系统性硬皮病患者皮肤成纤维细胞克隆与生长异质性

Growth Heterogeneity of Skin Fibroblast Clones from Patients with Systemic Sclerolerma

目的分离系统性硬皮病(SSc)和正常皮肤成纤维细胞(FB)克隆,观察其形态和生长特性。方法采用改良的有限稀释法分离SSc和正常皮肤FB克隆,光镜下观察克隆FB细胞形态,应用MTT法绘制细胞生长曲线,流式细胞仪检测细胞周期分布。结果SSc和正常皮肤克隆FB的细胞形态均有异质性,主要分为细小梭形细胞、粗长梭形细胞和大多角形细胞3种。细胞形态呈大多角形的克隆增殖很慢,难以传代和冻存。与细胞形态呈细小梭形的克隆相比,呈粗长梭形的克隆增殖速度较慢,细胞倍增时间较长,G0/G1期细胞比例较高(P<0.05),S+G2/M期细胞比例较低(P<0.05)。SSc和正常皮肤克隆FB的细胞形态和生长特性经数次传代保持稳定。结论SSc和正常皮肤克隆FB的细胞形态和生长特性均有异质性。

Objective To isolate skin fibroblast clones from controls and patients with systemic scleroderma（SSc） and to observe the morphology and growth characteristics of fibroblasts in clones. Methods Control and SSc-derived skin fibroblast clones were isolated by modified limiting dilution assays. Morphology and proliferation of cloned fibroblasts were evaluated by light microscopy and MTT assay, respectively. The distribution of cloned cells in different phases of cell cycle was assessed by flow cytometer. Results Both normal and SSc fibroblasts in clones exhibited phenotypic diversity in their morphologies. Clones were divided by cell morphology into three main categories： small spindle-shaped, stout spindle-shaped and large polygonal cells. Clones composed of large polygonal cells grew at considerably reduced rates, and were not further utilized. Compared with that of small spindleshaped cells in clones, stout spindle-shaped cells showed slower growth rates, longer doubling time, higher percentage in G0/G1 phase （P 〈0.05） and lower percentage in S ＋ G2/M phase （P 〈0.05）. Both normal and SSc fibroblasts in clones maintain their phenotypic characteristics of morphology and growth throughout several cell passages. Conclusion Fibroblasts in both control and SSc-derived clones are heterogeneous in their morphology and growth characteristics.