人真皮原代成纤维细胞的培养、鉴定及蛋白酶活性受体的表达

目的探索和建立人真皮原代成纤维细胞培养并鉴定纯度,检测蛋白酶活性受体在该细胞的表达状态。方法用胰酶消化法分离人包皮成纤维细胞并培养人真皮原代成纤维细胞,用免疫细胞化学法鉴定纯度;用RT-PCR、流式细胞分析和免疫细胞化学染色法检测蛋白酶活性受体的表达。结果胰酶消化法成功培养出人真皮原代成纤维细胞,纯度达99%;人真皮成纤维细胞高表达PAR1、PAR3 mRNA及蛋白,微弱表达PAR2 mRNA及蛋白,不表达PAR4 mRNA及蛋白。结论用胰酶消化法成功培养高纯度原代人真皮成纤维细胞;蛋白酶活性受体1、3在人真皮成纤维细胞上高表达,提示可能存在于炎症及损伤修复功能作用,为进一步研究打下基础。

Expression of proteinase activated receptors from primary cultured human dermal fibroblast

Objective To explore and establish the process of culturing primary human dermal fibroblasts (HDFs) and examine which protease activity receptor (PARs) are expressed on the cells. Methods Human dermal fibroblasts were isolated from prepuce by enzymatic digestion with 0.25% trypsin. The purity of the cell, which was assessed by a typical spindle-like configuration, was determined by indirect immunofluorescent staining with a mouse anti-human fibroblast monoclonal antibody. PARs in HDFs were detected by flow cytometry, RT-PCR and immunocytochemistry. Results The isolated and cultured cells were identified as fibroblasts. The purity of HDFs was at least 99%. Primary cultured HDFs express PAR-1 and PAR-3 mRNA and molecule. Expression of PAR2 on HDFs was weak and no any PAR-4 was expressed on the cells. Conclusion Purity of primary HDFs can be cultured by enzymatic digestion. The expression of PAR-1 and PAR-3 in HFDs indicate that PARs may play a functional role in HDFs.