【摘要】：目的： 探討硫酸鎂對(duì)人臍靜脈血管內(nèi)皮細(xì)胞（HUVEC）細(xì)胞經(jīng)60Co γ射線照射后細(xì)胞增殖、周期、凋亡的影響，并分析其可能的機(jī)制。同時(shí)觀察硫酸鎂對(duì)膠質(zhì)瘤U251細(xì)胞照射后細(xì)胞周期、凋亡的影響。研究結(jié)果為硫酸鎂的臨床應(yīng)用提供實(shí)驗(yàn)依據(jù)和支撐。 方法： 1、取對(duì)數(shù)生長(zhǎng)期的HUVEC細(xì)胞，采用CCK-8法檢測(cè)硫酸鎂對(duì)細(xì)胞存活率及增殖的影響，篩選合適濃度的硫酸鎂。用流式細(xì)胞儀檢測(cè)細(xì)胞周期分布和AnnexinV/PI雙染色法檢測(cè)細(xì)胞凋亡情況。 2、用NO試劑盒和SOD試劑盒分別測(cè)定NO含量和SOD活性，硫代巴比妥酸法（thiobarbituric acid,TBA）測(cè)定丙二醛（malondialdehyde,MDA）含量變化。 3、Real-Time PCR方法檢測(cè)ICAM-1mRNA、NF-κB mRNA表達(dá)。 4、不同濃度的硫酸鎂預(yù)處理對(duì)數(shù)生長(zhǎng)期的腦膠質(zhì)瘤U251細(xì)胞，經(jīng)γ射線照射后不同時(shí)間點(diǎn)收集細(xì)胞，，采用流式細(xì)胞儀檢測(cè)細(xì)胞周期、細(xì)胞凋亡率的變化。 5、數(shù)據(jù)分析采用SAS8.0統(tǒng)計(jì)軟件包，采用均數(shù)±標(biāo)準(zhǔn)差表示，組間差異進(jìn)行方差分析，P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1、 CCK-8檢測(cè)結(jié)果顯示：在1.25mg/mL~6.25mg/mL濃度范圍內(nèi)硫酸鎂對(duì)HUVEC細(xì)胞無(wú)毒性，其中6.25mg/mL硫酸鎂對(duì)γ射線照射或非照射的細(xì)胞增殖皆無(wú)影響，而大于6.25mg/mL的硫酸鎂對(duì)照射或非照射的HUVEC細(xì)胞皆有不同程度的增殖抑制作用。 2、6.25mg/mL及大于6.25mg/mL硫酸鎂能不同程度的緩解HUVEC細(xì)胞經(jīng)照射后產(chǎn)生的G2/M期阻滯。照射組細(xì)胞凋亡率較空白對(duì)照組均有不同程度的增加，但48h時(shí)，6.25mg/mL、12.5mg/mL硫酸鎂處理組HUVEC細(xì)胞凋亡率較24h有所恢復(fù)，說(shuō)明硫酸鎂能部分緩解HUVEC細(xì)胞凋亡。 3、HVEC細(xì)胞經(jīng)γ射線照射后，在不同的時(shí)間點(diǎn)收集細(xì)胞，細(xì)胞內(nèi)NO含量、MDA含量增加，SOD活性降低，較空白對(duì)照組皆有統(tǒng)計(jì)學(xué)差異（p0.05）。6.25mg/mL硫酸鎂能提高細(xì)胞內(nèi)SOD活性，降低細(xì)胞內(nèi)自由基水平。 4. Real-Time PCR結(jié)果顯示，γ射線能增加細(xì)胞內(nèi)ICAM-1mRNA、NF-κB mRNA的表達(dá),6.25mg/mL硫酸鎂能在一定程度上降低二者的表達(dá)水平。 5、6.25mg/mL、12.5mg/mL的硫酸鎂能增加膠質(zhì)瘤U251細(xì)胞照射后12h、24h的凋亡率。 結(jié)論： 1、低濃度的硫酸鎂對(duì)HUVEC細(xì)胞無(wú)毒性，較高濃度的硫酸鎂對(duì)HUVEC細(xì)胞增殖具有抑制作用。 2、硫酸鎂能緩解HUVEC細(xì)胞γ射線照射后G2/M阻滯、細(xì)胞凋亡。 3、硫酸鎂保護(hù)HUVEC細(xì)胞輻射損傷的機(jī)制可能是通過(guò)降低ICAM-1mRNA、NF--κB mRNA表達(dá)來(lái)實(shí)現(xiàn)。 4、硫酸鎂能不同程度的增加受照后膠質(zhì)瘤U251細(xì)胞的凋亡，提高U251細(xì)胞輻射敏感性。
[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 緯 -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 緯 -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 鹵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 緯 -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 緯 -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 緯 -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 緯-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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

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