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目的:观察雷公藤红素对人骨肉瘤HOS细胞凋亡的影响,并探讨其可能的作用机制。方法 :用不同浓度(0、1.5、2.5、3.5和4.5μmol/L)的雷公藤红素处理骨肉瘤HOS细胞24 h后,采用CCK-8法检测细胞活性,确定最佳实验浓度。采用倒置相差显微镜观察细胞形态变化,Hoechst33258染色后相差显微镜观察细胞凋亡的形态学改变,FCM法检测细胞凋亡率和细胞内Ca2+浓度变化,蛋白质印迹法检测结合免疫球蛋白(binding immunoglobulin protein,BIP)、钙联蛋白(calnexin,CNX)、内质网氧化还原酶1-Lα(endoplasmic reticulum oxidoreductin 1-L alpha,Ero1-Lα)、蛋白质二硫键异构酶(protein disulfide isomerase,PDI)、肌醇依赖酶1(αinositol-requiring enzyme 1 alpha,IRE1α)、蛋白激酶样内质网激酶(protein kinase-like endoplasmic reticulum kinase,PERK)和磷酸化PERK(phosphorylatedPERK,p-PERK)、CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancerbinding protein homologous protein,CHOP)、剪切型caspase-12(cleavedcaspase-12,c-caspase-12)和c-caspase-3的表达变化。结果 :不同浓度的雷公藤红素均可抑制HOS细胞活性(P值均<0.05),且呈浓度依赖性。3μmol/L雷公藤红素处理24 h后,HOS细胞形态明显萎缩、紊乱,漂浮细胞增多,且出现明显核固缩和核碎裂等典型的细胞凋亡形态学改变。与未处理对照组相比,3μmol/L雷公藤红素处理24 h后细胞凋亡率和细胞内Ca2+浓度明显升高(P值均<0.05)。3μmol/L雷公藤红素处理24 h后,内质网应激相关标志蛋白BIP、CNX、Ero1-Lα、PDI、IRE1α和p-PERK以及内质网应激介导凋亡相关蛋白CHOP、c-caspase-12和c-caspase-3的表达水平明显升高(P值均<0.05)。结论 :雷公藤红素可诱导人骨肉瘤HOS细胞凋亡,其作用机制可能与内质网应激介导细胞凋亡通路有关。
Objective: To observe the effect of Tripterine on the apoptosis of human osteosarcoma HOS cells and to explore its possible mechanism. Methods: The osteosarcoma HOS cells were treated with different doses (0, 1.5, 2.5, 3.5 and 4.5 μmol / L) for 24 h. Cell viability was determined by CCK-8 assay and the optimal concentration was determined. The morphological changes of cells were observed by phase contrast microscopy after Hoechst33258 staining. The changes of apoptosis rate and intracellular Ca2 + concentration were detected by FCM. The expression of binding immunoglobulin protein BIP), calnexin (CNX), endoplasmic reticulum oxidoreductin 1-L alpha (Ero1-Lα), protein disulfide isomerase (PDI) Αinositol-requiring enzyme 1 alpha (IRE1α), protein kinase-like endoplasmic reticulum kinase (PERK) and phosphorylated PERK (p-PERK), CCAAT / enhancer The expressions of cleaved caspase-12 (c-caspase-12) and c-caspase-3 were detected by flow cytometry (CCAAT / enhancerbinding protein homologous protein, CHOP) Results: Tripterine inhibited the activity of HOS cells (P <0.05) in a concentration-dependent manner. After treatment with 3 μmol / L tripterine for 24 h, the morphology of HOS cells was significantly atrophic, disorganized, and the number of floating cells increased. Morphological changes such as nuclear condensation and nuclear fragmentation were observed. Compared with the untreated control group, the apoptotic rate and the intracellular Ca2 + concentration were significantly increased after 3 μmol / L tripterine treatment for 24 h (P <0.05). Endoplasmic reticulum stress-related markers BIP, CNX, Ero1-Lα, PDI, IRE1α and p-PERK and endoplasmic reticulum stress-mediated apoptosis-associated protein CHOP, c The expressions of caspase-12 and c-caspase-3 were significantly increased (P <0.05). Conclusion: Tripterine can induce apoptosis of HOS cells in human osteosarcoma cells. The mechanism may be related to the pathway of endoplasmic reticulum stress-mediated apoptosis.