乏氧诱导因子-1α抑制剂盐酸小檗碱和蜂毒肽对乏氧肿瘤细胞放射敏感性的影响及机制研究

发布时间：2018-04-21 09:44

本文选题：放射治疗 + 盐酸小檗碱　；参考：《南京医科大学》2016年博士论文

【摘要】：第一部分：乏氧诱导因子-1α抑制剂盐酸小檗碱对乏氧食管鳞癌细胞放射敏感性的影响及其机制研究目的：观察盐酸小檗碱对乏氧食管鳞癌细胞乏氧诱导因子-1α (hypoxia inducible factor-1α, HIF-1 α)及其下游靶基因血管内皮生成因子(vascμ lar endothelial growth factor, VEGF)的作用,体内外实验研究盐酸小檗碱对多个乏氧食管鳞癌放射敏感性的影响,探讨盐酸小檗碱增加放射线对乏氧食管鳞癌的敏感性机制。方法：选用2种食管鳞癌细胞株ECA109、TE13,研究梯度浓度及不同时间点下盐酸小檗碱处理肿瘤细胞,采用四甲基偶氮唑盐比色法(MTT法)观察盐酸小檗碱对食管鳞癌细胞作用；采用免疫荧光技术观察乏氧前后ECA109细胞HIF-1α表达情况,并观察高低浓度盐酸小檗碱作用后HIF-1α表达；应用蛋白免疫印迹法(Western-blot)测定高低浓度盐酸小檗碱处理后细胞HIF-1α、VEGF表达变化；分别给予常氧细胞、乏氧细胞、乏氧细胞联合不同浓度下盐酸小檗碱、顺铂阳性参照组6MV-X线照射处理后,平板克隆形成实验观察盐酸小檗碱对乏氧食管鳞癌放射敏感性的影响；采用流式细胞术检测AnnexinV-PI双染色后各组细胞凋亡率；构建裸鼠食管鳞癌ECA109移植瘤模型,设立对照组、单纯照射组、腹腔注射盐酸小檗碱组、腹腔注射盐酸小檗碱联合照射组,予照射组皮下移植瘤8Gy单次大剂量放疗,放疗14天后处死裸鼠,测量肿瘤体积,免疫组化法检测肿瘤内HIF-1α、VEGF表达情况。结果：盐酸小檗碱抑制食管鳞癌细胞ECA109和TE13的生长,具有时间以及剂量依赖性；通过单击多靶模型拟合曲线,可见不同浓度的盐酸小檗碱预处理乏氧食管鳞癌细胞24小时后,与照射组相比,增加乏氧食管鳞癌细胞的辐射损伤,盐酸小檗碱作用乏氧细胞照射组的细胞凋亡率增加,且成剂量依赖性；盐酸小檗碱作用于乏氧食管癌细胞时,可以使乏氧相关蛋白HIF-1α、VEGF的表达降低,并且呈现量效关系,且减少ECA109细胞内核内HIF-1α表达；盐酸小檗碱可显著抑制放疗后ECA109细胞移植瘤模型裸鼠皮下肿瘤生长,免疫组化显示盐酸小檗碱可降低移植瘤内HIF-1α、VEGF表达。结论：盐酸小檗碱降低乏氧食管鳞癌细胞HIF-1α及VEGF表达,增加乏氧食管鳞癌细胞放射敏感性；抑制食管鳞癌裸鼠移植瘤放疗后肿瘤组织生长。第二部分：乏氧诱导因子-1α抑制剂蜂毒肽对乏氧鼻咽癌细胞CNE-2放射敏感性的影响及其机制研究目的：观察蜂毒肽对乏氧鼻咽癌细胞CNE-2乏氧诱导因子-1α (hypoxia inducible factor-1α, HIF-1α)及其下游靶基因血管内皮生成因子(vascμ lar endothelial growth factor, VEGF)的抑制情况,研究蜂毒肽对CNE-2细胞株放射敏感性的影响,探讨蜂毒肽增加乏氧鼻咽癌细胞放射敏感性的机制。方法：‘选用鼻咽癌细胞CNE-2进行研究,梯度浓度及不同时间点蜂毒肽处理肿瘤细胞后,采用CCK8法观察蜂毒肽对CNE-2细胞增殖能力的影响。应用蛋白免疫印迹法(Western-blot)测定不同浓度蜂毒肽处理后CNE-2细胞内HIF-1α、 VEGF表达变化；分别给予常氧细胞、乏氧细胞,乏氧细胞联合蜂毒肽、常氧细胞联合蜂毒肽6MV-X线照射处理后,平板克隆形成实验观察蜂毒肽对乏氧CNE-2细胞放射敏感性的影响；AnnexinV-PI双染色后采用流式细胞术检测各组细胞凋亡率；构建裸鼠CNE-2移植瘤模型,设立对照组、单纯照射组、腹腔注射蜂毒肽组、腹腔注射蜂毒肽联合照射组,予照射组皮下移植瘤6Gy单次大剂量放疗,放疗14天后处死裸鼠,对移植瘤拍照并测量肿瘤体积。结果：蜂毒肽抑制CNE-2细胞生长,具有时间以及剂量依赖性；通过单击多靶模型拟合曲线,可见蜂毒肽对常氧和乏氧鼻咽癌细胞均有增敏作用,且在乏氧组增敏效果更为明显。相比于常氧细胞照射组,可以显著增加常氧CNE-2细胞的辐射敏感性,而蜂毒肽对乏氧CNE-2细胞照射组中的细胞凋亡率增加更为明显。蜂毒肽作用于乏氧CNE-2细胞,诱导乏氧相关蛋白HIF-1α、VEGF的表达降低,呈现量效关系。蜂毒肽抑制放疗后CNE-2细胞移植瘤模型裸鼠皮下肿瘤生长。结论：蜂毒肽抑制乏氧鼻咽癌细胞CNE-2内HIF-1α及其VEGF的表达,增加乏氧和常氧下CNE-2细胞放射敏感性；同时抑制鼻咽癌裸鼠移植瘤放疗后肿瘤组织生长。蜂毒肽对增加常氧和乏氧情况下鼻咽癌细胞的放疗敏感性,且对乏氧细胞作用更为明显,这一现象可能与HIF-1 α相关。
[Abstract]:The effects of berberine on the radiosensitivity of hypoxia inducible factor - 1伪 , HIF - 1伪 and vasc 渭lar endothelial growth factor ( VEGF ) were studied .
The expression of HIF - 1伪 in ECA109 cells before and after hypoxia was observed by immunofluorescence technique , and the expression of HIF - 1伪 was observed after berberine hydrochloride .
Western - blot was used to determine the expression of HIF - 1伪 and VEGF after treatment with berberine hydrochloride .
The effects of berberine hydrochloride on the radiosensitivity of patients with hypoxic esophageal squamous cell carcinoma were observed by using berberine hydrochloride and cisplatin positive reference group ( 6MV - X ) .
Flow cytometry was used to detect the apoptosis rate of each group after AnnexinV - PI double staining .
The growth , time and dose dependence of ECA109 and TE13 of esophageal squamous cell carcinoma were detected by using berberine hydrochloride combined with 8 Gy single - dose radiation therapy . The results showed that berberine hydrochloride could inhibit the growth of ECA109 and TE13 in esophageal squamous carcinoma cells , and had time and dose - dependent manner .
After 24 hours of pretreatment of hypoxic esophageal squamous cell carcinoma cells with berberine hydrochloride pretreatment with different concentrations of berberine hydrochloride , the apoptosis rate of cells irradiated by berberine hydrochloride was increased compared with the irradiation group , and the dose - dependent manner was observed .
When berberine hydrochloride acts on hypoxic esophageal cancer cells , the expression of HIF - 1伪 and VEGF in hypoxia - related protein can be reduced , and the expression of HIF - 1伪 in the inner core of ECA109 cells is reduced .
Conclusion : berberine hydrochloride can decrease the expression of HIF - 1伪 and VEGF and increase the radiosensitivity of hypoxic esophageal squamous carcinoma cells .
Objective : To investigate the effect of bee venom peptide on the radiosensitivity of CNE - 2 cell line CNE - 2 and its mechanism , and to investigate the effect of bee venom peptide on the radiosensitivity of CNE - 2 cell line CNE - 2 . The expression of HIF - 1伪 and VEGF in CNE - 2 cells was investigated by Western - blot .

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R73-36

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