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目的:分离培养类风湿关节炎(RA)滑膜细胞、检测其表面标志及研究其对软骨的侵蚀特性。方法:取6例RA患者滑膜,分别采用组织块、胰蛋白酶消化及胶原酶、胰蛋白酶先后消化培养后,用相差显微镜观察细胞的形态、用流式细胞术(FCM)检测培养细胞表面标记CD3、CD19、CD14、CD68、CD44、CD55、CD90及细胞内脯氨酰-4-羟基化酶(Prolyl-4-hydroxylase)的表达并与传统的免疫组化染色法检测波形蛋白的表达相比较。并用SCID小鼠研究成纤维样滑膜细胞(FLS)对软骨的侵蚀作用。结果:(1)3种方法均分离出FLS,胶原酶与胰蛋白酶先后消化法分离细胞所需时间少、获得的细胞数多、纯度较高。(2)第2代细胞与第3代细胞的均质性分别达90%及98%以上。(3)在第3~6代的细胞比较稳定,增殖活跃;第7代以后增长缓慢,逐渐老化。(4)流式细胞术分析显示,原代细胞随着传代次数的增加逐渐纯化,第3~6代CD90+细胞>98%,脯氨酰4-羟基化酶+细胞>98%;与免疫组化检测波形蛋白的表达相一致。(5)将第4~5代的细胞和软骨一起植入SCID小鼠,或单独注入SCID小鼠的膝关节,都可导致软骨的浸蚀性破坏。结论:以胶原酶与胰蛋白酶先后消化分离FLS,效果较好,CD90、脯氨酰4-羟基化酶可作为鉴定FLS的标志。用SCID小鼠可研究FLS对软骨的侵蚀特性。
OBJECTIVE: To isolate and culture rheumatoid arthritis (RA) synoviocytes, detect their surface markers and study their erosive properties on cartilage. Methods: Synovial membrane was taken from 6 cases of RA patients. Tissue blocks, trypsin digestion and collagenase and trypsin digestion and culture were performed respectively. The morphology of the cells was observed by phase contrast microscopy. The surface markers of cultured cells were detected by flow cytometry (FCM) CD3, CD19, CD14, CD68, CD44, CD55, CD90 and Prolyl-4-hydroxylase expression compared with conventional immunohistochemical detection of vimentin expression . SCID mice were used to study the erosive effect of fibroblast-like synoviocytes (FLS) on cartilage. Results: (1) FLS were isolated from all three methods. The separation of collagenase and trypsin by digestion method required less time, more cells were obtained and purity was higher. (2) The second generation of cells and third generation of cells were homogeneity of 90% and 98% respectively. (3) The cells in the 3rd to 6th generation were stable and proliferated; the growth was slow and gradually aged after the 7th generation. (4) Flow cytometry analysis showed that the primary cells were gradually purified with the increase of the number of passages, the percentage of CD90 + cells was 98% in the 3rd to 6th generation and> 98% in the Prolyl 4-hydroxylase + cells; The detection of vimentin expression is consistent. (5) The fourth to fifth generation of cells and cartilage implantation with SCID mice, or SCID mice injected into the knee, can cause erosion of cartilage destruction. Conclusion: The digestion and separation of FLS by collagenase and trypsin is better, and CD90 and prolyl 4-hydroxylase can be used as markers to identify FLS. SCID mice can be used to study FLS erosion of cartilage characteristics.