重组人内抑素对佐剂性关节炎大鼠成纤维样滑膜细胞内钙离子稳态的影响

Effects of recombinant human endostatin on intracellular Ca2+ homeostasis in fibroblast-like synoviocytes in rats with adjuvant arthritis

目的观察重组人内抑素（rhEndostatin）对佐剂性关节炎大鼠成纤维样滑膜细胞（AAFLSs）内钙离子（Ca2＋）稳态的影响，探讨rhEndostatin促进AAFLSs凋亡的离子机制，为RA药物治疗及寻找其治疗新靶点提供实验依据。方法雄性sD大鼠12只，体质量140～160g，分为正常组（n=3）和AA模型组（n=9）。模型组大鼠制备AA模型，体外培养AAFLSs，应用Ca2＋荧光指示剂Fluo-3／AM孵育培养的细胞，激光扫描共焦显微镜检测有、无细胞外Ca2＋，而rhEndostatin作用所致AAFLSs胞内Ca2＋荧光强度发生动态变化，可以判断rhEndostatin对AAFLSs胞内Ca2＋浓度（[Ca2＋]i）的影响。结果在胞外有Ca2＋的情况下，rhEndostatin可引起静态AAFLS[Ca2＋]i快速增加，rhEndostatin作用10s后，[Ca2＋]i急剧增加达峰值，继之随时间缓慢下降，停止加药50s后，[Ca2＋]i尚未回复到加药前基础水平；而在无细胞外钙环境中，rhEndostatin未引起AAFLSs[Ca2＋]i变化。结论rhEndostatin可促进AAFLSs胞外Ca2＋内流，引起胞内Ca2＋超载，从而促进AAFLSs凋亡。

Objective To observe the effects of recombinant human endostatin （rhEndostatin） on intracellular Ca2＋ homeostasis in fibroblast-like synovioeytes（FLSs） in rats with adjuvant arthritis（AA）, approach ionic mechanisms of AA FLSs apoptosis induced by rhEndostatin, and provide experimental data for possible drug treatment and their targets in rheumatoid arthritis （RA）. Methods Twelve male Sprague-Dawley（SD） rats weighing 140-160 g were used for these studies. The rats were divided at random into a normal group （ n = 3） and an AA model group （ n = 9）. Adjuvant arthritis was induced in rats in the AA model group and the FLSs obtained from AA rats were cultured in vitro. The cultured AA FLSs were incubated with the calcium ion-sensitive fluorescent indicator fluo-3/AM. A confoeal laser scanning microscope was used to measure changes of fluorescence intensity of intracellular Ca2＋ in the cells caused by rhEndostatin when the cells were immersed in buffer （with or without Ca2＋ ）, and to investigate the effect of rhEndostatin on intraeellular free calcium concentration [Ca2＋]i in AA FLS. Results Recombinant human endostatin could cause rapid [Ca2＋]i elevation in static AA FLSs in Ca2＋ -containing buffer. Within 10s after treatment with rhEndostatin, the elevated levels of [ Ca2＋ ]i ran rapidly to peaked and was followed by a plateau period. The elevated [ Ca2＋ ]i did not return to baseline until 50 s after rhEndostatin was removed. Recombinant human endostatin did not cause [Ca2＋]i elevation when AA FLSs were immersed in Ca2＋ -free buffer. Conclution Recombinant human endostatin evoked an influx of Ca2＋ from the extracellular environment, which led to intraeellular Ca2＋ overload and promoted AA FLSs apoptosis.