心脏成纤维细胞生物表型多样性

Multiple phenotypes in cardiac fibroblasts

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。

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 ＋/sea-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.