PC12神经细胞糖氧剥夺模型的建立

Oxygen-glucose deprivation model establishment in PC12 nerve cell

目的建立PC12神经细胞缺氧缺糖模型,为探索神经元损伤的机制以及防治药物研究提供可靠的工具。方法将PC12神经细胞置于三气培养箱中(1%O2—5%CO2—94%N2)分别缺氧缺糖lh.2h,3h和4h,采用H·E染色观察细胞形态学变化,MTT法检测细胞代谢活力,分光光度法检测LDH外漏量,台盼蓝染色计数测定细胞存活率,Hochest/PI染色和流式细胞检测技术测定细胞凋亡率,系统考察糖氧剥夺的PC12神经细胞随缺氧时间延长,细胞的损伤变化规律。结果缺氧缺糖2h时,PC12神经细胞即已经明显损伤,此后随着缺氧时间的延长,PC12神经细胞活力逐渐下降,LDH外漏逐渐增加,细胞凋亡率逐渐升高,细胞存活率逐渐降低,其中,缺氧缺糖1h时,早期凋亡细胞占多数.缺氧缺糖4h时则以晚期凋亡及坏死细胞占多数。结论本实验所建立的PC12神经细胞缺氧模型稳定可靠,重现性良好。

Objective To establish the oxygen-glucose deprivation (OGD) model of PC 12 nerve cell in vitro, and provide potential tool for drug research. Methods Tri-gas CO2 incubator (l%O2-5%CO2-94%N2) was used to make it lack of oxygen and glucose for 1 h、2 h、3 h、4 h respectively. The changes of cell morphology was observed by H·E staining, the metabolism vitality was determined by MTT method, the extracellular leakage of LDH was detected by spectrophotometry, the cell survival rate was measured by trypan blue staining counting, the cell apoptosis rate was detected by Hochest/PI staining and flow cytometry. Results PC12 nerve cells were obviously damaged at 2h OGD time. With the prolongation of the anoxic time, the vitality of PC12 nerve cell was gradually decreased;LDH leakage was gradually increased, the apoptosis rate was increased gradually;and the cell survival rate was gradually decrease. The flow cytometry data showed that the early apoptotic cells were observed at 1h OGD;late apoptosis and necrosis cells were detected at the 4h OGD.Conclusion The OGD model of PC12 nerve cell is simple, reliable and reproducible.