【摘要】：第一部分低氧微環(huán)境加放療對Hep-2細胞系CD133+細胞的富集作用 目的：觀察體外低氧和常氧培養(yǎng)下喉癌Hep-2細胞系在放療前后CD133+細胞比例及生長抑制率的變化，探討腫瘤干細胞在低氧介導(dǎo)的喉癌放療抵抗中的作用。方法：體外常氧和低氧環(huán)境下培養(yǎng)人喉癌Hep-2細胞，分別給予不同劑量的射線照射，檢測各組細胞放療后不同時間細胞生長抑制率和CD133+細胞比例的變化。結(jié)果：常氧組各個劑量和時間點的生長抑制率均高于低氧組，24小時、10Gy時差異最大，具有顯著性（P0.05）。放療后CD133+細胞低氧和常氧組均有不同程度的富集，低氧組在各個劑量和時間點的CD133+細胞比例均高于常氧組，24小時、10Gy時差異最大，具有顯著性（P0.05）。結(jié)論：喉癌干細胞在低氧介導(dǎo)的喉癌放療抵抗中有重要作用，并提示阻斷低氧因素可能會增強喉癌的放療敏感性。 第二部分喉癌干細胞在低氧介導(dǎo)的喉癌放療抵抗中作用機制 目的：探討腫瘤干細胞在低氧介導(dǎo)的喉癌放療抵抗中的作用機制。 方法：體外培養(yǎng)人喉癌Hep-2細胞和HIF-1α沉默的Hep-2細胞，分別用流式細胞儀分選出CD133+細胞，并分別在低氧和常氧條件下行無血清培養(yǎng)，由此分為四組:（1）低氧培養(yǎng)的CD133+細胞為A組；（2）常氧培養(yǎng)的CD133+細胞為B組；（3）低氧培養(yǎng)的HIF-1α沉默的CD133+細胞為C組（4）常氧培養(yǎng)的HIF-1α沉默的CD133+細胞為D組。各組細胞分別給予不同劑量的照射，照射后不同時間行MTT檢測。行軟瓊脂克隆形成實驗，并給予10Gy照射，14天后觀察四組細胞克隆形成率。四組細胞分別給予10Gy照射，24小時后用流式細胞儀分別檢測DNA-PKcs、ATM、Survivin、P53的表達。結(jié)果：流式細胞儀分選后獲得CD133+細胞純度是92.8%和94.1%，CD133+細胞B、C、D三組放療后各個劑量和時間點的生長抑制率均高于A組。放療前后CD133+細胞克隆形成率均高于CD133-細胞。A組的克隆形成率均明顯高于B、C、D組（P0.05）。A組放療后DNA-PKcs、Survivin表達均較其余三組高（P0.05），而ATM、P53表達無明顯差別（P0.05）。結(jié)論：喉癌干細胞在低氧介導(dǎo)的喉癌放療抵抗中有重要作用，其放療抵抗的機制是通過激活以DNA-PK為主的NHEJ和以ATM為主的HR兩種途徑來修復(fù)損傷的DNA，而低氧導(dǎo)致的喉癌干細胞放療抵抗能力的增強則主要是通過DNA-PK的活性增強實現(xiàn)的。 第三部分喉癌干細胞對低氧介導(dǎo)的喉癌放療抵抗作用的動物實驗 目的：通過觀察喉癌干細胞在裸鼠體內(nèi)成瘤情況，及放療后腫瘤內(nèi)的CD133+細胞比例、各種蛋白表達情況，進一步證實喉癌干細胞在低氧介導(dǎo)的喉癌放療抵抗中的作用。方法：取HIF-1α沉默和未沉默的CD133+細胞分別注入裸鼠背部皮下成瘤，成瘤后，隨機將12只裸鼠分為4組：HIF-1α未沉默放射組（A組），HIF-1α未沉默對照組（Ac組）HIF-1α沉默放射組（B組）、HIF-1α沉默對照組（Bc組）,每組3只。2周后A組和B組給予10Gy放療，Ac組和Bc組給予0Gy。每隔2日測量腫瘤大小，2周后殺鼠取瘤，測腫瘤重量，計算抑瘤率，測CD133+細胞比例，和DNA-PKcs、ATM、P53、Survivin蛋白的表達。結(jié)果：CD133+細胞只需4000-10000就能成瘤。放療后抑瘤率A組較B組低（P0.05）；CD133+細胞比例放療組均大于相應(yīng)對照組，，A組大于B組（P0.05）。DNA-PKcs、ATM、P53、Survivin蛋白的表達放療組均大于相應(yīng)對照組（P0.05），DNA-PKcs、Survivin表達A組大于B組（P0.05），而ATM、P53的表達A組和B組無明顯差異。結(jié)論：CD133+細胞對放療有抵抗作用，其放療抵抗的機制是通過激活NHEJ和HR兩種途徑來修復(fù)損傷的DNA，而低氧導(dǎo)致的其抵抗能力的增強則主要是通過DNA-PK的活性增強和干細胞數(shù)量增多實現(xiàn)的。
[Abstract]:The enrichment of the first part of low-oxygen micro-environment plus radiotherapy on the CD133 + cells of the Hep-2 cell line Objective: To observe the changes of the ratio of CD133 + cells and the rate of growth inhibition before and after radiotherapy for laryngeal carcinoma Hep-2 cell line under low oxygen and normal oxygen culture in vitro Study on the role of tumor stem cells in the hypoxia-mediated radiotherapy resistance of laryngeal carcinoma Methods: Human laryngeal carcinoma (Hep-2) cells were cultured in vitro and in low-oxygen environment, and different doses of radiation were given respectively. The inhibition rate of cell growth and the proportion of CD133 + cells were detected in different time after the irradiation of each group of cells. Results: The inhibition rate of each dose and time point of the normal oxygen group was higher than that of the hypoxia group, and the difference was the most significant at 24 hours and 10 Gy (P0.05). The CD133 + cells in the hypoxic group and the normal oxygen group had different levels of enrichment. The proportion of CD133 + cells in the hypoxic group was higher than that of the normal oxygen group, 24 hours and 10 Gy, and the difference was significant (P0.05). Conclusion: Laryngeal cancer stem cells play an important role in the hypoxia-mediated radiotherapy resistance of laryngeal carcinoma, and it is suggested that the blocking of hypoxia may be sensitive to the radiotherapy of laryngeal carcinoma. The second part of laryngeal cancer stem cells in the hypoxia-mediated resistance of laryngeal cancer Objective: To investigate the effect of tumor stem cells in the resistance of hypoxia-mediated radiotherapy for laryngeal carcinoma Methods: The human laryngeal carcinoma (HIF-2) and HIF-1 (HIF-1) silent Hep-2 cells were cultured in vitro, and the CD133 + cells were selected by flow cytometry. 3 + cells were group A; (2) CD133 + cells in normal oxygen culture were group B; (3) HIF-1 and silent CD133 + cells in hypoxia culture were HIF-1 and silence CD13 in C group (4) The 3 + cells were group D. The cells of each group were given different doses of irradiation, and the cells were not at the same time after irradiation. Inter-line MTT assay was performed. A soft agar clone was used to form an experiment, and irradiated with 10Gy, and four groups were observed after 14 days. DNA-PKcs, ATM, and Survivin were detected by flow cytometry, respectively. The results showed that the purity of CD133 + cells was 92.8% and 94.1% after sorting by flow cytometry, and the growth and inhibition of each dose and time point in the three groups of CD133 + cells B, C and D were inhibited. The rate of CD133 + cell clone before and after radiotherapy was higher than that of group A. The expression of DNA-PKcs and Survivin in group A was significantly higher than that in group B, C and D (P0.05). The expression of DNA-PKcs and Survivin in group A was higher than that of the other three groups (P0.05). Conclusion: Laryngeal cancer stem cells play an important role in the hypoxia-mediated resistance of laryngeal cancer. The mechanism of radiotherapy resistance is to repair by activating NHEJ, which is mainly DNA-PK, and HR-based HR. DNA of complex injury, while the enhancement of radiation resistance of laryngeal cancer stem cells caused by hypoxia is mainly through DNA-PK The effect of the third part of laryngeal cancer stem cells on hypoxia-mediated laryngeal squamous cell carcinoma The purpose of animal experiments in the treatment of resistance to treatment: by observing the tumor of laryngeal cancer stem cells in the nude mice, and the CD133 + cells in the tumor after radiotherapy The expression of various proteins and the expression of various proteins further confirm that the stem cells of the laryngeal carcinoma are mediated by hypoxia Methods: The HIF-1 silent and unsilent CD133 + cells were injected into the back of the nude mice to form a tumor, and 12 bare mice were randomly divided into 4 groups: the HIF-1, the non-silent radiation group (group A), the HIF-1 and the silent control group (Ac group) HIF-1 group of silent radiation (group B), HIF-1 and silence control group (Bc group), 3 rats in each group. The tumor size was measured every 2 weeks after 2 weeks, the tumor weight was measured, the tumor-inhibiting rate was calculated, the proportion of CD133 + cells, and the DNA-PKcs, ATM, P53 and Su were measured. Expression of survivin protein. Results: CD133 + cells only need 400 The tumor-inhibiting rate in group A was lower in group A than in group B (P0.05). The group of CD133 + cells was more than that in group B (P0.05). The expression of DNA-PKcs, ATM, P53 and Survivin was greater than that of the control group (P0.05). The expression of DNA-PKcs and Survivin in group A was greater than that of group B (P0.05). P0. 05), and the table of ATM and P53 Conclusion: CD133 + cells are resistant to radiotherapy. The mechanism of radiotherapy resistance is to repair the damaged DNA by activating both NHEJ and HR, while the enhancement of resistance of CD133 + cells is mainly enhanced by the activity of DNA-PK.
【學(xué)位授予單位】：中國人民解放軍軍醫(yī)進修學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R739.65

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