Abstract:

Objective To explore the multiple phenotypes in cardiac fibroblasts (CFs). Methods CFs of neonatal rats were isolated from twelve 1-3-day neonatal SD rats and cultured using combined type I collagenase(w/v, 0.1%) trypsin (w/v, 0.25%) digestion method . Morphological characteristics were observed between primary and subcultured CFs. Common molecular markers for fibroblasts such as vimentin, fibroblast specific protein(FSP1) and discoidin domain receptors 2(DDR2) were detected by immunohistochemical staining. Nanog, c-kit, sca-1, CD73 and CD90 recognized as stem cell markers were evaluated in CFs by immunofluorescence staining, and co-expression of those markers was investigated by three-color immunofluorescence technology. Results By the combined enzyme digestion method , the isolated fibroblasts had large volume. Passage 3 cells exhibited good biological activity, fast proliferation and high purity. Vimentin, FSP1 and DDR2 were all expressed and DDR2 was strongly expressed in CFs. Nanog, c-kit, sca-1, CD73 and CD90 were detected in some CFs. Interestingly, CD73, CD90 and sca-1 were co-expressed with nanog positive CFs respectively and the result showed the positive expression rate of nanog⁺ /CD73 ⁺ was the highest than other three groups (59.02%±8.39%). It had significant different (P<0.01). while the positive rate of nanog⁺ /CD90⁺ in CFs had no different with the rate of nanog⁺ /sca-1⁺ (P>0.05). And the rate of nanog ⁺ /CD90⁺ and nanog⁺ /sca-1⁺had extremely significant different with the rate of CD90⁺ /sca-1⁺ (P<0.01). But the positive rate of CD90⁺ /sca-1⁺ was significantly lower than that in the other three groups (P<0.01). Conclusion CFs have several subpopulations with stem cell characteristics and multiple phenotype.

Key words: Cardiac fibroblast, Stem cell marker, Cell culture, Immunohistochemical staining, Neonatal rat