本文選題：金雀異黃酮 + 輻射增敏　； 參考：《中國科學院研究生院（近代物理研究所）》2014年博士論文

【摘要】：目的： 研究金雀異黃酮在體內、體外的輻射增敏作用并探討其作用機制。研究內容包括兩部分：第一部分，研究金雀異黃酮對雌激素受體和p53狀態(tài)不同的兩株乳腺癌細胞的輻射增敏作用及機理；第二部分，體內、體外實驗相結合，研究金雀異黃酮對小鼠肉瘤的輻射增敏作用及機理。 材料與方法： 流式細胞術法檢測細胞周期的分布及細胞凋亡情況；MTT法和細胞計數(shù)法檢測金雀異黃酮對細胞增殖的影響；克隆存活法檢測細胞存活率；免疫熒光法研究γ-H2AX、Rad51、Ku70/80、DNA-PKcs在細胞內的分布及數(shù)量；Western blot法檢測G2/M期檢驗點蛋白及細胞凋亡相關蛋白的表達水平；Tunel法檢測石蠟包埋組織切片的細胞凋亡情況。 結果： 第一部分： 1．MTT法測得金雀異黃酮濃度在5-20μM時，對乳腺癌細胞MCF-7和MDA-MB-231的毒性較小。因此，以0，5,10，20μM的金雀異黃酮預處理細胞24h，再給予4Gy的X-射線照射，隨著金雀異黃酮濃度的增加，細胞克隆存活率有了明顯的下降趨勢。當用10μM的金雀異黃酮預處理細胞24h，再給予不同劑量的X射線照射，在存活率為10%（IC10）時，MCF-7和MDA-MB-231細胞的輻射增敏比分別為1.43和1.36。并且，金雀異黃酮聯(lián)合X-射線處理使γ-H2AX foci數(shù)量隨著金雀異黃酮濃度的增加而增加，說明金雀異黃酮加重了DNA損傷。以上結果表明，金雀異黃酮對兩株乳腺癌細胞都具有很好的輻射增敏效果。 2．金雀異黃酮聯(lián)合X-射線處理乳腺癌細胞，處理后12h，隨著金雀異黃酮濃度的增加，細胞發(fā)生了明顯的G2/M期阻滯現(xiàn)象，但處理后24h，G2/M期細胞比率卻隨著金雀異黃酮濃度的增加而顯著降低；γ-H2AX和Rad51foci共定位的結果表明，金雀異黃酮可抑制Rad51結合到DNA損傷位點，使同源重組修復受到抑制，并且隨著聯(lián)合處理后時間的延長，細胞凋亡率有了急劇增大。也就是說，聯(lián)合處理后12h，大量細胞被阻滯在G2/M期，但由于DNA損傷嚴重，被阻滯的細胞沒有得到修復，而是以凋亡的形式發(fā)生了死亡。 3．金雀異黃酮聯(lián)合X-射線處理激活了G2/M期檢驗點蛋白ATM/Chk/Cdc25c/Cdc2信號通路，使細胞周期阻滯在G2/M期；并且，隨著金雀異黃酮濃度的增加，兩種細胞Bcl-2/Bax值逐漸降低、野生型p53的表達幾乎沒有差異（MCF-7細胞中），但突變型p53的表達則隨之降低（MDA-MB-231細胞中）、兩株細胞p73表達量均有顯著上升。因此，我們推斷，聯(lián)合處理可能使這兩株乳腺癌細胞通過依賴于p73的線粒體凋亡途徑發(fā)生了死亡。 第二部分： 1.細胞計數(shù)結果顯示，10μM的金雀異黃酮對S180細胞的生長無明顯抑制作用。因此，以10μM的金雀異黃酮預處理S180細胞24h，再給予不同劑量的X-射線照射，在存活率為10%（IC10）時，輻射增敏比為1.38；并且，金雀異黃酮聯(lián)合X-射線顯著增加了細胞凋亡率。 2.以接種S180肉瘤的Balb/c小鼠為研究對象，末次照射后24h斷頸處死并剝離小鼠腫瘤組織，，對照組腫瘤組織有大量的血管叢生，加藥+輻照組（D+IR）腫瘤組織的血管數(shù)量明顯減少。D+IR組小鼠腫瘤體積、重量比其它各組都有極顯著降低。HE染色的結果顯示，D+IR組腫瘤組織細胞密度較低，tunel檢測的結果說明聯(lián)合處理組腫瘤組織中有大量細胞以凋亡方式發(fā)生了死亡。 3.金雀異黃酮聯(lián)合X-射線處理使線粒體中Bax表達上升，Bcl-2表達下降，并且線粒體中的細胞色素c大量轉移至胞質中。隨著照射后時間的延長，聯(lián)合處理組Rad51的表達顯著降低，而不同時間點γ-H2AX在穩(wěn)定表達，說明HR修復受阻而損傷持續(xù)存在。Ku70/80、DNA-PKcs、Rad51共定位的結果說明，金雀異黃酮聯(lián)合X-射線處理抑制了DNA-PKcs的活性，導致Ku70/80長期占據DNA損傷位點，使NHEJ修復和HR修復都不能進行，最終細胞發(fā)生了凋亡。 結論： 1.金雀異黃酮對雌激素受體和p53狀態(tài)不同的乳腺癌細胞MCF-7（ER+，wild-p53）和MDA-MB-231（ER-，mut-p53）都具有輻射增敏作用，其增敏機理在于通過激活周期檢驗點蛋白，將細胞阻滯在輻射最為敏感的G2/M期，最終使受損的細胞沒有得到修復，而是通過p73誘導的線粒體細胞凋亡途徑發(fā)生死亡。 2. S180肉瘤的體內、體外實驗結果顯示，金雀異黃酮對小鼠S180肉瘤具有輻射增敏效應，增敏機理在于金雀異黃酮抑制了DNA-PKcs的活性，從而限制了NHEJ修復，并且Ku70/80長期占據DNA損傷位點，導致HR修復也不能進行，最終受損的細胞不能得到修復而發(fā)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 緯 - 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 緯 - 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 .
【學位授予單位】：中國科學院研究生院（近代物理研究所）
【學位級別】：博士
【學位授予年份】：2014
【分類號】：Q691;R730.55

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