| Regeneration has always been a hot topic in life science research.Vortex is regarded as the best biological model for studying regeneration because of its unique regeneration ability.Our previous research found that the dynamic balance between Drp1 and Mfn1 played a decisive role in the formation of embryonic base on the second day and the formation of nervous system and tissue differentiation on the fifth day.It indicated that more mitochondria were needed to provide energy for stem cell differentiation in the critical stage of regeneration of planarian.In the regeneration process,the number of mitochondria changes mainly through the mitochondrion division and fusion caused by the dynamic balance between Drp1 and Mfn1.Previous studies have shown that Wnt/?-Catenin signaling pathway is closely related to mitochondrion division and fusion in tissue and nerve regeneration,and Smed-tcfl is a key regulator in nerve regeneration of planarian,which is also closely related to Wnt/?-Catenin signaling pathway.However,it is not clear how Wnt/?-Catenin signaling pathway and Drpl interact to regulate cell division and programmed cell death,and how Smed-tccf1 interacts with Wnt/?-Catenin signaling pathway and Drpl to regulate nerve regeneration in planarian.To study the role of Wnt/?-Catenin signaling pathway and Smed-tcf1 in the regeneration of planarian,the polyclonal antibody against?-Catenin of planarian was prepared.On the basis of this antibody,the relationship between the expression changes of ?-Catenin,Smed-tcfl and mitochondrion fusion protein and its influence on the regeneration of planaria japonica after head injury was studied.In this study,pET-28a-?-Catenin recombinant plasmid was constructed,the expression of ?-Catenin protonuclear protein was induced,and the purified recombinant protein was used to immunize mice to prepare ?-Catenin polyclonal antibody.Secondly,biological techniques such as qRT-PCR and Western blotting were used to explore the changes of the contents of ?-Catenin,TCF1,Drpl and Mfn2 in gene and protein level during the regeneration of planarian after head injury,so as to explore the interaction between ?-Catenin,Drpl and Smed-tcfl in the regeneration of planarian after head injury.The main results of this study are as follows:1.The ?-Catenin protein of planaria japonica was predicted and analyzed by means of bioinformatics.the size of the protein was 78.35 kDa,and the isoelectric point was 5.69.the predicted secondary structure had regular curly structure at the n end of peptide chain,and the protein did not have transmembrane structure.There are 27 antigenic peptide segments and 7 surface accessible peptide segments in this protein.Two best peptide segments of B cell epitopes were obtained by comprehensive analysis based on the predicted results of other data:68MVRMQKPAQR77 and 306DYSSNINDMQKLIK319.2.The recombinant plasmid pET-28a-?-Catenin was successfully constructed.After sequencing,the BLAST results of gene sequence and ?-Catenin original sequence were 100%.3.The titer of the prepared polyclonal antibody against ?-Catenin of planaria japonica reached 1:200 000 by ELISA.The results showed that the prepared antibody could effectively bind to the protein of planaria japonica,indicating that the prepared antibody had good binding ability.The prepared antibody can be used in experimental study.4.The expression changes of drpl and mfn2 mRNA content in the formation of embryo base on the second day and the formation of nerve and tissue differentiation on the fifth day of regeneration of planarian indicate that mitochondrial division plays an important role in cell differentiation.5.The mRNA expressions of ?-Catenin and Smed-tcf1 were down-regulated simultaneously during the formation of embryo base on the second day and the development of nervous system on the fifth day,which had the same trend as the mitochondrial fusion protein Mfn2 mRNA.After Wnt/?-Catenin signaling pathway was activated,the mRNA levels of ?-Catenin and smed-tcf1 were significantly increased on the 2nd and 5th day,while the mRNA content of drp1 was significantly decreased at this time point,and the regeneration of planarian was significantly inhibited.The results indicated that ?-Catenin/TCF1 can regulate the expression of mitochondrion fusion protein during the regeneration of the head of planaria,and its expression is closely related to the dynamic balance of mitochondrion fusion,and directly affects the regeneration of planaria.Conclusion:In this study,the polyclonal antibody against ?-Catenin of planaria japonica was prepared for the first time,and it was used to further study how ?-Catenin and Smed-tcf1 regulate mitochondrion division and fusion,and how they interact with Drp1 and Mfn1/2.In this study,it was found that the expression changes of ?-Catenin and Smed-tcfl were consistent with mitochondrial fusion protein Mfn2,but contrary to mitochondrial mitogen Drp1.Activation of Wnt/?-Catenin signaling pathway can inhibit the expression of Drpl,which leads to obstacles in embryo formation and regeneration of head and nervous system in the regeneration process of planarian,indicating that?-Catenin and Smed-tcfl can regulate the dynamic balance of mitochondrion division and fusion,and then regulate the key steps of regeneration.Wnt/?-Catenin signaling pathway and Smed-tcf1 play an important role in the regeneration process of planarian. |
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