Regulatory T Cells Regulate Myocardial Regeneration In Neonatal Mice

Objective:To investigate the role of regulatory T cells and its mechanism of action in the regeneration of myocardial injury in neonatal mice.Methods:1.A model of myocardial regeneration in neonatal mice was established.The experimental group underwent apical resection and the sham operation group only performed thoracotomy.The immunofluorescence co-staining of pH3 and Ki67 with cardiomyocyte cytoplasma antigen?-actinin was used to observe cardiomyocyte proliferation at seven days post surgery.2.Myocardial regeneration was observed by Masson staining at 21 days post surgery,.3.The expression of Foxp3 protein in heart and spleen was detected by Western blotting at 7,14 and 21 days post surgery,the enrichment of T_reg cells after apical resection was detected by immunohistochemistry at the same time points.4.The number of T_reg cells in heart tissue and spleen tissue was measured by flow cytometry at seven days post surgery.5.Foxp3^DTR newborn mice were injected with DT to delete T_regeg cells,real-time quantitative PCR was used to detect the expression of anti-inflammatory factors?IL-10,IL-13 and TGF-??,as well as pro-inflammatory factors?IL-6,IL-1?and TNF-??at seven days post resection.6.The effect of knocking T_reg cells on cardiomyocyte proliferation after myocardial injury in neonatal mice was detected by immunofluorescence at seven days post surgery.7.The number of T_reg cells was detected by flow cytometry after T_regeg cells knockout at 7 days post surgery.The influence exerted by T_reg cells knockout on regeneration of myocardial injury in neonatal mice was observed by Masson staining at21 days post surgery.Results:1.Immunofluorescence staining demonstrated that the number of pH3⁺cardiomyocytes and Ki67⁺cardiomyocytes in AR group was significantly increased.2.Masson staining showed that the myocardium fully regenerated at 21 days post resection?dpr?.3.Western blotting results showed that the expression of Foxp3 in heart and spleen of AR group was significantly higher than that in SH group at 7 and 14 days post surgery?P<0.05?.We also applied immunohistochemical staining for Foxp3,and the results revealed that a large amount of T_reg cells were enriched in the apex of AR group compared with the SH group at 7 and 14 days post surgery.4.The number of T_reg cells measured by flow cytometer showed that T_regeg cells was increased in AR group than that in SH group?P<0.01?.5.We found that the expression of anti-inflammatory factors?interleukin-10,interleukin-13,transforming growth factor-??was reduced?P<0.05,P<0.01,P<0.01?,while the expression of pro-inflammatory factors?interleukin-6,interleukin-1?and tumor necrosis factor-??was increased?P<0.01,P<0.001,P<0.01?using real-time quantitative PCR when T_reg cells were deleted.6.Immunofluorescence co-staining of pH3 and Ki67 with a-actinin indicated that the number of pH3⁺cardiomyocytes and Ki67⁺cardiomyocytes in AR+DT hearts was significantly reduced relative to the AR+PBS hearts at 7 dpr.7.The number of T_reg cells in heart tissue and spleen tissue measured by flow cytometer showed that T_regeg cells in AR+DT group was significantly decreased relative to the AR+PBS group?P<0.01,P<0.05?.Masson staining also showed that the myocardium could not regenerated in AR+DT group at 21 dpr.Conclusions:1.A model of apical resection in neonatal mice was established successfully.2.T_regeg cells participate in the regulation of myocardial regeneration after AR in neonatal mice.3.Deletion of T_reg cells aggravates the inflammatory response,inhibits cardiomyocyte proliferation,and impairs myocardial regenerative capability in neonatal mice after AR.