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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。
OBJECTIVE: To investigate the mechanism of apoptosis induced by cyclooxygenase-2 (COX-2) inhibitor celecoxib (CXC) in human polycystic kidney cyst lining epithelial cells. Methods (1) Primary cultured cyst-lined epithelial cells were divided into control group and CXB group. Brdu method was used to detect cell proliferation status. (2) Transmission electron microscopy was used to observe the ultrastructure of cells. (3) TUNEL method was used to detect apoptosis and apoptosis rate. (4) Annexin V + flow cytometry was used to detect CXC-induced apoptosis and apoptosis rate. (5) The expression of Bcl-2 and Bax protein was detected by Western blot. Results (1) The results of Brdu showed that CXB could inhibit the proliferation of epithelial cells in the cyst lining of polycystic kidney in a range of (0.25 ~ 2) × 10 ~ (-5) mol / L at 24 ~ 72 h Time and dose-dependent. (2) Under electron microscope, typical apoptotic signs such as nuclear condensation, chromatin aggregation, cytoplasmic vacuolization, appearance of apoptotic bodies, ditch-cleavage and nude nucleus formation were observed. (3) TUNEL assay showed that the rate of apoptotic cells in control group was (2.8 ± 0.2)%, and the apoptosis rate of cells treated with 2 × 10 -5 mol / L CXB for 24 and 48 h was (28. 5 ± 1.6)% and (48.5 ± 1.2)%, respectively, with statistical significance (P <0.05). (4) The results of Annexin V + flow cytometry showed that the apoptotic rate of CXB treated with different concentrations (0.5, 1, 2 × 10 -5 mol / L) for 24 h were (7.15 ± 0.11 ), (7.76 ± 0.08)% and (12.15 ± 0.07)%, respectively, which were significantly higher than those in the serum-free control (3 15 ± 0.05)% The mortality rates were (18.36 ± 0.17)%, (24.87 ± 0.25)% and (53.66 ± 0.32)%, respectively, which were significantly higher than those in the serum-free control group (13.53 ± 0 .21)% respectively. There was significant difference between the two groups (all P <0.01). (5) Western blotting showed that the expression of Bcl-2 gradually decreased and the expression of Bax gradually increased, while the ratio of Bcl-2 / Bax decreased gradually with the prolongation of CXB. Conclusion Celecoxib inhibits the proliferation of cyst-lining epithelial cells in a time-and dose-dependent manner, and induces apoptosis by down-regulating Bcl-2, up-regulating Bax and decreasing Bcl-2 / Bax ratio. The results of this study provide an experimental basis for the use of CXB in the treatment of autosomal dominant polycystic kidney disease.