谷氨酰胺对过氧化氢诱导氧化应激人结肠癌HT-29细胞损伤和凋亡的影响

发布时间：2018-05-06 03:06

本文选题：谷氨酰胺 + 氧化损伤　；参考：《动物营养学报》2017年08期

【摘要】：本试验旨在通过研究谷氨酰胺对过氧化氢(H_2O_2)诱导氧化应激人结肠癌HT-29细胞损伤和凋亡的影响,阐明谷氨酰胺的抗氧化效果和作用机理。HT-29细胞经不同浓度[0(对照组)、0.5、2.0、10.0 mmol/L]谷氨酰胺和0.35 mmol/L H_2O_2分别处理12、24、32 h后,测定细胞的超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,并采用荧光定量PCR方法分析谷氨酰胺对H_2O_2诱导的细胞凋亡相关基因的mRNA相对表达量,以及采用膜联蛋白V-异硫氰酸荧光素/碘化丙啶双染法对HT-29细胞染色,并用流式细胞仪检测细胞的凋亡情况。结果表明:1)处理24 h后,0.5、2.0 mmol/L Gln组SOD活性显著高于对照组(P0.05);处理32 h后,0.5、2.0 mmol/L Gln组SOD活性显著高于对照组(P0.05),MDA含量显著低于对照组(P0.05)。2)处理12 h后,各组天冬氨酸蛋白水解酶-3(Caspase-3)和B淋巴细胞瘤-2相关X蛋白(Bax)mRNA相对表达量无显著差异(P0.05)。与对照组比较,0.5 mmol/L Gln组核转录因子κB(NF-κB)mRNA相对表达量显著降低(P0.05),0.5与2.0 mmol/L Gln组B淋巴细胞瘤-2(Bcl-2)mRNA相对表达量显著提高(P0.05)。处理24 h后,与对照组比较,0.5、2.0和10.0 mmol/L Gln组Caspase-3、NF-κB和Bax mRNA相对表达量均显著降低(P0.05),Bcl-2mRNA相对表达量显著升高(P0.05)。处理32 h后,与对照组比较,2.0 mmol/L Gln组细胞表面诱导凋亡分子(FAS)、Caspase-3、NF-κB、Bax mRNA相对表达量均显著降低(P0.05),Bcl-2mRNA相对表达量显著升高(P0.05);10.0 mmol/L Gln组FAS、Caspase-3、NF-κB、Bax mRNA相对表达量均显著升高(P0.05)。3)处理24 h后,与对照组比较,Gln处理使活细胞数量提高了5.32%~11.97%,坏死细胞数量降低了6.75%~12.66%。处理32 h后,与对照组比较,Gln处理使活细胞数量提高了1.39%~7.63%,坏死细胞数量降低了3.40%~4.57%。由此可见,谷氨酰胺可抑制氧化应激反应,降低H_2O_2诱导的HT-29细胞凋亡。
[Abstract]:The purpose of this study was to investigate the effects of glutamine on oxidative stress induced HT-29 cell damage and apoptosis in human colon cancer cells induced by H _ 2O _ 2 / H _ 2O _ 2 / H _ 2O _ 2. To elucidate the antioxidation effect and mechanism of glutamine. HT-29 cells were treated with glutamine and 0.35 mmol/L H_2O_2 at different concentration [0 (control group) 0.52.0 ~ 10.0 mmol/L] for 32 h respectively. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in HT-29 cells were measured. The relative expression of glutamine on apoptosis-related genes induced by H_2O_2 was analyzed by fluorescence quantitative PCR, and HT-29 cells were stained by fluorescein isothiocyanate / pyridine iodide double staining. Apoptosis was detected by flow cytometry. The results showed that the activity of SOD was significantly higher in 0.5 mmol/L Gln group than that in control group after 24 h treatment, and significantly higher in 0.55 mmol/L Gln group than that in control group after 32 h treatment. There was no significant difference in the relative expression of aspartate proteolytic enzyme-3 (caspase-3) and B lymphocytoma-related X protein (Baxo) mRNA in each group (P 0. 05). Compared with the control group, the relative expression of NF-魏 B)mRNA in the 0.5 mmol/L Gln group was significantly lower than that in the 2. 0 mmol/L Gln group. The relative expression of NF-魏 B)mRNA in the B lymphocytoma of group 2. 0 was significantly higher than that in the group of 2. 0 mmol/L Gln. After treatment for 24 h, the relative expression of Caspase-3 NF- 魏 B and Bax mRNA in the 0. 5 mmol/L Gln group was significantly lower than that in the control group (P 0. 05) and the relative expression of Bcl-2 mRNA was significantly increased (P 0. 05). After treatment for 32 h, the relative expression of apoptosis-inducing Caspase-3NF- 魏 BnBax mRNA was significantly decreased in the 2. 0 mmol/L Gln group compared with the control group. The relative expression of Bcl-2 mRNA in the P0. 05 mmol/L Gln group was significantly higher than that in the control group. The relative expression of Caspase-3 NF- 魏 B Bax mRNA in the P0. 05 mmol/L Gln group was significantly higher than that in the P0. 05 mmol/L Gln group (P 0. 05. 3) after treatment, the relative expression of Caspase-3NF- 魏 B Bax mRNA was significantly increased at 24 h after treatment. Compared with the control group, GLN treatment increased the number of living cells by 5.32 and 11.97, and decreased the number of necrotic cells by 6.75 and 12.66. After 32 hours of treatment, compared with the control group, GLN treatment increased the number of living cells by 1.39 and 7.63, and the number of necrotic cells decreased by 3.40 and 4.57. Therefore, glutamine can inhibit oxidative stress reaction and reduce H_2O_2 induced apoptosis of HT-29 cells.
【作者单位】：浙江省农业科学院农产品质量标准研究所;浙江省植物有害生物防控省部共建国家重点实验室培育基地;贵州大学动物科学学院;浙江大学动物科学学院;
【基金】：国家自然科学基金(31402083) 浙江省自然科学基金重点项目(LZ15C170001)
【分类号】：S816

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