系统性硬皮病(systemic scleroderma，SSc)是以皮肤硬化和内脏器官纤维化为特征的结缔组织病，目前该病发病机制尚不清楚，成纤维细胞胶原合成异常增多是导致疾病发生的重要环节。 SSc患者和正常人皮肤成纤维细胞(fibroblasts)在细胞形态、生长特性和胶原代谢方面均具有异质性(heterogeneity)。SSc患者皮肤成纤维细胞中具有高胶原合成能力的细胞比例远比正常人皮肤成纤维细胞高，但原因尚不清楚。以整群成纤维细胞研究SSc患者皮肤成纤维细胞高胶原合成克隆的形成机制较为困难，也无法对影响因素进行细致的分析。采用克隆成纤维细胞来研究这一问题，探讨这些异质性克隆的细胞形态、生长特性和胶原代谢的特点，关注TGFβ1、 CTGF和IFN-γ对SSc患者和正常人具有不同胶原合成能力的成纤维细胞克隆胶原代谢的影响，有助于了解SSc患者皮肤成纤维细胞高胶原合成克隆的形成机制。 首先，分离SSc患者和正常人皮肤成纤维细胞克隆，观察其形态和生长异质性。采用改良的有限稀释法(limiting dilution assays)，分离皮肤成纤维细胞克隆，光镜下观察克隆成纤维细胞的细胞形态，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。结果显示：SSc患者和正常人皮肤成纤维细胞克隆的细胞形态和生长特性均具有异质性，两者形态和生长异质性的特点在克隆水平上未见差异。按细胞形态划分，SSc患者和正常人皮肤克隆成纤维细胞主要有3种：细小梭形细胞、粗长梭形细胞、大多角形细胞。与细胞呈细小梭形的克隆相比，细胞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)。细胞呈大多角形的克隆增殖很慢，难以传代和冻存。SSc患者和正常人皮肤成纤维细胞克隆的细胞形态和生长特性经数次传代保持稳定。 其次，以克隆成纤维细胞作为研究对象，进一步研究SSc患者和正常人皮肤成纤维细胞胶原代谢异质性的特点。采用实时荧光定量RT-PCR法检测其Ⅰ、Ⅲ型前胶原(procol lagen)和基质金属蛋白酶(matrix metalloproteinase， MMP)(MMP-1、MMP-3)mRNA水平；采用免疫印迹法检测其细胞内Ⅰ型前胶原的表达；采用酶联免疫吸附法检测其细胞培养上清中Ⅰ型胶原的表达，筛选出具有不同胶原合成能力的异质性克隆，分析其胶原代谢的特点。结果显示：(1)SSc患者和：正常人皮肤克隆成纤维细胞胶原合成能力均具有异质性，SSc患者克隆成纤维细胞Ⅰ、Ⅲ型前胶原mRNA水平的异质性较正常人大，SSc患者Ⅰ型前胶原mRNA高表达克隆的比例高于正常人(48.0% vs 14.3%)。(2)不同细胞形态(细小和粗长梭形)的克隆成纤维细胞Ⅰ型前胶原mRNA高、中、低表达组的总体构成比未见不同(P＞0.05)。(3)SSc患者和正常人克隆成纤维细胞细胞培养上清中Ⅰ型胶原蛋白水平与其mRNA水平之间均存在正相关关系。 最后，观察多种细胞因子对SSc患者和正常人皮肤成纤维细胞胶原合成异质性克隆胶原代谢的影响，以实时荧光定量RT-PCR法检测细胞因子对其Ⅰ型前胶原和MMP-1 mRNA水平的影响。结果显示：TGF β1、CTGF和IFN-γ对SSc患者和正常人具有不同胶原合成能力的克隆成纤维细胞Ⅰ型前胶原、MMP-1 mRNA水平的影响具有不尽相同的特点：(1)对于SSc患者克隆成纤维细胞，TGFβ1 (5ng/ml)作用48小时仅使Ⅰ型前胶原mRNA低表达组的Ⅰ型前胶原mRNA的水平上升； CTGF(20ng/ml)作用48小时，仅下调其具有高胶原合成能力的克隆的MMP-1 mRNA水平。(2)对于SSc患者克隆成纤维细胞，其Ⅰ型前胶原mRNA的水平对CTGF的反应下降。(3)IFN-γ(100ng/ml)作用48小时同时下调SSc患者和正常人克隆成纤维细胞Ⅰ型前胶原、MMP-1 mRNA水平，对于SSc患者克隆成纤维细胞，其“优先”下调具有高胶原合成能力的克隆的MMP-1 mRNA水平。 综上所述，SSc患者和正常人皮肤成纤维细胞克隆的细胞形态、生长特性和胶原代谢均具有异质性，细胞因子TGF β1、CTGF和IFN-γ对SSc患者和正常人具有不同胶原合成能力的克隆成纤维细胞Ⅰ型前胶原、MMP-1 mRNA水平的影响具有不尽相同的特点，提示异常的细胞外微环境可能参与SSc患者皮肤成纤维细胞高胶原合成克隆的形成，有助于进一步了解SSc胶原代谢异常的机制。 关键词 系统性硬皮病 成纤维细胞 克隆细胞 异质性 有限稀释法 前胶原 基质金属蛋白酶 转化生长因子-β 结缔组织生长因子 干扰素 中图分类号：R75

 Systemic scleroderma (SSc) is a connective tissue disease with unknown etiology characterized by fibrosis of skin and other internal organs. Fibroblasts isolated from SSc lesions and cultured in vitro are characterized by increased synthesis of collagen and other extracellular matrix proteins. Among skin fibroblasts derived from controls and patients with SSc, there is marked heterogeneity in morphology, proliferation and collagen metabolism. SSc favors high-collagen-producing skin fibroblast clones with unknown reason. It is difficult to study the mechanism and to analyze the results with fibroblast populations. Focusing on clonal heterogeneity of fibroblasts in morphology, growth characteristics, the synthesis of collagen and the effects of cytokines on collagen metabolism, this study may help to better understand the mechanism of SSc-derived high-collagen-producing fibroblast clones. Firstly, control and SSc-derived skin fibroblast clones were isolated by modified limiting dilution assays, and the morphology and growth characteristics of fibroblasts in clones were observed. Cloned fibroblasts' cell morphology and proliferation were evaluated by light microscopy and MTT assay, respectively. The distribution of cloned cells in different phases of the cell cycle was assessed by flow cytometer. fibroblasts in both control and SSc-derived clones were heterogeneous in their morphology and growth characteristics. There was no difference in their morphology and proliferation characteristics between control and SSc-derived Clones. Clones were divided by cell morphology into three main categories : small spindle shafted, stout spindle-shaped and large polygonal cells. Compared with small spindle shaped cells in clones, stout spindle shaped cells showed slower growth rates, longer doubling time, higher percent age in G₀/G₁ phase (P＜0.05) and lower percentage in S+G₂/M phase (P＜0.05). Clones composed of large polygonal cells grew at considerably reduced rates, and wore not further utilized. Both normal and SSc fibroblasts in clones maintained their phenotypic characteristics of morphology and growth throughout several cell passages. Secondly, in order to study the heterogeneity in collagen metabolism, it was compared between normal and SSc fibroblasts in clones. By means of real-time RT-PCR, Western blotting and ELISA assays, type Ⅰ/Ⅲ procollagen and MMP-1/3 mRNA expressions were determined, the production of type Ⅰ procollagen and type Ⅰ collagen were evulated, so that clones wi th marked heterogeneity in the synthesis of collagen were discriminated. The results showed that: (1) Fibroblasts in both control and SSc-derived clones were heterogeneous in the synthesis of collagen. The hetergeneity of SSc derived clones in type Ⅰ/Ⅲ procollagen mRNA levels were much higher than control -derived clones. Compared with controls, SSc had a larger proportion of high-mRNA-expression clones (48. 0% vs 14.3%). (2)There was no difference in the distribution of high, middle and low collagen producing clones between small spindle-shaped and stout spindle shaped cloned fibroblasts (P＞0.05). (3) The production levels of type Ⅰ collagen had a positivo correlation with their mRNA levels among cloned fibroblasts. Finally, the effects of cytokines on type Ⅰ procollagen and MMP-1mRNA expressions of cloned fibroblasts with heterogeneity in the synthesis of collagen were studied by real-time RT-PCR. The results showed that fibroblasts in both control and SSc-derived clones were heterogeneous in their response to TGFβ1, CTGF and IFN-γ. (1) In SSc derived clones, only fibroblasts in low collagen-producing clones, their mRNA levels of type Ⅰ proeollagen were upregulated by TGFβ1(5ng/ml, for 48 hours), and only fibroblasts in high collagen producing clones, their mRNA levels of MMP 1 were downregulated by CTGF (20ng/ml, for 18 hours). (2) In SSc-derived clones, fibroblasts' type Ⅰ proeollagen mRNA levels had decreased response to CTGF(20ng/ml, for 18 hours). (3)In control and SSc-derived clones, the mRNA expressions of type Ⅰ procollagen and MMP-1 were downregulated by IFN-γ (100ng/m I, for 18 hours); In SSc-derived clones, the downregulation of MMP-1 mRNA levels favored high collagen producing clones. In conclusion, fibroblasts in both control and SSc-derived clones ace heterogeneous in their morphology, growth characteristics, collagen metabolism and response to TGFβ1, CTGF and IFN-γ. This study indicated that high collagen-producing skin fibroblast clones may be due to abnormal extracellular microenvironment around them. Key words: scleroderma, systemic fibroblasts clone cells heterogeneity limiting dilution assays procollagen matrix metalloproteinase transforming growth factor-β connective tissue growth factor interferon CLC number: R75