改良组织块法培养细针活检滑膜组织成纤维样滑膜细胞

Modified Tissue Culture for Fibroblast-like Synoviocytes from Needle Biopsied Synovium Tissue

【目的】探讨体外分离培养细针活检滑膜组织成纤维样滑膜细胞(FLS)的方法。【方法】滑膜组织来自接受盲式细针滑膜活检术的活动期类风湿关节炎患者,分别采用消化酶培养法、组织块培养法和改良组织块培养法分离培养FLS。采用台盼蓝染色进行细胞计数及活性鉴定,并应用倒置相差显微镜、透射电镜、免疫细胞化学染色、流式细胞术对第3～4代FLS进行鉴定。【结果】3种原代分离培养的方法均可成功培养出FLS。台盼蓝染色示FLS细胞计数约(8.30±1.65)×105/瓶,活细胞百分率>95%。传代可使FLS达到形态学上的纯化要求,倒置相差显微镜、透射电镜观察均符合FLS的形态结构特征,免疫细胞化学染色示Vimentin阳性、CD68阴性、CK阳性,流式细胞术检测示CD55+细胞占(95.34±2.47)%。改良组织块法培养FLS成功率较高,细胞游出时间较早,所需传代时间较短。【结论】改良组织块法特别适合于细针滑膜活检标本FLS的培养,第3～4代细胞的数目、活性及纯度符合进一步实验的要求。

【Objective】 To explore the culture method for fibroblast-like synoviocytes （FLS） from needle biopsied synovium tissue. 【Methods】 Synovium tissue obtained from patients with active rheumatoid arthritis by blind needle synovium biopsy were cultured with three different methods （digestive enzyme culture,tissue culture and modified tissue culture）. Cell count and the percentage of viable cells were observed by trypan blue staining. FLS were identified by morphology,immunocytochemical staining and flow cytometry. 【Results】 All three methods could culture FLS successfully,of which modified tissue culture method was better than the other two methods. FLS cell count was （8.30 ± 1.65） × 105 per flask with more than 95% viable cells by Trypan blue staining. FLS of the third or forth generation showed typical morphological characters under inverted phase contrast microscope and transmission electron microscope with Vimentin＋/ CD68-/ CK＋ staining and （95.34 ± 2.47）% CD55＋ cells. Modified tissue culture method has higher successful rate with earlier and more quickly cell emigration from synovium tissue. 【Conclusion】 Modified tissue culture method is especially suitable for FLS culture in vitro from needle biopsied synovium tissue. The cell count,the percentage of viable cells,and purity of FLS of the third or forth generation meet the requirement for molecular biology experiments.