黑蒜提取液对lewis肺癌及Ht-29结肠癌放射增敏研究

发布时间：2018-02-11 10:10

本文关键词： 黑蒜 Lewis Ht-29 放射增敏分子机制　出处：《滨州医学院》2014年硕士论文　论文类型：学位论文

【摘要】：目的：观察黑蒜提取液(Aged Black Garlic Extract-ABGE)对Lewis市癌细胞株及结肠癌Ht-29小鼠移植瘤的放射增敏作用并探讨其可能机制。方法：常规培养lewis肺癌细胞,用不同浓度的黑蒜提取液联合6oCoγ线照射进行体外实验。MTT法测定黑蒜提取液对肺癌lewis细胞株生长抑制率；平板集落形成试验观察黑蒜提取液联合放疗对细胞克隆存活的影响；Hoechst荧光染色观察细胞形态的变化；用流式细胞仪检测黑蒜提取液作用后肿瘤细胞周期变化；Western Blot分析黑蒜提取液对lewis细胞Bcl-2、Bax蛋白表达影响。建立结肠癌Ht-29小鼠皮下移植瘤模型,将40只荷瘤小鼠随机分为4组,空白对照组,γ照射组,黑蒜组,黑蒜+γ照射组。分别给予生理盐水、γ射线照射、黑蒜提取液及黑蒜提取液联合γ射线照射的处理,观察各组移植瘤生长速度和各治疗组移植瘤生长延迟时间。实验结束后摘取眼球取血后脱臼处死小鼠,剥离瘤体称重,计算肿瘤生长抑制率；计算肿瘤生长延迟时间及放射增敏比；流式细胞术Annexin V-FITC/PI双染法检测肿瘤组织细胞凋亡情况；Realtime-PCR检测Bcl-2、Bax、Caspase-9的表达情况；外周血白细胞计数、紫外分光光度计法检测各组小鼠血清中SOD及CAT的活力变化。结果：(1)黑蒜提取液对肺癌lewis细胞株生长有明显抑制作用,黑蒜提取液联合60 Co γ射线照射能够促进lewis细胞凋亡,影响细胞周期及Bcl-2、Bax蛋白表达。(2)黑蒜提取液联合γ线照射对结肠癌Ht-29小鼠移植瘤有生长抑制作用,可延迟肿瘤生长时间,提高放疗增敏比,促进肿瘤细胞凋亡,并且能够下调移植瘤组织中Bcl-2,上调Bax,提高Caspase-9蛋白表达。黑蒜提取液可提高小鼠的外周血白细胞总数,增加红细胞超氧化物歧化酶SOD及CAT的活力。结论：(1)黑蒜提取液对肺癌lewis细胞具有放射增敏作用,其增敏机制可能与其改变细胞周期及影响B cl-2、B ax蛋白表达有关。(2)黑蒜提取液可通过下调Bcl-2上调Bax及活化Caspase-9蛋白的表达提高γ射线对结肠癌Ht-29小鼠移植瘤放射敏感性,并通过提高外周血白细胞计数和机体抗氧化能力提高对机体的辐射保护作用。
[Abstract]:Objective: to observe the radiosensitizing effect of Aged Black Garlic extract (Aged Black Garlic Extract-ABGE) on Lewis cancer cell line and colonic cancer Ht-29 mouse transplanted tumor and to explore its possible mechanism. Methods: lewis lung cancer cells were cultured routinely. Different concentrations of black garlic extract combined with 6o Co 纬 -ray irradiation were used to determine the growth inhibition rate of black garlic extract on lung cancer lewis cell line in vitro. The effect of black garlic extract combined with radiotherapy on cell clone survival was observed by plate colony forming test. The morphological changes of cells were observed by Hoechst fluorescence staining. Flow cytometry was used to detect the changes of tumor cell cycle after the treatment of black garlic extract. Western Blot was used to analyze the effect of black garlic extract on the expression of Bcl-2P Bax protein in lewis cells. A subcutaneous transplanted tumor model of Ht-29 mice with colon cancer was established and 40 mice were randomly divided into 4 groups. Blank control group, 纬 irradiation group, black garlic group and black garlic 纬 irradiation group were treated with normal saline, 纬 -ray irradiation, black garlic extract and black garlic extract combined with 纬 -ray irradiation, respectively. The growth rate of transplanted tumor in each group and the growth delay time of each treatment group were observed. After the experiment, the mice were killed after eyeball extraction, the tumor was weighed and the tumor growth inhibition rate was calculated. The tumor growth delay time and radiosensitization ratio were calculated, the apoptosis of tumor tissue was detected by flow cytometry with Annexin V-FITC / Pi double staining, and the expression of Bcl-2BaxCaspase-9 was detected by Realtime-PCR. The activity of SOD and CAT in serum of each group of mice was detected by ultraviolet spectrophotometer. Results the extract of black garlic significantly inhibited the growth of lung cancer lewis cell line, and the combination of black garlic extract and 60Co 纬 -ray irradiation could promote the apoptosis of lewis cells. The cell cycle and the expression of Bcl-2P Bax protein.) Black garlic extract combined with 纬 -ray irradiation can inhibit the growth of transplanted tumor of colon cancer Ht-29 mice, delay the tumor growth time, increase the radiosensitization ratio and promote the apoptosis of tumor cells. It can down-regulate Bcl-2, up-regulate Baxand increase the expression of Caspase-9 protein. The extract of black garlic can increase the total number of white blood cells in peripheral blood of mice. Conclusion the extract of RBC superoxide dismutase (SOD) and CAT can enhance the radiosensitivity of lung cancer lewis cells. The mechanism of its sensitizing mechanism may be related to its alteration of cell cycle and its effect on the expression of Bcl-2 Bax protein. The extract of black garlic can increase the radiosensitivity to colonic cancer Ht-29 transplanted tumor by down-regulating the expression of Bax and activating Caspase-9 protein by down-regulating Bcl-2, and increasing the radiosensitivity of 纬 -ray to colonic cancer Ht-29 mice. The radiation protection of the body was improved by increasing the white blood cell count and anti-oxidation ability of the body.
【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R730.55

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