Expression Of Genes Involved In Zebrafish Fin Regeneration And Effect Of Sanguinine On Its Regeneration

Regenerated,that is,mature individuals,still have the ability to develop into an intact individual on the original basis after the tissue or organ is damaged.In contrast to mammals,teleost fishes fin tissue has a strong regeneration potential after amputation,the process of the caudal fin regeneration is an important model to study the molecular basis of tissue regeneration.The zebrafish(Danio rerio)have recently emerged as a new vertebrate model for genetic studies of tissue or organ regeneration.In response to injury,major fin structures may regenerate very rapidly in zebrafish,all kinds of regeneration related genes expressed,which caused by cascade.And then the loss of caudal fin structures can still be restored to its size and shape.Since matrix metalloproteinases are involved in the degradation and remodeling of extracellular matrix,which could influence the migration of cells and are the key enzymes in the turnover of extracellular matrix during health,disease,development and regeneration.Therefore,we carried out bioinformatics analysis of the two genes mmp9 and mmp13.In the NCBI GeneBank,we find the full-length of mmp9 cDNA sequence is 2801 bp,there are 10 2bp 5? untranslation region(5?UTR)and 656 bp 3?UTR,the Open Reading Frame(ORF)is 2043 bp,encode 680 amino acids,molecular weight 76.589 kD,theoretical isoelectric point 5.63.We found that the protein is composed of 21.32% alpha helix,accounting for 145,11.32% beta angle,accounting for 77,45.44% random coil,accounting for 309.The full-length of mmp13 cDNA sequence is 1653 bp,there are 12 bp 5?UTR and 213 bp 3?UTR,the ORF is 1428 bp,encode 475 amino acids,molecular weight 53.792 kD,theoretical isoelectric point 5.97.The protein is composed of 23.37% alpha helix,accounting for 111,9.26% beta angle,accounting for 44,41.89% random coil,accounting for 199.We did qPCR for mmp9 and mmp13,find that the two genes were significantly increased after the fins were cut.According to in situ hybridization technique,we made different periods of caudal fin with zebrafish into frozen sections,that were hybridized wit prefabricated sense and antisense RNA probes,the result demonstrated that in the caudal fin of regenerated zebrafish,mmp9 gene could express in skeletal precursor cells at 3 dpa.The expression of MMP13 was strongest in the third days of regeneration,and the expression signal was weakened for fourth days.In addition,a full-length cDNA sequence of thirteen regeneration-related genes were searched in GeneBank.Pairs of Fluorescent quantitative Real Time PCR-specific primers were designed according to the full-length sequence and ?-actin was used as a reference gene for qRT-PCR.Detection of the thirteen regeneration related genes at different critical stages of caudal fin regeneration,we found that compared with the uncut fins,these genes expression levels were significantly increased after amputation.Therefore,in this article,we also studied the effect of Sanguinarine on the expression of regenerative-related genes,such as mmp9,mmp13,lef1,msxc,Igf2 b,osterix,col2a1 b,evx1,fgf20 a,wnt10a,and1,bmp2 b at different critical periods(2 dpa,3 dpa,4 dpa,5 dpa,6 dpa,7 dpa,8 dpa)of caudal fin regeneration.First of all,according to the laboratory experimental data and pre-experimental results,determine the final concentration of Sanguinarine is 0.05 mg/L.The experimental group is the zebrafish which have been dealt with Sanguinarine solution,but the freshwater and ethanol treated groups were used as the control groups.The caudal fin of the experimental group and the control group were stained with hematoxylin and eosin(HE),we find that sanguinarine could significantly increase the growth of fin regeneration.Then we did qPCR on these thirteen genes,it proved that sanguinarine could obviously upregulated the expression of regeneration-related genes.Through the above studies,we found that Sanguinarine plays an important role in the regeneration of zebrafish caudal fin,and it also plays an important regulatory role in the related genes expression.In summary,the results of this experiment revealed that after the zebrafish caudal fin injury,by using morphological and gene expression analyses and compared experimental group with the control,we get the relationship between related-genes.It helped us elucidate the molecular mechanism of caudal fin regeneration.These results also provide a certain amounts of data evidences for the regulation relationship between genes and unlimited fin regeneration about zebra-fish caudal fin regeneration.