RESUMO
Sinoatrial node (SAN) is the pacemaker of the heart in charge of initiating spontaneous electronical activity and controlling heart rate. Myocytes from SAN can generate spontaneous rhythmic action potentials, which propagate through the myocardium, thereby triggering cardiac myocyte contraction. Acutely, the method for isolating sinoatrial node myocytes (SAMs) is critical in studying the protein expression and function of myocytes in SAN. Currently, the SAMs were isolated by transferring SAN tissue directly into the digestion solution, but it is difficult to judge the degree of digestion, and the system was unstable. Here, we present a modified protocol for the isolation of SAMs in mice, based on the collagenase II and protease perfusion of the heart using a Langendorff apparatus and subsequent dissociation of SAMs. The appearance and droplet flow rate of the heart could be significantly changed during enzymatic digestion via perfusion, which allowed us to easily judge the degree of digestion and avoid incomplete or excessive digestion. The SAMs with stable yield and viability achieved from our optimized approach would facilitate the follow-up experiments.
Assuntos
Separação Celular , Miócitos Cardíacos , Nó Sinoatrial , Animais , Nó Sinoatrial/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Separação Celular/métodos , Camundongos , Camundongos Endogâmicos C57BL , Masculino , PerfusãoRESUMO
BACKGROUND: Rhododendron pudingense, firstly discovered in Puding county of Guizhou province in 2020, have adapted to living in rocky fissure habitat, which has important ornamental and economic values. However, the genetic diversity and population structure of this species have been rarely described, which seriously affects the collection and protection of wild germplasm resources. RESULTS: In the present study, 13 pairs of primers for polymorphic microsatellite were used to investigate the genetic diversity of 65 R. pudingense accessions from six different geographic populations. A total of 254 alleles (Na) were obtained with an average of 19.5 alleles per locus. The average values of polymorphic information content (PIC), observed heterozygosity (Ho), and expected heterozygosity (He) were 0.8826, 0.4501, and 0.8993, respectively, These results indicate that the microsatellite primers adopted demonstrate good polymorphism, and the R. pudingense exhibits a high level of genetic diversity at the species level. The average genetic differentiation coefficient (Fst) was 0.1325, suggested that moderate divergence occurred in R. pudingense populations. The average values of genetic differentiation coefficient and gene flow among populations were 0.1165 and 3.1281, respectively. The analysis of molecular variance (AMOVA) indicated that most of the population differences (88%) were attributed to within-population variation. The PCoA results are consistent with the findings of the UPGMA clustering analysis, supporting the conclusion that the six populations of R. pudingense can be clearly grouped into two separate clusters. Based on Mantel analysis, we speculate that the PD population may have migrated from WM-1 and WM-2. Therefore, it is advised to protect the natural habitat of R. pudingense in situ as much as possible, in order to maximize the preservation of its genetic diversity. CONCLUSIONS: This is the first comprehensive analysis of genetic diversity and population structure of R. pudingense in Guizhou province. The research results revealed the high genetic diversity and moderate population diferentiation in this horticulture plant. This study provide a theoretical basis for the conservation of wild resources of the R. pudingense and lay the foundation for the breeding or cultivation of this new species.