不同浓度缺氧预适应小鼠脑匀浆提取液对PC12细胞缺氧耐受性的影响

Effects of brain tissue extract of hypoxia-preconditioned mice at different concentrations on tolerance of PC12 cells to hypoxia

目的:探讨缺氧预适应小鼠脑匀浆提取液(HP extract)对PC12细胞缺氧耐受性的影响。方法:复制小鼠急性重复缺氧模型,制备HP extract。在培养PC12细胞中加入HP extract使其终浓度分别为0.2、0.8、3.2、6.4、12.8g/L(HP组)。以同浓度正常小鼠脑匀浆提取液(N extract)作为对照组(N组)。缺氧(2%O2)培养24、48、72h后,采用四唑盐(MTT)比色法检测各组细胞活力(A570值)并检测缺氧24、48、72h乳酸脱氢酶(LDH)透出率、缺氧24h早期凋亡率(Annexin V-FITC/PI双染流式细胞仪检测)、缺氧72h晚期凋亡率(Hoechst33258染色荧光显微镜检测)。结果:HP extract浓度低于6.4g/L(包括6.4g/L),缺氧培养24h时,HP组A570值显著高于同浓度N组,其LDH透出率显著低于同浓度N组。随缺氧时间延长,高浓度HP extract逐渐失去细胞保护作用。至72h时浓度高于6.4g/L(包括6.4g/L)HP extract有促细胞凋亡作用,此时HP组A570值显著低于同浓度N组,其LDH透出率和晚期凋亡率均显著高于同浓度N组。结论:缺氧预适应小鼠脑匀浆提取液对PC12细胞缺氧耐受性的影响呈浓度依赖性和时间依赖性。

AIM： To investigate the effect of brain tissue extract of hypoxia -preconditioned mice （HP extract） on tolerance of PC12 cells to hypoxia. METHODS： The mice model of acute repetitive hypoxia was reproduced and brain tissue extracts were prepared. HP extract was added into the cultures of PC12 cells and the final concentrations of HP extracts were 0.2, 0.8, 3.2, 6. 4 or 12. 8 g/L （ HP group）, respectively. Brain tissue extract of normal mice （ N extract） at the same five concentrations were used as controls （ N group）. The PC12 cells were cultured in hypoxia （2% O2 ）. After hypoxia for 24 h, 48 h or 72 h, colorimetric method （A570 ） of tetrazolium salt MTr （3- [4,5- dimethyhhiazol- 2- yl] -2,5 - diphenyl tetrazolium bromid ） staining was adopted to determine the cell viability and lactate dehydrogenase （LDH） release percentage assay was also conducted after 24 h, 48 h or 72 h hypoxia. Besides, apoptotic percentages at early stage （24 h hypoxia） and late stage （72 h hypoxia） were detected respectively by means of annexin V - FITC/PI double - stained flow cytometry and Hoechst 33258 stained fluorescence microscopy. RESULTS： HP extract at the concentrations lower than 6.4 g/L （including 6. 4 g/L） showed protective effect on PC12 cells in early stage of hypoxia （24 h）. A570 values in HP group were significantly higher than those in N group, but LDH release percentages were significantly lower than those in N group after 24 h hypoxia. With hypoxia prolonging, HP extract at high concentrations gradually lost the protective effect. At the time point of 72 h hypoxia, HP extract at concentrations higher than 6. 4 g/L （ including 6. 4 g/L）had pro - apoptotic effect. At this time point, A570 values of HP groups at these concentrations were significantly lower than those in the corresponding N group, both LDH release percentages and apoptotic percentages were significantly higher than those in the N group. CONCLUSION： The effects of HP extract on tolerance of PC12 cells to hypoxia depend on its concentrations and on the time of treatment.