金雀异黄酮的辐射增敏作用及机理研究

发布时间：2018-05-27 04:15

本文选题：金雀异黄酮 + 辐射增敏　；参考：《中国科学院研究生院（近代物理研究所）》2014年博士论文

【摘要】：目的：研究金雀异黄酮在体内、体外的辐射增敏作用并探讨其作用机制。研究内容包括两部分：第一部分，研究金雀异黄酮对雌激素受体和p53状态不同的两株乳腺癌细胞的辐射增敏作用及机理；第二部分，体内、体外实验相结合，研究金雀异黄酮对小鼠肉瘤的辐射增敏作用及机理。材料与方法：流式细胞术法检测细胞周期的分布及细胞凋亡情况；MTT法和细胞计数法检测金雀异黄酮对细胞增殖的影响；克隆存活法检测细胞存活率；免疫荧光法研究γ-H2AX、Rad51、Ku70/80、DNA-PKcs在细胞内的分布及数量；Western blot法检测G2/M期检验点蛋白及细胞凋亡相关蛋白的表达水平；Tunel法检测石蜡包埋组织切片的细胞凋亡情况。结果：第一部分： 1．MTT法测得金雀异黄酮浓度在5-20μM时，对乳腺癌细胞MCF-7和MDA-MB-231的毒性较小。因此，以0，5,10，20μM的金雀异黄酮预处理细胞24h，再给予4Gy的X-射线照射，随着金雀异黄酮浓度的增加，细胞克隆存活率有了明显的下降趋势。当用10μM的金雀异黄酮预处理细胞24h，再给予不同剂量的X射线照射，在存活率为10%（IC10）时，MCF-7和MDA-MB-231细胞的辐射增敏比分别为1.43和1.36。并且，金雀异黄酮联合X-射线处理使γ-H2AX foci数量随着金雀异黄酮浓度的增加而增加，说明金雀异黄酮加重了DNA损伤。以上结果表明，金雀异黄酮对两株乳腺癌细胞都具有很好的辐射增敏效果。 2．金雀异黄酮联合X-射线处理乳腺癌细胞，处理后12h，随着金雀异黄酮浓度的增加，细胞发生了明显的G2/M期阻滞现象，但处理后24h，G2/M期细胞比率却随着金雀异黄酮浓度的增加而显著降低；γ-H2AX和Rad51foci共定位的结果表明，金雀异黄酮可抑制Rad51结合到DNA损伤位点，使同源重组修复受到抑制，并且随着联合处理后时间的延长，细胞凋亡率有了急剧增大。也就是说，联合处理后12h，大量细胞被阻滞在G2/M期，但由于DNA损伤严重，被阻滞的细胞没有得到修复，而是以凋亡的形式发生了死亡。 3．金雀异黄酮联合X-射线处理激活了G2/M期检验点蛋白ATM/Chk/Cdc25c/Cdc2信号通路，使细胞周期阻滞在G2/M期；并且，随着金雀异黄酮浓度的增加，两种细胞Bcl-2/Bax值逐渐降低、野生型p53的表达几乎没有差异（MCF-7细胞中），但突变型p53的表达则随之降低（MDA-MB-231细胞中）、两株细胞p73表达量均有显著上升。因此，我们推断，联合处理可能使这两株乳腺癌细胞通过依赖于p73的线粒体凋亡途径发生了死亡。第二部分： 1.细胞计数结果显示，10μM的金雀异黄酮对S180细胞的生长无明显抑制作用。因此，以10μM的金雀异黄酮预处理S180细胞24h，再给予不同剂量的X-射线照射，在存活率为10%（IC10）时，辐射增敏比为1.38；并且，金雀异黄酮联合X-射线显著增加了细胞凋亡率。 2.以接种S180肉瘤的Balb/c小鼠为研究对象，末次照射后24h断颈处死并剥离小鼠肿瘤组织，，对照组肿瘤组织有大量的血管丛生，加药+辐照组（D+IR）肿瘤组织的血管数量明显减少。D+IR组小鼠肿瘤体积、重量比其它各组都有极显著降低。HE染色的结果显示，D+IR组肿瘤组织细胞密度较低，tunel检测的结果说明联合处理组肿瘤组织中有大量细胞以凋亡方式发生了死亡。 3.金雀异黄酮联合X-射线处理使线粒体中Bax表达上升，Bcl-2表达下降，并且线粒体中的细胞色素c大量转移至胞质中。随着照射后时间的延长，联合处理组Rad51的表达显著降低，而不同时间点γ-H2AX在稳定表达，说明HR修复受阻而损伤持续存在。Ku70/80、DNA-PKcs、Rad51共定位的结果说明，金雀异黄酮联合X-射线处理抑制了DNA-PKcs的活性，导致Ku70/80长期占据DNA损伤位点，使NHEJ修复和HR修复都不能进行，最终细胞发生了凋亡。结论： 1.金雀异黄酮对雌激素受体和p53状态不同的乳腺癌细胞MCF-7（ER+，wild-p53）和MDA-MB-231（ER-，mut-p53）都具有辐射增敏作用，其增敏机理在于通过激活周期检验点蛋白，将细胞阻滞在辐射最为敏感的G2/M期，最终使受损的细胞没有得到修复，而是通过p73诱导的线粒体细胞凋亡途径发生死亡。 2. S180肉瘤的体内、体外实验结果显示，金雀异黄酮对小鼠S180肉瘤具有辐射增敏效应，增敏机理在于金雀异黄酮抑制了DNA-PKcs的活性，从而限制了NHEJ修复，并且Ku70/80长期占据DNA损伤位点，导致HR修复也不能进行，最终受损的细胞不能得到修复而发生凋亡。
[Abstract]:Purpose :

The effects of genistein on radiosensitization in vivo and in vitro and its mechanism of action were studied . The study included two parts : the first part , the effect and mechanism of genistein on the radiosensitization of two breast cancer cells with different estrogen receptor and p53 status ;
In the second part , in vivo and in vitro experiments , the radiosensitizing effect and mechanism of genistein on mouse sarcoma were studied .

Materials and Methods :

Flow cytometry was used to detect cell cycle distribution and cell apoptosis .
The effect of genistein on cell proliferation was detected by MTT method and cell counting method .
cloning survival method to detect cell survival rate ;
The distribution and quantity of 纬 - H2AX , Rad51 , Ku70 / 80 , DNA - PKcs in cells were studied by immunofluorescence .
Western blot was used to detect the level of protein and apoptosis - related protein in G2 / M phase .
The apoptosis of paraffin - embedded tissue sections was detected by Tunel method .

Results :

Part I :

1 . The cytotoxicity of genistein on MCF - 7 and MDA - MB - 231 cells was less than that of genistein . The results showed that genistein combined with X - ray treatment increased the dose of 纬 - H2AX . The results showed that genistein combined with X - ray treatment increased the DNA damage . The results showed that genistein had good radiosensitivity to both breast cancer cells .

2 . Treatment of breast cancer cells with genistein combined with X - ray showed obvious G2 / M arrest at 12 h after treatment , but at 24 h after treatment , the ratio of G2 / M cells decreased with the increase of genistein concentration .
The results showed that genistein could inhibit the binding of Rad51 to DNA damage sites , and the repair of homologous recombination was inhibited , and the apoptosis rate increased sharply as the time of joint treatment increased . In other words , the cells were blocked in G2 / M phase after treatment for 12 h , but the blocked cells were not repaired , but death occurred in the form of apoptosis .

3 . The G2 / M phase checkpoint protein ATM / Chk / Cdc25c / Cdc2 signaling pathway was activated by genistein combined with X - ray treatment , and the cell cycle was blocked in G2 / M phase ;
In addition , with the increase of genistein concentration , the Bcl - 2 / Bax values of both cells decreased gradually , and the expression of wild type p53 was almost no difference ( in MCF - 7 cells ) , but the expression of mutant p53 was decreased ( MDA - MB - 231 cells ) .

Part Two :

1 . The results of cell count showed that 10 渭M genistein did not significantly inhibit the growth of S180 cells . Therefore , the S180 cells were pretreated with 10 渭M genistein for 24 h , then irradiated with different doses of X - ray , and the radiosensitivity ratio was 1.38 when the survival rate was 10 % ( IC10 ) .
Moreover , genistein combined with X - rays significantly increased the rate of apoptosis .

2 . Balb / c mice inoculated with S180 sarcoma were studied . After the last irradiation , the tumor tissues of mice were sacrificed and the tumor tissues of mice were peeled off .

3 . The expression of Bax in mitochondria increased , the expression of Bcl - 2 in mitochondria decreased , and the expression of cytochrome c in mitochondria decreased significantly . The results of co - localization of Ku70 / 80 , DNA - PKcs and Rad51 showed that genistein combined with X - ray treatment inhibited the activity of DNA - PKcs .

Conclusion :

1 . All breast cancer cells MCF - 7 ( ER + , wild - p53 ) and MDA - MB - 231 ( ER - , mut - p53 ) with different estrogen receptor and p53 status have radiosensitizing effect .

2 . In vivo and in vitro experiments of S180 sarcoma show that genistein has radiosensitizing effect on S180 sarcoma of mice . The mechanism of sensitization is that genistein suppresses the activity of DNA - PKcs , thus limiting NHEJ repair , and Ku70 / 80 occupies DNA damage site for a long time , which leads to the repair of HR , which can not be repaired and apoptosis .
【学位授予单位】：中国科学院研究生院（近代物理研究所）
【学位级别】：博士
【学位授予年份】：2014
【分类号】：Q691;R730.55

【参考文献】