类风湿关节炎滑膜CD68阳性细胞筛选的意义

Isolation of CD68-positive synoviocytes of rheumatoid arthritis

目的:探讨用CD68作为细胞标志筛选类风湿关节炎和正常滑膜组织中的滑膜巨噬细胞CD68+细胞,并观察筛选后细胞的活性及特性。方法:滑膜组织来源于2003-02/2004-03南方大学南方医院脊柱骨病科收治的12例住院患者。类风湿关节炎组6例源自全膝关节置换术或经关节镜滑膜切除术的类风湿关节炎患者,正常滑膜组6例来自无关节炎病史的半月板损伤患者,均经病理诊断证实。从滑膜组织中分离滑膜细胞,免疫磁珠法筛选滑膜CD68+和CD68-细胞,用流式细胞仪检测筛选效能;用免疫组化染色观察类风湿关节炎组和正常滑膜组筛选前后滑膜CD68+细胞所占比例;以四甲基偶氮唑盐比色法观察筛选后细胞体外培养增殖活性。结果:所获12例滑膜组织全部进入结果分析。①滑膜细胞分离后观察:滑膜细胞24h后贴壁生长,滑膜巨噬样细胞在类风湿关节组的比例较正常滑膜组高。②筛选后滑膜细胞的观察结果:免疫磁珠法筛选的CD68+和CD68-细胞均在2h内开始贴壁,24h后CD68-细胞呈极向排列生长,贴壁能力强于CD68+细胞。筛选后的滑膜CD68+细胞与磁珠结合在一起,具有巨噬样细胞形态,胞周可有突起,分裂增殖较慢,但在体外能传数代;CD68-细胞未与磁珠结合,表现为长梭形成纤维细胞样形态,细胞分裂增殖迅速。③CD68+细胞的纯度分析:经流式细胞仪分析细胞纯度为93.6%,主峰右移,细胞平均体积增大。滑膜CD68-细胞不能被FITC标记。④滑膜细胞CD68的表达观察:筛选后CD68+细胞均为棕黄色。细胞核大、较疏松,细周有突起。CD68-细胞基本为成纤维样细胞,胞浆未被黄染,细胞核位于细胞中央,呈卵圆形,核仁明显。⑤两组滑膜细胞体外增殖速度:在体外均有不同程度增殖活性。类风湿关节炎和正常滑膜CD68-细胞增殖速度快于同一组织来源滑膜CD68+细胞的增殖速度[(0.680±0.047),(0.236±0.014);(0.605±0.031),(0.195±0.019);t=21.989,P<0.05]。类风湿关节炎滑膜CD68+细胞增殖速度快于正常滑膜CD68+细胞的增殖速度[(0.236±0.014),(0.195±0.019);t=4.149,P<0.05]。结论:CD68可作为一种标志来筛得滑膜细胞中的巨噬细胞和其他滑膜细胞,筛选后滑膜细胞均具有良好增殖活性。CD68+细胞具有典型巨嗜样细胞形态,CD68-细胞主要为成纤维样细胞,两种细胞区分程度好有助于临床对疾病活动期和严重程度判断。

AIM: To isolate the synovial macrophage CD68+ synoviocytes from rheumatoid arthritis(RA) synovial tissue and normal synovial tissues using CD68 cell as the cell marker, and observe the activity and characters of the isolated synoviocytes. METHODS: Synovium was obtained from 12 patients hospitalized in the Department of Orthopaedics and Spine Surgery, Nanfang Hospital, Southern Medical University between February 2003 and March 2004. Six RA patients following total knee joint replacement or arthroscopic synovectomy were selected for the RA group, and 6 patients with meniscus injury but without history of arthritis were for the normal synovium group. All the patients were verified pathologically. Synoviocgtes were isolated from synovial tissues. CD68+ and CD68- synoviocytes were sorted from synovial cells by immunomagnetic beads, and screened in a flow cytometer. Immunohistochemistry was employed to observe the ratio of CD68+ synoviocytes from rheumatoid arthritis and normal synovium before and after screening. The activities of proliferation of all types of cells cultured in vitro were assessed by the methyl-thiaxolyl-tetvaxolium assay after screening. RESULTS: All the 12 cases of synovial tissues were analyzed in the result. ①Observation after separation of synoviocytes: The synoviocytes grew on the wall after 24 hours, and macrophage-like synoviocytes were more in the RA group than in the normal synovium group. ②Observation after screening of synoviocytes: The CD68+ and CD68- synoviocytes screened by immunomagnetic beads began to grow on the wall within 2 hours. After 24 hours, CD68- synoviocytes grew in the same direction, and their adhesive ability was stronger than the CD68+ synoviocytes'. The screened CD68+ synoviocytes combined with beads in macrophage-like forms with processes around them. The division and proliferation of CD68+ synoviocytes was slow, but could passage in vitro again and again; CD68- synoviocytes did not combine with beads, in a fibrocyte-like form with long fusiform shape, but the division and proliferation of them was fast. ③The purity of CD68+ synoviocytes was 93.6% by flow cytometry. The main peak of them moved right, and the mean volume was enlarged. The CD68- synoviocytes could not be labeled by fluorescein isothiocyanate(FITC). ④Observation on the expression of CD68 synoviocytes: The screened CD68+ synoviocytes were yellow buffy, and nuclei were big and rare with processes around them. CD68- synoviocytes were nearly fibroblast-like and their plasm was not stained yellow. The nuclei located in the central cells in a elliptical form and the nucleoli were obviously found. ⑤The proliferation speed of synoviocytes in the two groups: The synoviocytes proliferated in vitro differently. In the RA and normal synovium group, the proliferation speed of CD68+ synoviocytes(0.680±0.047,0.236±0.014) was faster than that of CD68- synoviocytes(0.605±0.031, 0.195±0.019)(t=21.989, P < 0.05), and the proliferation speed of CD68+ synoviocytes was faster than that of CD68- synoviocytes in the RA group (t=4.149, P < 0.05). CONCLUSION: CD68 can be used as a marker to screen the macrophages of synovial tissue and other synoviocytes, and the screened synoviocytes are of well-proliferating ability. CD68+ synoviocytes are typically macrophage-like, and CD68- synoviocytes fibroblast-like, so both CD68+ and CD68- synoviocytes are beneficial to the clinical judgment of active phase and severity of RA attributing to their easy-screening.