蜂毒注射液对类风湿关节炎成纤维样滑膜细胞凋亡影响的研究

Study of Apistoxin Injection on Apoptosis of Fibroblast-like Synovial Cells of Rheumatoid Arthritis Patients

目的 ：探究蜂毒注射液对类风湿关节炎成纤维样滑膜细胞（FLS-RA）凋亡的影响。方法 ：原代培养FLS-RA,MTT法测定蜂毒注射液对细胞增殖及细胞毒性作用,hoechst染色法观察凋亡小体,Annexin V/PI双染法检测细胞凋亡率。结果：组织块培养法培养2周后细胞向心性排列,呈梭形及三角形。经过3-5次传代,FLS-RA细胞逐渐纯化,以FLS为主（〉95%）,体外生长稳定。倒置相差显微镜观察细胞形态：细胞排列整齐,向心性排列,呈梭形或三角形,偶见圆形细胞,细胞核成卵圆形,居细胞中央,胞质均匀透亮。干预24 h：各组生存率低于空白对照组（P〈0.05）,浓度越高,生存率越低;蜂毒注射液处理48 h：各组生存率低于空白对照组（P〈0.05）,浓度越高,生存率越低;同浓度蜂毒注射液处理24 h生存率与48 h比较：48 h组小于24h组（P〈0.05）。利用ORIGIN 5.0软件计算蜂毒注射液的半数抑制浓度（IC50）为（16.79±0.13）mg/L。实验组与阳性对照组中凋亡小体形态相似,细胞核呈致密浓染,颜色发白,而阴性对照组未见明显凋亡小体,细胞核未见固缩的染色质。B1区为细胞碎片,B2区为晚期凋亡和死亡的细胞,B3区为正常细胞,B4区为早期凋亡的细胞。由于B2区包含死亡的细胞,多选择B4区比较早期凋亡率。低浓度组蜂毒注射液作用FLS-RA细胞24 h,早期凋亡率大于空白对照组（P〈0.05）;高浓度组蜂毒注射液早期凋亡率明显高于空白对照组（P〈0.01）,且大于低浓度组（P〈0.05）。结论：蜂毒注射液可诱导成纤维样滑膜细胞凋亡,这可能是蜂毒治疗类风湿关节炎的机理之一。

Objective： To explore the influence of Apistoxin injection on the apoptosis of fibroblast-like synovial cells of rheumatoid arthritis patients. Methods： Rheumatoid arthritis fibroblast-like synovial cells （FLS-RA） was concentrated by the primary culture in vitro. Effect of Apistoxin injection on cell proliferation and cell toxicity was measured by MTT technique, and apoptotic body was observed by Hoechst staining, apoptotic rate was detected after Annexin V/PI double staining. Results： After primary culture for 2 weeks, the cells were fusiform or triangle-like, arranged around the center. After passage culture for 3-5 generations, FLS-RA became pure and the percentage of FLS was over 95%, growing steady in vitro. The results under inverted phase contrast microscope showed that the ceils were arranged around the center in order, were fusiform or triangle-like, and seldom were round with oval nucleus in the center of the cell. The cytoplasm was well-distributed and lucency. After intervention with Apistoxin injection for 24 and 48 hours, the surviva（ rate of FLS-RA was lower in the intervention groups than that in the blank control group （P 〈 0.05）, and the survival rate decreased with the increase of Apistoxin injection concentration（P 〈 0.05）. The survival rate induced by the same concentration of Apistoxin injection was lower after intervention for 48 hours than that after 24 hours （P 〈 0.05）. The 50% inhibiting concentration （IC50）of Apistoxin injection was 16.79 ±0.13 mg/L. The apoptotic body was found in both observation group and positive control group, while was not shown in the negative control group. The cells in B4 region were chosen for the calculation of early apoptotic rate, and the results showed that low- and high-concentration Apistoxin injection groups had higher early apoptotic rate than the blank control groups （P 〈 0.05 or P 〈 0.01）, and the high-concentration groups had the highest early apoptotic rate（P 〈 0.05）. Conclusion： Apistoxin injection can induce the apoptosis of fibroblast-like synovial cells, which may be one of the therapeutic mechanisms of Apistoxin injection for rheumatoid arthritis.