硫酸镁对人脐静脉血管内皮细胞放射损伤的防护作用

发布时间：2018-10-26 20:56

【摘要】：目的：探讨硫酸镁对人脐静脉血管内皮细胞（HUVEC）细胞经60Co γ射线照射后细胞增殖、周期、凋亡的影响，并分析其可能的机制。同时观察硫酸镁对胶质瘤U251细胞照射后细胞周期、凋亡的影响。研究结果为硫酸镁的临床应用提供实验依据和支撑。方法： 1、取对数生长期的HUVEC细胞，采用CCK-8法检测硫酸镁对细胞存活率及增殖的影响，筛选合适浓度的硫酸镁。用流式细胞仪检测细胞周期分布和AnnexinV/PI双染色法检测细胞凋亡情况。 2、用NO试剂盒和SOD试剂盒分别测定NO含量和SOD活性，硫代巴比妥酸法（thiobarbituric acid,TBA）测定丙二醛（malondialdehyde,MDA）含量变化。 3、Real-Time PCR方法检测ICAM-1mRNA、NF-κB mRNA表达。 4、不同浓度的硫酸镁预处理对数生长期的脑胶质瘤U251细胞，经γ射线照射后不同时间点收集细胞，，采用流式细胞仪检测细胞周期、细胞凋亡率的变化。 5、数据分析采用SAS8.0统计软件包，采用均数±标准差表示，组间差异进行方差分析，P0.05为差异有统计学意义。结果： 1、 CCK-8检测结果显示：在1.25mg/mL~6.25mg/mL浓度范围内硫酸镁对HUVEC细胞无毒性，其中6.25mg/mL硫酸镁对γ射线照射或非照射的细胞增殖皆无影响，而大于6.25mg/mL的硫酸镁对照射或非照射的HUVEC细胞皆有不同程度的增殖抑制作用。 2、6.25mg/mL及大于6.25mg/mL硫酸镁能不同程度的缓解HUVEC细胞经照射后产生的G2/M期阻滞。照射组细胞凋亡率较空白对照组均有不同程度的增加，但48h时，6.25mg/mL、12.5mg/mL硫酸镁处理组HUVEC细胞凋亡率较24h有所恢复，说明硫酸镁能部分缓解HUVEC细胞凋亡。 3、HVEC细胞经γ射线照射后，在不同的时间点收集细胞，细胞内NO含量、MDA含量增加，SOD活性降低，较空白对照组皆有统计学差异（p0.05）。6.25mg/mL硫酸镁能提高细胞内SOD活性，降低细胞内自由基水平。 4. Real-Time PCR结果显示，γ射线能增加细胞内ICAM-1mRNA、NF-κB mRNA的表达,6.25mg/mL硫酸镁能在一定程度上降低二者的表达水平。 5、6.25mg/mL、12.5mg/mL的硫酸镁能增加胶质瘤U251细胞照射后12h、24h的凋亡率。结论： 1、低浓度的硫酸镁对HUVEC细胞无毒性，较高浓度的硫酸镁对HUVEC细胞增殖具有抑制作用。 2、硫酸镁能缓解HUVEC细胞γ射线照射后G2/M阻滞、细胞凋亡。 3、硫酸镁保护HUVEC细胞辐射损伤的机制可能是通过降低ICAM-1mRNA、NF--κB mRNA表达来实现。 4、硫酸镁能不同程度的增加受照后胶质瘤U251细胞的凋亡，提高U251细胞辐射敏感性。
[Abstract]:Aim: to investigate the effects of magnesium sulfate on proliferation, cell cycle and apoptosis of human umbilical vein endothelial cell (HUVEC) cells irradiated by 60Co 纬 -rays, and to analyze its possible mechanism. To observe the effect of magnesium sulfate on cell cycle and apoptosis after irradiation on U251 glioma cells. The results provide experimental basis and support for the clinical application of magnesium sulfate. Methods: 1. The effects of magnesium sulfate on cell survival and proliferation were detected by CCK-8 assay in logarithmic growth phase of HUVEC cells, and the appropriate concentration of magnesium sulfate was screened. Cell cycle distribution was detected by flow cytometry and apoptosis was detected by AnnexinV/PI double staining. 2. The content of NO and the activity of SOD were determined by NO kit and SOD kit, and the content of malondialdehyde (malondialdehyde,MDA) by thiobarbituric acid (thiobarbituric acid,TBA). The expression of ICAM-1mRNA,NF- 魏 B mRNA was detected by Real-Time PCR. 4. Different concentrations of magnesium sulfate pretreated U251 glioma cells at logarithmic growth stage. The cells were collected at different time points after 纬 -ray irradiation. The cell cycle and apoptosis rate were detected by flow cytometry. 5. SAS8.0 statistical software package was used to analyze the data, and the mean 卤standard deviation was used to analyze the variance between groups. Results: 1. The results of CCK-8 showed that magnesium sulfate had no toxicity to HUVEC cells in the range of 1.25mg/mL~6.25mg/mL concentration, and 6.25mg/mL magnesium sulfate had no effect on the proliferation of cells irradiated by 纬 -rays or non-irradiated cells. Magnesium sulfate larger than 6.25mg/mL inhibited the proliferation of HUVEC cells in different degrees. (2) 6.25 mg / mL and larger than 6.25mg/mL magnesium sulfate could relieve the G _ 2 / M phase arrest of HUVEC cells after irradiation to varying degrees. The apoptosis rate of HUVEC cells in irradiation group was higher than that in blank control group, but at 48 h, the apoptosis rate of HUVEC cells in 6.25 mg / mL magnesium sulfate group was significantly higher than that in 24 h, indicating that magnesium sulfate could partly alleviate the apoptosis of HUVEC cells. 3After 纬 -ray irradiation, the NO content, MDA content and SOD activity of HHVEC cells were increased and decreased at different time points. Compared with the control group, there was significant difference between the two groups (p0.05). Magnesium sulfate of 6.25mg/mL could increase the activity of SOD and decrease the level of intracellular free radical. 4. Real-Time PCR results showed that 纬 -rays could increase the expression of ICAM-1mRNA,NF- 魏 B mRNA in the cells, and that 6.25mg/mL magnesium sulfate could decrease the expression level of ICAM-1mRNA,NF- 魏 B mRNA to some extent. (5) magnesium sulfate of 12.5 mg / mL at 6.25 mg / mL could increase the apoptosis rate of U251 glioma cells at 12 h and 24 h after irradiation. Conclusion: 1. Low concentration of magnesium sulfate has no toxicity to HUVEC cells, and high concentration of magnesium sulfate can inhibit the proliferation of HUVEC cells. 2, magnesium sulfate can relieve G 2 / M arrest and apoptosis of HUVEC cells after 纬-ray irradiation. 3. The mechanism of magnesium sulfate protecting HUVEC cells from radiation damage may be by decreasing the expression of ICAM-1mRNA,NF-- 魏 B mRNA. 4. Magnesium sulfate could increase the apoptosis of U251 cells and enhance the radiosensitivity of U251 cells.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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