塞来昔布诱导多囊肾囊肿衬里上皮细胞凋亡的实验研究

目的 通过观察特异性环氧酶2(COX-2)抑制剂塞来昔布(celecoxib，CXB)诱导人多囊肾囊肿衬里上皮细胞凋亡，初步探讨该药诱导细胞凋亡的作用机制。 方法 (1)原代培养囊肿衬里上皮细胞，分为对照组、CXB组，采用Brdu法检测细胞增殖状态。(2)应用透射电镜观察细胞超微结构。(3)TUNEL法检测细胞凋亡及凋亡率。(4)应用AnnexinV+流式细胞术检测CXB诱导细胞凋亡及凋亡率。(5)应用免疫印迹法检测Bcl-2、Bax蛋白的表达。 结果 (1)Brdu结果显示，CXB能抑制多囊肾囊肿衬里上皮细胞的增殖，在24~72 h，浓度为(0.25~2)×10^-5 mol/L的范围内呈时间和剂量依赖性。(2)电镜下可见细胞核浓缩、染色质聚集、胞浆空泡化、凋亡小体出现、沟裂和裸核形成等典型凋亡征象。(3)TUNEL法显示，对照组细胞凋亡率为(2.8±0.2)%，2×10^-5 mol/L CXB作用24、48 h细胞凋亡率为(28.5±1.6)%和(48.5±1.2)%，两者差异有统计学意义(P < 0.05)。(4) AnnexinV+流式细胞仪法结果显示，不同浓度(0.5、1、2×10^-5 mol/L)CXB处理24 h凋亡率分别为(7.15±0.11)%、(7.76±0.08)%和(12.15±0.07)%，显著高于无血清对照组(3.15±0.05)%；不同浓度CXB处理48 h的凋亡率分别为(18.36±0.17)%、(24.87±0.25)%和(53.66±0.32)%，显著高于无血清对照组(13.53±0.21)%，组间差异均有统计学意义(P 均< 0.01)。(5)Western 印迹结果显示，随CXB作用时间延长，Bcl-2的表达逐渐减弱而Bax的表达逐渐增强，Bcl-2/Bax的比值逐渐减少。结论 塞来昔布呈时间和剂量依赖性抑制囊肿衬里上皮细胞增殖，通过下调Bcl-2，上调Bax，减少Bcl-2/Bax的比值诱导细胞凋亡。本研究结果为将CXB用于治疗常染色体显性多囊肾病提供了实验依据。

Experimental study of celecoxib inducing the apoptosis of cyst liner epithelial cells of polycystic kidney

Objective To initially investigate the mechanism of COX-2 inhibitor inducing cell apoptosis through the observation of celecoxib(CXB)， a specific COX-2 inhibitor，inducing apoptosis of cyst lining epithelial cells of human polycystic kidney. Methods (1)Primarily cultured cell was divided into control group and CXB group to evaluate the proliferative state by Brdu assay.(2)The cell apoptosis was observed by transmitted electronic microscope after being cultured in CXB 2×10^-5 mol/L for 24，48 hours.(3)The cell apoptosis and apoptotic rate were detected by TUNEL assay.(4)The cell apoptotic rate were measured by AnnexinV， PI-labeled flow cytometry after being cultured in CXB 2×10^-5 mol/L for 0， 24， 48 hours.(5)Protein expression of Bax，Bcl-2，caspase 3 was examined by Western blotting. Results (1)The Brdu assay revealed that CXB inhibited cell growth in a concentration-dependent manner， with the maximum growth inhibition ratio of 63.9% when treated by CXB 2×10^-5 mol/L for 24 h.(2)Typical morphological changes of apoptotic cell were apoptotic body， nuclear concentration，chromatin aggregation， endochylema vacuolization and ravinement under eletrou microscope. (3)TUNEL assay showed that the apoptotic rate was (2.8±0.2)% in control group， and (28.5±1.6)%， (48.5±1.2)% in CXB group for 24，48 hours respectively，with significant differences to control group(P < 0.05). (4) AnnexinV， PI-labeled flow cytometry showed that，in 0，0.5，1，2×10^-5 mol/L CXB group，the apoptotic rates were(3.15±0.05)%，(7.15±0.11)%，(7.76±0.08)%，(12.15±0.07)% for 24 hours respectively，and(13.53±0.21)%，(18.36±0.17)%，(24.87±0.25)%，(53.66±0.32)% for 48 hours respectively. Significant differences were found among corresponding groups(all P < 0.01). (5) Extracted total cell protein in every group and more protein of Bax，Bcl-2 expressed in CXB-treated group was detected by Western blotting than that in control group.Conclusions CXB can inhibit the proliferation of cyst liner epithelial cells in a time- and concentration-dependent manner， and induce cell apoptosis through increasing the ratio of Bax/Bcl-2. CXB is hopeful to become an effective drug to treat ADPKD.