放射线对小鼠组织线粒体基因和鼻咽癌细胞生物学行为影响的初步研究

发布时间：2018-05-27 19:39

本文选题：VEGF + 放射敏感性　；参考：《华中科技大学》2010年博士论文

【摘要】： 第一部分：放射线与缬沙坦联合对鼻咽癌细胞生物学效应的影响目的：研究血管紧张素Ⅱ(AngⅡ)及其受体(AT1R)阻滞剂缬沙坦(valsartan)和gamma射线联合对人鼻咽癌细胞系(CNE-2)的VEGF基因表达,细胞增生与侵袭,放射敏感性的影响。方法：用RT-PCR和ELISA分别检测在干预前后CNE-2细胞血管内皮生长因子(VEGF)的基因和蛋白水平表达变化。用MTT检测AngⅡ对CNE-2细胞增殖的作用。用体外侵袭实验测定AngⅡ及缬沙坦对鼻咽癌细胞CNE-2侵袭力的影响。用成克隆分析法观察缬沙坦与放射联合对CNE-2细胞体外存活效应的影响,用流式细胞术和体外侵袭实验(小室法)检测对细胞凋亡和侵袭能力的影响。结果：AngⅡ可以诱导CNE-2表达VEGF上调,对照组与10-9,10-8,10-7mol/L AngⅡ组VEGF分泌分别为246和350,521.5,595.5 pg/105 cell。AngⅡ还能诱导CNE-2增殖和侵袭,经10-9,10-8,10-7mol/L AngⅡ处理后,侵袭细胞数分别为103,111,124,较对照组均有所增高。缬沙坦可以抑制AngⅡ的这些作用(p0.05)。在与gamma射线联合的实验中,CNE-2细胞经10-9、10-8、10-7mol/L缬沙坦与放射联合作用后,放射增敏比(SER)分别为1.10、1.20、1.36。细胞侵袭能力在6Gy gammma射线和缬沙坦作用下,抑制率分别为8.11%、16.77%、16.49%。经10-7mol/L缬沙坦和8Gy gamma放射线处理并孵育24h后,CNE-2细胞凋亡率为6.17%±0.22%,与单纯放射组2.44%±0.72%相比有所提高(P0.05)。结论：AngⅡ可以诱导鼻咽癌细胞CNE-2增殖和侵袭,AT1R阻滞剂缬沙坦能抑制这种作用,其机制可能涉及对VEGF的表达调控。不仅如此,AT1R阻滞剂还能在体外对细胞有放射增敏作用,与放射联合能抑制细胞侵袭能力和在一定程度诱导细胞凋亡,这些可为缬沙坦体内实验的放射增敏效应提供基础。第二部分：放射线诱导下线粒体基因在小鼠组织中的变化目的：研究小鼠在放射线下线粒体基因拷贝数的改变。方法：所有石蜡组织来源于全身接受gamma和中子线照射的小鼠,相似的总剂量(550cGy)和三种不同水平的剂量率以及照射分次为我们选择的实验分组。来源于5Gy gamma射线照射后的新鲜小鼠组织在24小时后被取出同样应用于该实验。实时定量PCR中绝对定量的方法被选择检测线粒体编码的线粒体基因COX1, ND1, MTATP6和ATPCYB,同时与之相对应的细胞核编码的线粒体基因COX6B, NDUFV1, ATP5A1和CYB5B作为参照基因也会被检测。它们的配对比值最终参与统计计算。结果：石蜡组织中,小鼠不同组织对放射线有不同的反应。中子射线在相同总剂量条件下较gamma射线对组织线粒体基因组有更大的损伤作用,并且它会诱导下调线粒体基因的拷贝数,这与gamma射线的上调作用是完全相反的。在单个组织分析中,脾脏组织,心脏组织和肾脏单次照射和较高剂量率的gamma射线实验组才能使线粒体基因的拷贝显著性升高。类似的结果在新鲜小鼠实验中也同样被观察到。除了放射线,在三种被检测的组织中,多种疾病和小鼠死亡年龄都能显著性改变线粒体基因拷贝数。结论：小鼠线粒体基因拷贝数会因放射线的作用,所患多种疾病和死亡年龄的改变而发生改变。仅急性gamma放射线才会产生显著影响,所涉及的机制可能与线粒体基因组的复制和修复机制有关。
[Abstract]:Part I: the effect of radiation combined with valsartan on the biological effects of nasopharyngeal carcinoma cells
Objective: To study the effect of angiotensin II (Ang II) and its receptor (AT1R) blocker valsartan (valsartan) and gamma rays on the expression of VEGF gene, cell proliferation and invasion, and radiosensitivity of human nasopharyngeal carcinoma cell line (CNE-2).
Methods: the expression of gene and protein level of vascular endothelial growth factor (VEGF) in CNE-2 cells before and after intervention was detected by RT-PCR and ELISA. The effect of Ang II on the proliferation of CNE-2 cells was detected by MTT. The effect of Ang II and valsartan on the CNE-2 invasion of nasopharyngeal carcinoma cells was measured by MTT in vitro. The effect of combination of Tan and radiation on the survival of CNE-2 cells in vitro was detected by flow cytometry and in vitro invasion assay (chamber method).
Results: Ang II could induce the up-regulated CNE-2 expression of VEGF. The secretion of VEGF in the control group and the 10-9,10-8,10-7mol/L Ang II group was 246 and 350521.5595.5 pg/105 cell.Ang II, respectively, which could induce the proliferation and invasion of CNE-2 respectively. After 10-9,10-8,10-7mol/L Ang II treatment, the number of invasive cells was 103111124, respectively, compared with the control group. To inhibit these effects of Ang II (P0.05). In the combination of gamma rays with gamma rays, CNE-2 cells were combined with 10-9,10-8,10-7mol/L valsartan and radiation, and the radiation sensitization ratio (SER) was the 1.10,1.20,1.36. cell invasiveness, respectively, under the action of 6Gy gammma ray and valsartan, and the inhibitory rates were 8.11%, 16.77%, and 16.49%. via 10-7mol/L valerine, respectively. When Sartan and 8Gy gamma were irradiated and incubated with 24h, the apoptotic rate of CNE-2 cells was 6.17% + 0.22%, which was higher than that of the simple radiation group (2.44% + 0.72%) (P0.05).
Conclusion: Ang II can induce the proliferation and invasion of CNE-2 in nasopharyngeal carcinoma cells. The AT1R blocker valsartan can inhibit this effect. The mechanism may involve the regulation of the expression of VEGF. Not only so, the AT1R blocker can also have radiosensitization to the cells in vitro, and the combination with radiation can inhibit cell invasion and induce cell withering to a certain extent. These can provide the basis for the radiosensitizing effect of valsartan in vivo.
The second part: the change of mitochondrial gene induced by radiation in mouse tissues.
Objective: To study the changes of mitochondrial gene copy number in mice under irradiation.
Methods: all paraffin tissues were derived from gamma and neutron irradiated mice. Similar total dose (550cGy) and three different levels of dose rate and irradiation were selected for the experimental group. Fresh mice derived from 5Gy gamma ray were removed after 24 hours and were also applied to the experiment. The absolute quantitative method in the quantitative PCR is selected to detect mitochondrial encoded mitochondrial genes, COX1, ND1, MTATP6 and ATPCYB, and the mitochondrial gene COX6B, NDUFV1, ATP5A1 and CYB5B, corresponding to the corresponding nuclear coded genes, are also detected as reference genes. Their pairing ratio finally participates in the statistical calculation.
Results: in paraffin tissues, different tissues of mice have different responses to radiation. The neutron rays have more damage to the mitochondrial genome than the gamma rays at the same total dose, and it induces the down regulation of the copy number of the mitochondrial genes, which is completely opposite to the up regulation of gamma rays. In the analysis, the spleen tissue, the heart tissue, the single irradiation of the kidney and the high dose rate gamma ray experiment group could make the copy of the mitochondrial gene significantly increase. The similar results were also observed in the fresh mice. In addition to the radiation, many diseases and the death age of the mice could be significant in the three detected groups. Sex changes the number of copies of the mitochondrial gene.
Conclusion: the copy number of mitochondrial gene in mice can be altered by the action of radiation and changes in many diseases and age of death. Only acute gamma radiation will have a significant effect. The mechanism involved may be related to the mechanism of the replication and repair of the mitochondrial genome.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.63

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