The Role Of Aj-msi1,Aj-lgr5,Aj-sdf2 And Aj-kif3a In The Intestinal Regeneration Of Apostichopus Japonicus

Apostichopus japonicus is an important economic echinoderm.When the external environment is uncomfortable or when it encounters enemies,the body will strongly contract and expel some or all of its internal organs.After vomiting,the A.japonicus has a strong regeneration ability,and it can regenerate a fully functional internal organ under appropriate environmental conditions.In recent years,MSI1(Musashi1),LGR5(leucine-rich repeat-containing g-protein coupled receptor5),SDF2(stromal cell derived factor 2)and KIF3A(kinesin family member 3A)have been deeply studied as a typical intestinal stem cell marker and cell proliferation and migration regulation gene.MSI1 protein affects cell proliferation of stem / progenitor cells by regulating Notch,Wnt / ?-catenin signaling pathway.LGR5 protein also enhances the expression of Wnt / ?-catenin signaling pathway,accelerates the cell cycle and promotes cell proliferation.SDF2 and KIF3 A play important roles in proliferation and migration,and they participate in the regulation of Wnt / ?-catenin signaling,and the Wnt / ?-catenin signaling pathway plays a role in cell proliferation,apoptosis and cell cycle.However,the role of the above genes in the regeneration process of A.japonicus is still unclear.In this study,the role of MSI1,LGR5,SDF2 and KIF3 A in the regeneration of A.japonicus intestine was studied.Therefore,this study will provide basic data for invertebrate regenerative and regenerative medicine research.It has important theoretical significance and has potential application value in the production and breeding of A.japonicus.In this study,RACE technology was used to clone partial sequences of Aj-msi1,Aj-lgr5,Aj-sdf2 and Aj-kif3 a,and bioinformatics analysis was performed on them,Real-time q PCR method was used to analyze the expression level of each gene during intestinal regeneration,Western Blotting method was used to analyze the regulation relationship of each gene during intestinal regeneration,immunohistochemistry method was used to analyze the expression pattern of each gene.The experimental results show that the ORF sequence of Aj-msi1 is1023 bp,encoding 341 amino acids,the ORF sequence of Aj-lgr5 is 2412 bp,encoding 804 amino acids,the ORF sequence of Aj-sdf2 is 456 bp,encoding 152 amino acids,and the ORF sequence of Aj-kif3 a is 1704 bp,encoding 568 amino acids.At the genetic level,the expression levels of Aj-msi1,Aj-lgr5,and Aj-kif3 a genes in the A.japonicus intestinal regeneration showed a trend of first increasing and then decreasing,reaching a peak on the12 th day.The expression level of Aj-sdf2 gene showed a trend of decreasing and then increasing,with the highest expression level on day 0.At the protein level,the expressions ofabove genes were all the same as the gene expression pattern,and In addition,the expression patterns of stem cell markers Aj-CD44 and cell migration-related proteins Aj-CDC42 are similar to the above-mentioned proteins,and they increase first and then decrease.The only difference is that the expression level of Aj-CDC42 protein peaks on the 6th day.The above research results prove that in the early stage of imitation of the A.japonicus,Aj-MSI1,Aj-LGR5,Aj-CD44,Aj-CDC42 and Aj-KIF3 A protein are up-regulated,and the expression of Wnt /?-catenin signaling pathway is enhanced and the stem cells are enriched,thereby going on preparation of materials for cell proliferation required for subsequent intestinal regeneration.With the formation of the intestinal prototype,the above-mentioned protein expression level was down-regulated,cell division activity was reduced,and cell differentiation began to dominate,thereby forming a functional intestine,and the final expression level was maintained at a normal level.In all stages of the regeneration process of the A.japonicus intestine,the above-mentioned protein expression levels in the intestinal mucosa and submucosa are relatively high,proving that the regeneration-related genes are mainly expressed in the cells here,and these cells continue to proliferate and differentiate to form new type cells.After forming a fully functioning intestine,the above proteins are still mainly expressed in the intestinal mucosa,so as to continuously proliferate to supplement cells that died due to erosion of intestinal contents,and maintain the structure and function of the intestine.In addition,the results of stem cell labeling of the mesentery confirmed that the stem cells that proliferate continuously during the process of intestinal regeneration are derived from the mesentery,which is essential in the regeneration of the A.japonicus.