mechanism Of Salivary Gland Cells In Hirudo Nipponia

Hirudo nipponia is a highly specialized annelid animal.Its salivary glands can secrete a large number of active substances,which can promote blood circulation and remove blood stasis,and resist blood coagulation.It has extremely high medicinal value.In this study,the morphological observation,cell culture and transcriptome analysis of Hirudo nipponia were carried out to explore its secretion mechanism.The main research results are analyzed as follows:(1)Through the morphological observation of Hirudo nipponiaes during starvation and different periods after feeding,the salivary glands of Hirudo nipponiaes were observed by light microscope and electron microscope,and the results showed that:1)Hirudo nipponiaes is a pharyngeal structure with triangular muscular jaws.The salivary glands are attached to the jaws in a grape shape,and the glands are white and symmetrically distributed around the jaws.2)Hematoxylin-eosin staining(HE staining)showed that salivary gland cells were composed of ovoid somatic cells and elongated ducts,and the nucleus was located at the bottom or edge of the cell.The unfed salivary gland cells were lightly stained with full cytoplasm;the fed salivary gland cells had dark staining and loose cytoplasmic structure.3)Based on transmission electron microscopy(TEM)imaging,it was observed that there are spherical secretory granules in salivary gland cells,and there are dense components with high electron density inside the secretory granules.The unfed salivary gland cells had compact space,secretory granules squeezed each other,and a large number of secretory granules contained dense components;after fed salivary gland cells had loose space,there were gaps between secretory granules,and the dense components in secretory granules disappeared.4)Based on scanning electron microscope imaging(SEM),it was observed that the salivary gland cells were arranged in a grape-like arrangement.The unfed salivary gland cells had smooth surface and rounded cells;the fed salivary gland cells had a large amount of granular material on the surface and the cells were concave.(2)By culturing the salivary gland cells of Hirudo nipponiaes in vitro and observing their secretory activities by microscope.1)After the salivary gland tissue is enzymatically hydrolyzed,the salivary gland cells just separated by enzymatic hydrolysis are spherical or ellipsoid,and some salivary gland cells have extended pipes at the tail and are tadpole-shaped.The salivary gland cells have low brightness and poor refractive index.Suspension in liquid does not adhere to the wall.During the culture process,it was found that the brightness of salivary gland cells gradually decreased,and the refractive index became worse,but the cells would produce secretory activities and secrete transparent vesicles.2)These secretory vesicles are large or small,and the shape is very regular and spherical.We found that soma secretory vesicles are generally larger and duct-secreting secretory vesicles are generally smaller.The culture found that the small secretory vesicles will continue to grow,and the large secretory vesicles will continue to grow until they finally burst and burst into fragments.(3)Transcriptome sequencing of salivary gland tissues of Hirudo nipponia during starvation and different periods after eating was performed using a sequencing platform.1)After splicing and sorting the transcriptome,the Clean data was obtained,and the quality of the Clean data was evaluated.It was found that the percentage of Q10 bases in the sequencing data of each sample exceeded 99%,which indicates that the sorted Clean data is of good quality and can be used for the transcriptome.analyze.2)Compare the Unigenes genes obtained from the transcriptome with the CDD,KOG,NR,NT,PFAM,Swissprot,Tr EMBL,GO,and KEGG databases to obtain detailed annotation information.3)In terms of GO functional annotation and enrichment analysis of differential genes,after feeding Hirudo nipponia,the GO functional annotation of differential genes in extracellular components may be related to the extracellular activities of salivary glands;differential genes are involved in cytoamide metabolism,peptide Enzyme activity,metabolic process of organic nitrogen compounds,cytoplasmic part,mitochondrial GO function annotation,which all indicate that salivary gland cells are carrying out anabolism,and mitochondria provide energy to synthesize proteins and other substances for salivary gland cells after eating;4)In the KEGG pathway analysis of differential genes,after feeding Hirudo nipponia,the differential genes are in the ribosomal pathway,oxidative phosphorylation pathway,and citric acid cycle pathway,which indicates that the salivary gland cells carry out anabolism and the synthesis of protein substances is very active;the differential genes are in On the ECM-receptor interaction pathway,this may be related to the extracellular signal received by the salivary gland;the differential genes are in the myocardial contractility pathway and the protein digestion and absorption pathway,indicating that the Hirudo nipponia is in the digestion activity.(4)Analysis and summary of the salivary gland secretion mechanism of Hirudo nipponia: When Hirudo nipponia eats,the extracellular receptors of salivary glands will combine with signal substances to cause a secretion response.In the anabolic stage,various proteins in saliva are synthesized.