大鼠中性粒细胞与滑膜细胞粘附模型的建立

ESTABLISHMENT OF A MODEL FOR ADHESION OF POLYMORPHONUCLEAR LEUKOCYTE TO SYNOVIAL CELL

目的:建立大鼠中性粒细胞(PMN)与滑膜细胞(RSC)的粘附模型。方法:MTT比色法。结果:500U·mL-1rhTNFα与IL1刺激RSC12h对PMN有明显的粘附作用,MTTPMN细胞数与540nm吸光度值有良好的线形相关性;氢化考的松(hydrocortisone,Hy)和美洛昔康(meloxicam,Melo)均可抑制PMN与500U·mL-1rhTNFα/或IL1诱导的RSC粘附,提示Hy和Melo治疗类风湿性关节炎(RA)可能与其抑制PMN与RSC粘附作用有关。结论:以MTT比色法为基础建立的检测大鼠PMN与RSC的粘附模型,可用于PMN与RSC粘附抑制剂的筛选。

AIM: To establish a new and simple method of quantitating adhesion of neutrophil or polymorphonuclear leukocyte(PMN) to rat synovial cell(RSC) using MTT [3 (4,5 dimethylthiazol 2 yl) 2,5 diphemyl tetrazolium bromide] stained PMN cell. METHODS: PMN were incubated with MTT, and formed MTT labeled PMN(MTT PMN) with a blue dye of formazan. After incubation of the MTT PMN and RSC, non adhered cells were washed out. Adhered cells were lysed with dimethylsulfoxide and quantitated by measure the absorbance at 540 nm. The concentration and time courses of rhTNF α and IL 1 which stimulate RSC were studied. According to the positive cross test, moreover, it were also studied that the inhibitory effects of hydrocortisone (Hy) and meloxicam (Melo) on the adhesion of PMN to RSC which were stimulated by rhTNF α and IL 1 at 500 U·mL -1 for 12 h. RESULTS: The absorbance at 540 nm was shown to be well correlated with the cell numbers of MTT PMN(0 25～2×10 9 cell·L -1 ) adhesion to RSC which were stimulated by 500 U·mL -1 rhTNF α and IL 1 for 12 h. Hydrocortisone inhibited the adhesion of PMN to RSC stimulated by 500 U·mL -1 rhTNF α and IL 1, the IC 50 was 2 6×10 -7 mol·L -1 , 2 29×10 -9 mol·L -1 , respectively. But IC 50 of meloxicam were 6 1×10 -7 mol·L -1 and 1 25×10 -6 mol·L -1 , respectively. CONCLUSION: A new and simple method of quantitating adhesion of PMN to RSC was established, by using MTT stained PMN cell. This method can be used for screening inhibitors of PMN adhesion to RSC.