| Objectives:(1)To investigate the regeneration of Xenopus tropicalis and Xenopus laevies-albino heart after the apical resection(approximately 10%).(2)To construct the Dendra2-tagged transgenic Xenopus tropicalis.Methods:(1)Establish the model of heart injury and regeneration in Xenopus tropicalis and Xenopus laevies-albino via resecting the ventricle at the cardiac apex and then carry out the following investigations.(1)Observation of the regeneration of the cardiac myocardium at different time points after the apical resection using hematoxylin-eosin staining and Masson's Trichrome staining.(2)Investigation of proliferation of cardiomyocytes near the wound using immunofluorescence staining for PH3 and alpha skeletal muscle actin(?-SA)(3)Transmission electron microscopy for the distribution of blood vessels,cardiac Telocytes and collagen in 2-day and 8-day after apex amputation and normal group.(2)Construct the vectors of p BS-CMVDendra2 and p BS-Flk-Dendra2 using molecular cloning techniques and establish transgenic Dendra2 Xenopus tropicalis via microinjection manipulation.Results:(1)The ventricle at the cardiac apex,which approximately occupied 10% of the Xenopus tropicalis heart,was removed.Around two-thirds of the amputated Xenopus tropicalis heart had regenerated with a perfect heart-shaped morphology in 30 days after apical resection(daar).The regenerated hearts were scar-free and were without adhesion between the regenerated sites and the peripheral tissues.However,approximately one-third of the amputated Xenopus tropicalis hearts had regenerated with a nearly heart-shaped morphology,which was scar-free but with adhesion between the regenerated sites and the peripheral tissues.(1)H&E staining revealed that there were some red blood cells and inflammatory cells on the proximal surface of the wound in 0 daar.The wound site was filled with red blood cells and inflammatory cells,both of which gradually increased from 0 daar to 4 daar.In 4 daar,the density of red blood cells significantly decreased while the highest inflammatory cell infiltration was observed.However,in 8 daar,most of the infiltrated red blood cells and inflammatory cells disappeared from the scar tissue in the regenerated area.Additionally,novel cardiomyocytes with irregular cross-striated structures were found at the border of the regenerated area.In 16 daar,most of the scar tissue was disappear.For 30 daar and 60 daar,two thirds of the amputated hearts had regenerated with a perfect morphology which is similar to normal hearts.However,one third of injured heart regenerated with a nearly perfect morphology.Same,in 70 daar,the vast majority of amputated hearts had regenerated with a perfect morphology.(2)Masson's Trichrome staining showed that a fibrosis-like structure was present and gradually increased in scar tissue from 0 daar to 8 daar.The highest intensity of fibrosis was found from 4 daar to 8 daar,after that fibrosis decreased progressively from 16 to 30 daar.In 30 daar,the fibrotic structure was rarely observed in the fully regenerated myocardium.In addition,in nearly heart-shaped regenerated hearts,fibrotic tissue was only found in the peripheral adhesion tissue.(3)Immunofluorescence staining for PH3 and a-SA indicated that the density of PH3+/a-SA+-positive cells was significantly increased near the wound area between 0 daar and 16 daar.The peak of PH3+/a-SA+-positive cells was observed in 16 daar which was 27 times higher than the basal level in the normal heart.From 30 daar to 70 daar,the density of PH3+/a-SA+-positive cells in the regenerated myocardium had significantly reduced and similar to the normal control.In 70 daar,the density of PH3+/a-SA+-positive cells was 4 times as high as the basal level.(2)After apical resection(the resection area approximately occupied 10% of the Xenopus laevisalbino heart),half of the Xenopus laevis-albino had regenerated with a perfect heart-shaped and scar-free morphology in 160 daar.The other half of the Xenopus laevis-albino had regenerated with only a nearly heart-shaped morphology with adhesion between the regenerated sites and peripheral tissues.(1)H&E staining demonstrated that red blood cells and inflammatory cells gradually increased from 0 daar to 4 daar in the wound sites.In 4 daar,the density of red blood cells significantly decreased,and the number of inflammatory cells reached the highest level.Most of the infiltrated red blood cells disappeared from the scar tissue in the regenerated area in 8 daar while inflammatory cells disappeared in 16 daar.Additionally,neonatal cardiomyocytes were found at the border of the regenerated area after 8 daar.In 30 daar,an intact epicardium regenerated in the regenerated myocardium.Until 160 daar,half of the amputated hearts had regenerated with a perfect morphology,while the rest of the resected heart regenerated with a nearly perfect morphology.(2)Masson's Trichrome staining suggested that fibrosis gradually increased in the scar tissue from 0 daar to 8 daar.The highest intensity of fibrosis was found in 8 daar,after that fibrosis decreased progressively from 16 daar to 160 daar.In 160 daar,the fibrotic structure nearly disappeared in the fully regenerated myocardium.(3)The vector of p BS-CMV-Dendra2 can express green fluorescence in 293 T cells,embryos,tadpoles.The F0 Dendra2 transgenic Xenopus tropicalis was constructed.Conclusion:(1)In 30 days after apical resection,on the condition which there was no inflammatory adhesion,the amputeted Xenopus tropicalis heart is able to regenerate with a perfect heart-shaped morphology,which is scar-free and without adhesion between the regenerated site and peripheral tissues.However,if there is inflammatory adhesion after the surgery,the injured heart only regenerated with a nearly heart-shaped morphology,which is scar-free but with adhesion between the regenerated site and peripheral tissues.(2)The regeneration of the heart in Xenopus laevie-albino after apical resection(approximately 10%)need longer time to regenerate compared with compared with Xenopus tropicalis heart.(3)One of the possible mechanisms for the regeneration of amputated adult Xenopus tropicalis heart was attributed to the proliferation of normal cardiomyocytes near the wound area.(4)The Dendra2-tagged transgenic Xenopus tropicalis(F0)was constructed successfully. |
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