地塞米松介导胸腺细胞凋亡过程中线粒体和细胞结构蛋白的变化

The mitochondrial and structural protein changes in dexamethasone-induced mouse thymocyte apoptosis

目的:研究地塞米松(DEX)介导的小鼠胸腺细胞凋亡过程中线粒体质量和结构蛋白变化特点。方法:以地塞米松(DEX)诱导小鼠胸腺细胞凋亡为模型,利用AnnexinV-FITC/PI双染流式细胞术研究细胞凋亡和坏死,JC-1染色流式细胞术检测线粒体膜电势(△Ψm)和线粒体质量,利用CFDA-SE染色流式细胞术检测细胞结构蛋白变化。结果:在1×10-6mol/LDEX诱导下,小鼠胸腺细胞在6h凋亡比率为(51.25±5.51)%,对照组为(12.03±2.00)%,差异显著(P<0.01);DEX组坏死比率为(30.25±3.67)%,对照组为(10.11±1.11)%,差异显著(P<0.01)。DEX组在6h时点的线粒体质量显著低于对照组(P<0.01),FL1平均荧光强度分别为(561.62±54.27)和(900.25±38.80)。DEX同时引起线粒体膜电势的显著下降(P<0.01),对照组FL2平均荧光强度为(267.51±26.48),DEX组为(133.17±12.29)。成熟T细胞培养48h,CFDA-SE法仅检测到亲代单一细胞峰;而在ConA刺激条件下出现3个子代峰。对照组小鼠胸腺细胞在CFDA-SE染色培养6h条件下,存在(5.25±1.15)%的低荧光强度细胞群,而在DEX刺激下,该群细胞占(47.39±9.76)%,并且在直方图结果上形成明显的细胞峰。结论:DEX诱导小鼠胸腺细胞凋亡过程中,线粒体质量和细胞结构蛋白均有所下降;CFDA-SE染色流式细胞术可以作为基于细胞结构蛋白变化的凋亡定量检测方法。

AIM: To study mitochondrial mass and structural protein changes in dexamethasone (DEX)-mediated mouse thymocyte apoptosis process. METHODS: DEX-induced mouse thymocyte apoptosis model was established. Annexin V-FITC/PI double staining was used to identify apoptotic and necrotic cells by flowcytometry, JC-1 staining was adopted to test mitochondrial membrane potential (△Ψ_m), and cellular structural protein changes were studied with CFDA-SE staining. RESULTS: By 1×10^(-6) mol/L DEX stimulation, the apoptotic rate was 51.25%±5.51% and had significantly difference from control group (12.03%±2.00%); the necrotic rate in DEX group was 30.25%±3.67% and also had significantly difference from control group (10.11%±1.11%, P<0.01). Mitochondrial mass in DEX group [FL1 (561.62±54.27)] was significantly lower than that in control (900.25±38.80, P<0.01). DEX also caused significant down-regulation of △Ψ_m (P<0.01), FL2 in control group was 267.51±26.48, and in DEX group was 133.17±12.29. Mature T cells cultured for 48 h showed only one peak by CFDA-SE staining; while by Con A stimulation, there were three offspring peaks. Low FL1 cell cluster (5.25%±1.15%) exited in control group, while by DEX stimulation, this cluster significantly was larger (47.39%±9.76%, P<0.01). CONCLUSIONS: In mouse thymocytes, mitochondrial mass and cellular structural protein amount are reduced by DEX; CFDA-SE staining flowcytometry can served as an apoptosis detection method based on cellular structural protein amount change.