本文選題：VEGF + 放射敏感性��； 參考：《華中科技大學(xué)》2010年博士論文

【摘要】： 第一部分：放射線與纈沙坦聯(lián)合對(duì)鼻咽癌細(xì)胞生物學(xué)效應(yīng)的影響 目的：研究血管緊張素Ⅱ(AngⅡ)及其受體(AT1R)阻滯劑纈沙坦(valsartan)和gamma射線聯(lián)合對(duì)人鼻咽癌細(xì)胞系(CNE-2)的VEGF基因表達(dá),細(xì)胞增生與侵襲,放射敏感性的影響。 方法：用RT-PCR和ELISA分別檢測(cè)在干預(yù)前后CNE-2細(xì)胞血管內(nèi)皮生長(zhǎng)因子(VEGF)的基因和蛋白水平表達(dá)變化。用MTT檢測(cè)AngⅡ?qū)NE-2細(xì)胞增殖的作用。用體外侵襲實(shí)驗(yàn)測(cè)定AngⅡ及纈沙坦對(duì)鼻咽癌細(xì)胞CNE-2侵襲力的影響。用成克隆分析法觀察纈沙坦與放射聯(lián)合對(duì)CNE-2細(xì)胞體外存活效應(yīng)的影響,用流式細(xì)胞術(shù)和體外侵襲實(shí)驗(yàn)(小室法)檢測(cè)對(duì)細(xì)胞凋亡和侵襲能力的影響。 結(jié)果：AngⅡ可以誘導(dǎo)CNE-2表達(dá)VEGF上調(diào),對(duì)照組與10-9,10-8,10-7mol/L AngⅡ組VEGF分泌分別為246和350,521.5,595.5 pg/105 cell。AngⅡ還能誘導(dǎo)CNE-2增殖和侵襲,經(jīng)10-9,10-8,10-7mol/L AngⅡ處理后,侵襲細(xì)胞數(shù)分別為103,111,124,較對(duì)照組均有所增高。纈沙坦可以抑制AngⅡ的這些作用(p0.05)。在與gamma射線聯(lián)合的實(shí)驗(yàn)中,CNE-2細(xì)胞經(jīng)10-9、10-8、10-7mol/L纈沙坦與放射聯(lián)合作用后,放射增敏比(SER)分別為1.10、1.20、1.36。細(xì)胞侵襲能力在6Gy gammma射線和纈沙坦作用下,抑制率分別為8.11%、16.77%、16.49%。經(jīng)10-7mol/L纈沙坦和8Gy gamma放射線處理并孵育24h后,CNE-2細(xì)胞凋亡率為6.17%±0.22%,與單純放射組2.44%±0.72%相比有所提高(P0.05)。 結(jié)論：AngⅡ可以誘導(dǎo)鼻咽癌細(xì)胞CNE-2增殖和侵襲,AT1R阻滯劑纈沙坦能抑制這種作用,其機(jī)制可能涉及對(duì)VEGF的表達(dá)調(diào)控。不僅如此,AT1R阻滯劑還能在體外對(duì)細(xì)胞有放射增敏作用,與放射聯(lián)合能抑制細(xì)胞侵襲能力和在一定程度誘導(dǎo)細(xì)胞凋亡,這些可為纈沙坦體內(nèi)實(shí)驗(yàn)的放射增敏效應(yīng)提供基礎(chǔ)。 第二部分：放射線誘導(dǎo)下線粒體基因在小鼠組織中的變化 目的：研究小鼠在放射線下線粒體基因拷貝數(shù)的改變。 方法：所有石蠟組織來源于全身接受gamma和中子線照射的小鼠,相似的總劑量(550cGy)和三種不同水平的劑量率以及照射分次為我們選擇的實(shí)驗(yàn)分組。來源于5Gy gamma射線照射后的新鮮小鼠組織在24小時(shí)后被取出同樣應(yīng)用于該實(shí)驗(yàn)。實(shí)時(shí)定量PCR中絕對(duì)定量的方法被選擇檢測(cè)線粒體編碼的線粒體基因COX1, ND1, MTATP6和ATPCYB,同時(shí)與之相對(duì)應(yīng)的細(xì)胞核編碼的線粒體基因COX6B, NDUFV1, ATP5A1和CYB5B作為參照基因也會(huì)被檢測(cè)。它們的配對(duì)比值最終參與統(tǒng)計(jì)計(jì)算。 結(jié)果：石蠟組織中,小鼠不同組織對(duì)放射線有不同的反應(yīng)。中子射線在相同總劑量條件下較gamma射線對(duì)組織線粒體基因組有更大的損傷作用,并且它會(huì)誘導(dǎo)下調(diào)線粒體基因的拷貝數(shù),這與gamma射線的上調(diào)作用是完全相反的。在單個(gè)組織分析中,脾臟組織,心臟組織和腎臟單次照射和較高劑量率的gamma射線實(shí)驗(yàn)組才能使線粒體基因的拷貝顯著性升高。類似的結(jié)果在新鮮小鼠實(shí)驗(yàn)中也同樣被觀察到。除了放射線,在三種被檢測(cè)的組織中,多種疾病和小鼠死亡年齡都能顯著性改變線粒體基因拷貝數(shù)。 結(jié)論：小鼠線粒體基因拷貝數(shù)會(huì)因放射線的作用,所患多種疾病和死亡年齡的改變而發(fā)生改變。僅急性gamma放射線才會(huì)產(chǎn)生顯著影響,所涉及的機(jī)制可能與線粒體基因組的復(fù)制和修復(fù)機(jī)制有關(guān)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R739.63

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