脂多糖诱导SD大鼠膝关节成纤维样滑膜细胞炎症模型建立及特征分析

Establishment and characteristic analysis of a model of knee fibroblast-like synoviocytes inlipopolysaccharide-induced Sprague-Dawley rats

目的建立Sprague Dawley (SD)大鼠的正常膝关节滑膜细胞原代培养方法及脂多糖(Lipopolysaccharides,LPS)诱导成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)炎症模型。方法选取80~120 g SPF级幼年SD大鼠,分离其滑膜组织,原代培养至第3代,用组织化学染色法和Ed U法检测FLS蛋白标志物vimentin和增殖功能。同时,用滑膜组织作对照,检测第3代至第8代原代培养FLS的特征蛋白表达,筛选具有生理功能的高纯度且可用于后续实验的FLS细胞。LPS诱导FLS炎症后,检测LPS制激FLS不同时间点的IL-1β和TNF-α的mRNA和蛋白表达,根据实验结果确定LPS成功诱导FLS炎症模型的时间点。最后,检测FLS被LPS诱导前后的细胞因子、增殖功能和特征蛋白的表达,为分析FLS炎症模型提供实验数据。结果采用0.2%I型胶原酶消化法,成功培养了FLS原代细胞。经检测蛋白标志物vimentin,第3代FLS纯度达98%以上。通过FLS特征蛋白检测,筛选出具有生理功能且可用作后续实验的FLS为第3代至第7代原代细胞。通过对LPS诱导后的FLS的细胞因子和特征蛋白分析,确定1μg/m L LPS诱导FLS细胞3 h,能复制出FLS炎症模型。结论 LPS诱导的FLS炎症模型可作为体外研究炎性关节病的细胞模型。

Objective To establish synovial cell primary cultures and a lipopolysaccharide( LPS)-induced fibroblast-like synoviocyte( FLS) inflammatory model of a normal knee joint in Sprague-Dawley( SD) rats. Methods Normal knee joint synovium( 80-120 g) was separated from specific-pathogen-free SD rats in an ice bath with phosphatebuffered saline. The primary synovium was cultured to the 3 rd generation. Vimentin expression and FLS proliferation were detected via immunohistochemical staining and the 5-ethynyl-2’-deoxyuridine method. Synovial tissue was used as a control to detect characteristic protein expressions in the FLS in the 3 rd-8 th generations of primary culturing and to screen the FLS for high purity and physiological functions for use in subsequent experiments. After LPS-induced FLS inflammation,the mRNA and protein expressions of IL-1β and TNF-α were detected at different time points to determine the time point at which LPS could successfully induce an FLS inflammatory model. Finally,the cytokine expression,proliferative functions and characteristic proteins of the FLS before and after LPS induction were detected to provide experimental data to evaluate the FLS inflammatory model. Results FLS primary cells were successfully cultured with 0. 2% collagenase I digestion. The FLS purity in the 3 rd generation exceeded 98% for detecting vimentin. By detecting characteristic FLS proteins,FLS with physiological functions that could be used for subsequent experiments were selected as the primary cells from the 3 rd-7 th generations. Analyzing the cytokines and characteristic proteins in FLS before and after LPS induction revealed that1 μg/m L LPS is required to stimulate FLS cells for 3 h to replicate the FLS inflammatory model. Conclusion The LPSinduced FLS inflammatory model can be used to study inflammatory arthritis in vitro.