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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(AA FLSs)内钙离子(Ca2+)稳态的影响,探讨rhEndostatin促进AA FLSs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140~160 g,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AA FLSs,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AA FLSs胞内Ca2+荧光强度发生动态变化,可以判断rhEndostatin对AA FLSs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AA FLS[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50 s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AA FLSs[Ca2+]i变化。结论rhEndostatin可促进AA FLSs胞外Ca2+内流,引起胞内Ca2+超载,从而促进AA FLSs凋亡。
Objective To investigate the effect of rhEndostatin on the steady state of calcium ion (Ca2 +) in AA FLSs in adjuvant arthritis rats and to explore the ionic mechanism of rhEndostatin promoting the apoptosis of AA FLSs. RA drug therapy and find new targets for the treatment of experimental basis. Methods Twelve male SD rats weighing 140-160 g were divided into normal group (n = 3) and AA model group (n = 9). The AA model was prepared in the model group. AA FLSs were cultured in vitro. The cultured cells were incubated with Fluo-3 / AM, a fluorescent indicator of Ca2 +. The cells were detected by confocal laser scanning microscope with no extracellular Ca2 + The dynamic change of Ca2 + fluorescence intensity can determine the effect of rhEndostatin on intracellular Ca2 + concentration ([Ca2 +] i) in AA FLSs. Results In the presence of extracellular Ca2 +, rhEndostatin caused a rapid increase of [Ca2 +] i in FLT. After 10 s of rhEndostatin, [Ca2 +] i increased sharply, then decreased slowly with time. After 50 s , [Ca2 +] i has not yet returned to the baseline level before dosing, whereas rhEndostatin did not cause changes in AA FLSs [Ca2 +] i in acellular calcium-free environment. Conclusion rhEndostatin can promote influx of extracellular Ca2 + in AA FLSs, cause intracellular Ca2 + overload and promote apoptosis of AA FLSs.