HT22细胞糖氧剥夺／复氧模型建立条件的优化研究

Optimization of condition in oxygen-glucose deprivation/re-oxygenation models of HT22 cells

目的优化小鼠海马神经元HT22细胞糖氧剥夺／复氧模型的建立条件，探讨该模型建立的依据并测定相关指标。方法首先通过细胞计数法绘制HT22细胞的生长曲线并计算该细胞的倍增时间。其次，采用乳酸脱氢酶（LDH）细胞活性实验确认模型最佳的糖氧剥夺时长和复氧时长。最后，用CCK-8细胞活性实验、细胞计数法、细胞形态学观察以及流式细胞仪凋亡检测方法进一步确定模型建立的条件。结果HT22细胞的倍增时间为17h 18min。最佳模型的糖氧剥夺时长为18h，复氧时间长为20h。CCK-8检测结果显示，糖氧剥夺18h／复氧20h（OGD18h／R20h）细胞吸光度值为0．334±0．138，对照组细胞吸光度值为0．756±0．026，差异有统计学意义（P〈0．05）。在形态学方面，OGD18h／R20h细胞明显发生皱缩。流式细胞仪凋亡检测结果显示，0GD18h／R20h细胞中凋亡晚期和坏死细胞分布比例为24．56％±5．76％．早期凋亡细胞2．63％±0．55％，对照组细胞中凋亡晚期和坏死细胞分布比例为8．13％±0．87％，早期凋亡细胞0.80％±0．10％，差异均有统计学意义（P〈0．05）。结论HT22细胞糖氧剥夺／复氧模型的糖氧剥夺时长和复氧时长与细胞的倍增时间有关，糖氧剥夺18h后复氧20h可较好实现建模要求。

Objective To optimize the HT22 cell oxygen-glucose deprivation/re-oxygenation models in hippocampal neurons of mice, discuss the basis of model establishment and test the related parameters. Methods HT22 cell growth curve was plotted by cell counting and cell doubling time was calculated, and then, the oxygen-glucose deprivation time and re-oxygenation time were determined by LDH assay. The CCK-8 assay, cell counting, cell morphology and flow cytometry were performed to further optimize the parameters of the models. Results The HT22 cell doubling time was 17 h 18 min. The oxygen-glucose deprivation time was 18 h and the re-oxygenation was 20 h. CCK-8 assay indicated that absorbance in cells of oxygen-glucose deprivation 18 h/re-oxygenation 20 h （OGD 18 h/R20 h） group was 0.334±0.138, which was significantly lower than that in the control group （0.756±0.026, P〈0.05）; cells in the OGD18 h/R20 h group were shrank obviously. Flow cytometry indicated that the percentage of cells in terminal apoptosis or non-viable non-apoptotic cells was 24.56%±5.76% and that of viable apoptotic cells was 2.63%±0.55% in the OGD18 h/R20 h group, which were significantly higher than those in the control group, respectively （8.13%±0.87% and 0.80%±0.10%, P〈0.05）. Conclusion The HT22 cell oxygen-glucose deprivation/re-oxygenation models are successfully established; both oxygen-glucose deprivation time and re-oxygenation time are related to the cell doubling time, and OGD18 h/R20 h can preferably help the establishment.