心脏成纤维细胞生物表型多样性

目的 探讨心脏成纤维细胞（CFs）生物表型多样性。方法 新生1～3天SD大鼠12只，胰蛋白酶/胶原酶联合消化法分离培养新60只生大鼠CFs，比较原代和传代培养细胞形态学变化，免疫组织化学学检测鉴定常见成纤维细胞标记波形蛋白（vimentin）、成纤维细胞特异性蛋白1（FSP1）和盘状结构域受体（DDR2）的表达，细胞免疫荧光检测CFs干细胞标记nanog、c-kit、sca-1、CD73和CD90的表达，细胞免疫荧光三标技术检测CFs干细胞标记间的共表达情况。 结果 酶联合消化法分离培养的CFs收获细胞量大，第3代细胞活力好、纯度高。Vimentin、FSP1和DDR2在CFs中均表达，但DDR2在CFs中呈强表达。部分CFs分别表达nanog、c-kit、sca-1、CD73和CD90，部分nanog阳性的CFs分别和CD73、CD90和sca-1，及其CD90和sca-1存在共表达，nanog⁺ /CD73⁺共表达阳性率最高（59.02%±8.39%），与其他3组相比差异均有极显著性（P<0.01）；而 nanog⁺ /CD90⁺共表达阳性率与nanog⁺ /sca-1⁺之间差异无显著性（P>0.05），但这两组与CD90⁺ /sca-1⁺相比差异均有极显著性（P<0.01）；CD90⁺ /sca-1⁺共表达阳性率均低于其他3组，差异具有极显著意义（P<0.01）。 结论 CFs是具有干细胞特征混合细胞群，在表型上具有多样性。

Multiple phenotypes in cardiac fibroblasts

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.