外周血CD34^+细胞形貌在系统性红斑狼疮患者与健康人的差异比较

Differences in peripheral blood CD34^+ cell morphology between systemic lupus erythematosus patients and healthy controls

背景:系统性红斑狼疮患者CD34+细胞是否存在异常目前仍有争议。目的:以健康人为对照,应用原子力显微镜观察比较二者外周血CD34+细胞形貌特征。设计:病例-对照分析。对象:6例系统性红斑狼疮患者,男3例,女3例,平均年龄26.3岁,平均病程19.0个月,均符合1982年美国风湿病学会制定的诊断标准。3例健康志愿者为对照。两组对象基线资料具有可比性。方法:系统性红斑狼疮患者均静脉注射环磷酰胺4g/m2,白细胞<1.0×109L-1时给予粒细胞集落刺激因子5μg/kg,待白细胞>5.58×109L-1及CD34+>2%时采集外周血干细胞。3例健康志愿者采取单用粒细胞集落刺激因子为动员方案。向外周血干细胞中加入CD34MicroBeads FcR和FcR Blocking Reagent制备细胞悬液,离心弃上清,重悬后过滤,应用免疫磁珠细胞分选技术纯化CD34+细胞。主要观察指标:原子力显微镜观察细胞表面形貌,每例患者随机测20个细胞,每个细胞不同部位做2μm×2μm图像5幅,将5幅图像的直径、平均粗糙度、均方根粗糙度、平均高度的均值作为该细胞的参数。结果:成像范围在12μm×12μm时,可将细胞整体模拟成像。成像范围在2μm×2μm时,两组细胞表面均可见大小、深浅不一的凹陷及椭圆形球状突起,粗糙不平。与健康志愿者比较,系统性红斑狼疮患者外周血CD34+细胞的直径、平均粗糙度、均方根粗糙度、平均高度等参数经单向方差分析差异无显著性意义(F=0.203~4.553,P均>0.05)。结论:应用原子力显微镜选择成像范围在2μm×2μm时可清晰观测外周血CD34+细胞的表面结构,且系统性红斑狼疮患者和健康人的外周血CD34+细胞形貌基本相似。

BACKGROUND： There is a debate addressing the existence of CD34^＋ cells in systemic lupus erythematosus patients. OBJECTIVE： To observe the morphology of CD34^＋ cells from peripheral blood of systemic lupus erythematosus patients versus health human by atomic force microscopes. DESIGN： A case-controlled analysis. PARTICIPANTS： Six systemic lupus erythematosus patients （3 male and 3 female）, averagely aged 26.3 years old were selected. The disease course was averagely 19.0 months. They were accorded with the diagnostic criteria made by America Rheumatology Association in 1982. Three healthy volunteers were used as controls. Baseline data had comparability in both groups. METHODS： Systemic lupus erythematosus patients were injected with 4 g/m^2 cyclophosphamide via intravenous injection. When leukocytes reached 〈 1.0×10^9/L, granulocyte colony-stimulating factor （5 ug/kg） was administered. When leukocytes reached 〉 5.58×10^9/L and CD34^＋〉2%, peripheral blood stem cells were collected. Three healthy volunteers were subjected to mobilization of granulocyte colony-stimulating factor alone. CD34 MicroBeads FcR and FcR Blocking Reagent were added into the peripheral blood stem ceils, which was made into cell suspension. Supematant was removed. After resuspension, samples were filtered. CD34^＋ cells were purified by immunomagnetic bead cell sorting. MAIN OUTCOME MEASURES： Atomic force microscopes was used to observe the surface of the CD34^＋ cells. Twenty cells were observed randomly in two groups. The average of some parameters such as diameter, mean square root roughness, mean height and roughness of five images （2 um × 2 u m） in each cell represented as the whole cell. RESULTS： When the scope of images were 12 um×12 u m, the whole cells could be imaged. When the scope of images were 2 u m×2 u m, there were introcession of different sizes, oval process, and rough structure on the surface of the CD34^＋ cells. Compared with the healthy patients, there were no significant differences in the diameter, mean roughness, mean square root roughness, and mean height in systemic lupus erythematosus patients （F=0.203-4.553, P 〉 0.05）. CONCLUSION： Atomic force microscopes can map the surface of CD34^＋ cells at image range of 2 u m×2 u m. There are no significant differences in CD34^＋ cell morphology between systemic lupus erythematosus patients and health human.