喉癌干细胞在低氧介导的喉癌放疗抵抗中的作用
发布时间：2019-02-16 18:22
【摘要】：第一部分低氧微环境加放疗对Hep-2细胞系CD133+细胞的富集作用 目的：观察体外低氧和常氧培养下喉癌Hep-2细胞系在放疗前后CD133+细胞比例及生长抑制率的变化，探讨肿瘤干细胞在低氧介导的喉癌放疗抵抗中的作用。方法：体外常氧和低氧环境下培养人喉癌Hep-2细胞，分别给予不同剂量的射线照射，检测各组细胞放疗后不同时间细胞生长抑制率和CD133+细胞比例的变化。结果：常氧组各个剂量和时间点的生长抑制率均高于低氧组，24小时、10Gy时差异最大，具有显著性（P0.05）。放疗后CD133+细胞低氧和常氧组均有不同程度的富集，低氧组在各个剂量和时间点的CD133+细胞比例均高于常氧组，24小时、10Gy时差异最大，具有显著性（P0.05）。结论：喉癌干细胞在低氧介导的喉癌放疗抵抗中有重要作用，并提示阻断低氧因素可能会增强喉癌的放疗敏感性。 第二部分喉癌干细胞在低氧介导的喉癌放疗抵抗中作用机制 目的：探讨肿瘤干细胞在低氧介导的喉癌放疗抵抗中的作用机制。 方法：体外培养人喉癌Hep-2细胞和HIF-1α沉默的Hep-2细胞，分别用流式细胞仪分选出CD133+细胞，并分别在低氧和常氧条件下行无血清培养，由此分为四组:（1）低氧培养的CD133+细胞为A组；（2）常氧培养的CD133+细胞为B组；（3）低氧培养的HIF-1α沉默的CD133+细胞为C组（4）常氧培养的HIF-1α沉默的CD133+细胞为D组。各组细胞分别给予不同剂量的照射，照射后不同时间行MTT检测。行软琼脂克隆形成实验，并给予10Gy照射，14天后观察四组细胞克隆形成率。四组细胞分别给予10Gy照射，24小时后用流式细胞仪分别检测DNA-PKcs、ATM、Survivin、P53的表达。结果：流式细胞仪分选后获得CD133+细胞纯度是92.8%和94.1%，CD133+细胞B、C、D三组放疗后各个剂量和时间点的生长抑制率均高于A组。放疗前后CD133+细胞克隆形成率均高于CD133-细胞。A组的克隆形成率均明显高于B、C、D组（P0.05）。A组放疗后DNA-PKcs、Survivin表达均较其余三组高（P0.05），而ATM、P53表达无明显差别（P0.05）。结论：喉癌干细胞在低氧介导的喉癌放疗抵抗中有重要作用，其放疗抵抗的机制是通过激活以DNA-PK为主的NHEJ和以ATM为主的HR两种途径来修复损伤的DNA，而低氧导致的喉癌干细胞放疗抵抗能力的增强则主要是通过DNA-PK的活性增强实现的。 第三部分喉癌干细胞对低氧介导的喉癌放疗抵抗作用的动物实验 目的：通过观察喉癌干细胞在裸鼠体内成瘤情况，及放疗后肿瘤内的CD133+细胞比例、各种蛋白表达情况，进一步证实喉癌干细胞在低氧介导的喉癌放疗抵抗中的作用。方法：取HIF-1α沉默和未沉默的CD133+细胞分别注入裸鼠背部皮下成瘤，成瘤后，随机将12只裸鼠分为4组：HIF-1α未沉默放射组（A组），HIF-1α未沉默对照组（Ac组）HIF-1α沉默放射组（B组）、HIF-1α沉默对照组（Bc组）,每组3只。2周后A组和B组给予10Gy放疗，Ac组和Bc组给予0Gy。每隔2日测量肿瘤大小，2周后杀鼠取瘤，测肿瘤重量，计算抑瘤率，测CD133+细胞比例，和DNA-PKcs、ATM、P53、Survivin蛋白的表达。结果：CD133+细胞只需4000-10000就能成瘤。放疗后抑瘤率A组较B组低（P0.05）；CD133+细胞比例放疗组均大于相应对照组，，A组大于B组（P0.05）。DNA-PKcs、ATM、P53、Survivin蛋白的表达放疗组均大于相应对照组（P0.05），DNA-PKcs、Survivin表达A组大于B组（P0.05），而ATM、P53的表达A组和B组无明显差异。结论：CD133+细胞对放疗有抵抗作用，其放疗抵抗的机制是通过激活NHEJ和HR两种途径来修复损伤的DNA，而低氧导致的其抵抗能力的增强则主要是通过DNA-PK的活性增强和干细胞数量增多实现的。
[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.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.65
本文编号：2424716
[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.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R739.65
【参考文献】
相关期刊论文 前4条
1 吴俊;史宏灿;;低氧在肿瘤中的研究进展[J];肿瘤基础与临床;2006年06期
2 刘春玲;李晓明;路秀英;张立坤;;HIF-1α反义寡核苷酸对喉鳞状细胞癌hep-2细胞放疗的增效作用[J];临床耳鼻咽喉头颈外科杂志;2007年13期
3 王卫东,陈正堂,段玉忠,赵志强,钱桂生;一种用于定量研究细胞缺氧的简易模型[J];中国病理生理杂志;2003年08期
4 卫旭东;周梁;程磊;田洁;;CD133作为喉癌肿瘤起始细胞标志的实验研究[J];中华耳鼻咽喉头颈外科杂志;2006年12期
本文编号：2424716
本文链接：https://www.wllwen.com/yixuelunwen/wuguanyixuelunwen/2424716.html
最近更新
教材专著
