【作者】 刘亮；

【导师】 贺永明； 崔凤梅； 陈秋；

【作者基本信息】 苏州大学 ， 内科学（专业学位）， 2015， 硕士

【摘要】 背景:血管稳态对人体健康至关重要,血管内皮损伤导致血管稳态破坏,将会导致包括冠心病在内的多种疾病的发生。放射性核素可以产生β射线,广泛用于肿瘤疾病的治疗,但其伴随的副作用不容忽视。我们推测:β射线的副作用与其损伤血管内皮,破坏了血管稳态有关,具体机制,国内外尚未见报道。因此,建立氚水(HTO)内照射模型来探讨放射性核素所产生的β射线对血管内皮细胞的损伤及修复机制。方法:将HTO加入脐血管内皮细胞(HUVEC)培养基中,连续培养三天,期间每天用CCK8比色测定法及台盼蓝细胞计数法来测定HTO组以及对照组细胞的增殖能力以及细胞活力。12小时内分别抽取6个不同的时间点,用实时荧光定量PCR技术检测HTO组HUVEC细胞中mi R-34a-5p表达水平。在四个不同的时间点,分别对对照组和HTO组的细胞使用彗星分析法和γ-H2AX免疫荧光染色法来测量DNA单链断裂和DNA双链断裂情况。通过脂质体2000将mi R-34a-5p类似物(mi R-34a-5p mimics),mi R-34a-5p抑制物(mi R-34a-5p inhibitor)以及它们的对照组分别转染进入HUVEC细胞中。将这四组细胞都暴露于HTO环境下,连续培养三天,用同样的方法来测定每天各组细胞的增殖能力以及细胞活力。用同样的方法测定四个时间点(0h,0.5h,2h,4h)上各组细胞的DNA单链断裂和DNA双链断裂的情况。最后,我们用RNA提取和逆转录聚合酶链反应的实验方法来测定上述六组HUVEC细胞中c-Myc以及STAG-2基因在0.5h,2h的表达情况。结果:与对照组相比,HTO组HUVEC细胞增殖能力及细胞活力在连续3天测定中均明显低于对照组。HTO组细胞中mi R-34a-5p的表达发生明显动态变化,根据mi R-34a-5p表达的动态变化,我们选择mi R-34a-5p表达最高点、最低点以及急性期的两个时间点来进行研究,分别是0h,0.5h,2h以及4h。除了0h以外,其余三个时间点,HTO组HUVEC细胞DNA单链以及双链的损伤相对于对照均明显增高,且趋势一致。同样的,在0.5h、2h、4h三个时间点上,mi R-34a-5p mimics组HUVEC细胞DNA单链以及双链的损伤均高于mi R-34a-5p inhibitor组,相反其细胞增殖能力以及细胞活力均明显低于mi R-34a-5p inhibitor组。我们观察到mi R-34a-5p mimics组中c-Myc以及STAG-2基因的表达在0.5h、2h均低于mi R-34a-5p inhibitor组。结论:mi R-34a-5p能够调节基因c-Myc以及STAG-2的表达来参与HTO引起HUVEC细胞损伤及修复过程。本研究从分子水平证实了β射线对血管内皮细胞的损伤机制,并为防治放射性核素临床毒副作用提供了崭新的视角。更多还原

【Abstract】 Background: Vascular homeostasis is important to our health. Damages to vascular endothelial cells lead to many diseases including coronary heart disease. Radionuclides irradiation which can produce β rays, is an important means of clinical treatment of malignancies, but the side effects caused by β rays cannot been ignored. We hypothesized that the side effects caused by β ray was related to the injury of vascular endothelial, which lead to the imbalance of blood vessel homeostasis, but the specific mechanisms have not been reported both at home and abroad. Therefore, we set up a model by tritiated water(HTO) to explore the mechanism of β-ray injury on vascular endothelium.Methods: After HTO was added into HUVEC cell suspension within three consecutive days, Cell Counting Kit-8 was used to evaluate the cell proliferation capacity and Trypan blue exclusion was used to measure the cell viability in each day. The expression of mi R-34a-5p was detected by QT-PCR within 12 H in HTO group at6 different time points. Comet assay and γH2AX Immunofluorescence staining were used to measure DNA single-strand breaks or DNA double-strand breaks in HTO and control group at 4 time points. Mi R-34a-5p mimics, mi R-34a-5p inhibitor and their controls were transfected into HUVEC cells by using lipofectamine 2000. The 4groups were irradiated by HTO. The same methods were used to measure the cell proliferation capacity, cell viability, DNA single-strand breaks and DNA double-strand breaks in those 4 groups at same condition as before. At last, we use RNA extraction and reverse transcriptase polymerase chain reaction to measure the expression of c-Myc and STAG-2 in 6 groups as mentioned above at 0.5h and 2h.Results: In HTO group, the cell proliferation capacity and viability were significantly lower within 3 days as compare with the control group. Mi R-34a-5p was changing within12 H after HTO irradiated HUVEC cell. According to the dynamic expression of mi R-34a-5p in HUVEC cell within 24 H, we selected the highest time point, the lowest time point and the 2 time points of acute phase, namely 0h, 0.5h, 2h and 4h to observe the function of mi R-34a-5p in cell which irradiated by HTO. The DNA single-strand breaks or DNA double-strand breaks were higher in HTO group than the control group at0.5h, 2h and 4h. Also, the trend of the two results at 0.5h, 2h and 4h are consistent. After HTO irradiated HUVEC cell, we found that over expression of mi R-34a-5p lead to DNA damage higher than suppression of mi R-34a-5p and the proliferation capacity and viability was lower than suppression of mi R-34a-5p at 0.5h, 2h and 4h. We also found that over expression of mi R-34a-5p can lead to the expression of c-Myc and STAG-2 lower than suppression of mi R-34a-5p at 0.5h and 2h time points.Conclusion: In summary, mi R-34a-5p regulated the expression of c-Myc and STAG-2 gene to participate in the process of cell damage caused by β-rays.Our study illust rated the damage mechanisms of vascular endothelial cells caused by β rays from the level of molecular. Apart from this, the study also has shown a completely new sight of preventing and treating the clinical side effects of radionuclide therapy.更多还原