不同浓度葡萄糖对小鼠海马神经元细胞系HT-22细胞凋亡的影响

目的 探讨不同浓度葡萄糖对小鼠海马神经元细胞系 HT-22细胞凋亡的作用及机制。方法 体外培养小鼠海马神经元 HT-22细胞，使用不同浓度的高糖培养液（25、35、45、55、65、75 mmol/L）分别作用细胞 24、48、72 h，以 25 mmol/L 糖浓度为对照组，其余组为实验组。采用 CCK-8 法检测各组细胞活力变化，筛选出最佳作用时间。HT-22细胞经不同浓度葡萄糖作用 48 h后，微板法检测细胞培养上清液中乳酸脱氢酶（LDH）释放率；光学显微镜观察细胞形态变化；流式细胞术检测各组细胞凋亡情况；蛋白免疫印迹法（Western blot）检测 Bcl-2、Bax蛋白表达量的变化。结果 CCK-8结果显示，高糖可抑制 HT-22细胞的活力，其抑制作用具有剂量和时间依赖性。高糖作用时间48 h时，细胞活力≥80%，可满足后续实验要求。随着葡萄糖浓度的增高，出现细胞数目减少，胞体变大，部分胞核溶解，突触断裂等改变，并且 LDH释放率及凋亡率也明显增高（P＜0.05），Western blot结果显示凋亡蛋白 Bcl-2表达下降（P＜0.05），Bax表达增高（P＜0.05）。结论 高糖能显著抑制 HT-22细胞生长和活力，诱导细胞凋亡，其作用机制可能与 Bcl-2、Bax表达有关。

Effects of different concentrations of glucose on apoptosis of mouse hippocampal neuron cell line HT-22

Objective To investigate the effect and mechanism of different concentrations of glucose on apoptosis of mouse hippocampal neuron cell line HT-22. Methods The mouse hippocampal neuron HT-22 cells were cultured in vitro.The cells were treated with different concentrations of sugar medium (25, 35, 45, 55, 65 and 75 mmol/L) for 24 h, 48 h and 72 h, respectively. The reference concentration of 25 mmol/L was used as the control group, and the other groups were the experimental groups. The cell activity was measured by cell counting kit (CCK)-8, and the optimal action time was screened.After HT-22 cells were treated with different sugar concentrations for 48 h, the release rate of lactate dehydrogenase (LDH)in cell culture supernatant was detected by microplate assay. The cell morphology was observed by optical microscope, and the apoptosis rate was determined by flow cytometry. The protein expressions of Bcl-2 and Bax were detected by Western blot assay. Results The results of CCK8 assay showed that high glucose can inhibit the viability of HT-22 cells in a doseand time -dependent manner. When the high glucose action time was 48 h, the cell viability≥80%, which can meet the requirements of subsequent experiments. With the increase of glucose concentration, the number of cells decreased, the cell body became larger, part of the nucleus dissolved and synapse rupture, and the release rate of LDH and apoptosis rate were also increased significantly (P＜0.05). Western blot analysis showed that the expression of apoptosis protein of Bcl-2 decreased (P＜0.05), and the expression of Bax increased gradually (P＜0.05). Conclusion High glucose can significantly inhibit the growth and viability of HT-22 cells and induce apoptosis, which may be related to the expressions of Bcl-2 and Bax.