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目的研究突变型人胰高血糖素样肽-1(mutated human glucagon-like peptide-1,mGLP-1)对谷氨酸诱导的人神经母细胞瘤细胞SH-SY5Y细胞损伤的影响。方法环磷酸腺苷(cAMP)试剂盒检测细胞内cAMP含量,比较mGLP-1和天然人胰高血糖素样肽-1(natural human glucagon-like peptide-1,nGLP-1)与GLP-1受体结合的能力;以谷氨酸诱导SH-SY5Y细胞损伤,同时用mGLP-1处理SH-SY5Y细胞,采用MTT法检测细胞存活率;乳酸脱氢酶(LDH)试剂盒检测细胞LDH的释放量;DAPI染色法观察细胞凋亡形态学特征;钙流法检测胞内钙离子浓度变化;通过检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、总谷胱甘肽(total GS)含量的变化判断细胞氧化损伤程度。结果 mGLP-1与nGLP-1类似,都能提高细胞内cAMP水平;mGLP-1能缓解谷氨酸诱导的细胞损伤,增加细胞存活率,降低LDH释放量;mGLP-1对谷氨酸导致的细胞内钙离子变化没有修复作用;mGLP-1能抑制氧化损伤指标MDA含量升高,促进抗氧化系统指标SOD活性增强和total GS含量增加。结论 mGLP-1可导致细胞内cAMP含量增加。mGLP-1对谷氨酸所致神经细胞损伤具有保护作用,这可能是mGLP-1通过对神经细胞的抗氧化损伤作用实现的,而不是改善谷氨酸导致的钙稳态失衡。mGLP-1的保护作用与nGLP-1类似,而mGLP-1在体内稳定性更强,半衰期更长;提示mGLP-1可能对相关神经退行性疾病的治疗具有更佳的潜在价值。
Objective To investigate the effect of mutated human glucagon-like peptide-1 (mGLP-1) on the injury of human neuroblastoma cells SH-SY5Y induced by glutamate. Methods Cyclic cAMP was detected by cAMP kit. Compared with mGLP-1 and natural human glucagon-like peptide-1 (nGLP-1) and GLP-1 SH5-SY5Y cells were induced by glutamate, while SH-SY5Y cells were treated with mGLP-1. Cell viability was measured by MTT assay. LDH release was detected by lactate dehydrogenase (LDH) kit The morphological features of apoptotic cells were observed by DAPI staining. The changes of intracellular calcium concentration were detected by calcium flow assay. The contents of malondialdehyde (MDA), superoxide dismutase (SOD), total glutathione (total glutathione GS) content changes to determine the degree of cell oxidative damage. Results Compared with nGLP-1, mGLP-1 could increase intracellular cAMP level; mGLP-1 could relieve glutamate-induced cell injury, increase cell survival and decrease LDH release; mGLP- MGLP-1 could inhibit the increase of MDA content in the index of oxidative damage, promote the increase of SOD activity and increase the content of total GS in the antioxidant system. Conclusion mGLP-1 can lead to the increase of intracellular cAMP content. mGLP-1 has a protective effect on glutamate-induced neuronal injury, which may be due to the anti-oxidative damage of mGLP-1 to nerve cells rather than the improvement of glutamate-induced imbalance of calcium homeostasis. The protective effect of mGLP-1 is similar to that of nGLP-1, while mGLP-1 is more stable and has longer half-life in vivo. It suggests that mGLP-1 may have better potential value for the treatment of neurodegenerative diseases.