【摘要】：目的：放射治療是惡性腫瘤綜合治療的重要手段之一，分析腫瘤細(xì)胞放射抵抗性的內(nèi)在機(jī)制有助于設(shè)計(jì)和改進(jìn)治療策略，提高放射治療療效。細(xì)胞的放射敏感性與細(xì)胞周期調(diào)控有著密切的聯(lián)系。細(xì)胞受照后發(fā)生DNA損傷,在通過G1和G2檢查點(diǎn)時(shí)會(huì)被阻滯在G1和G2期。細(xì)胞周期停滯使細(xì)胞有足夠的時(shí)間修復(fù)受損DNA，避免受損遺傳信息被復(fù)制，，DNA修復(fù)后的細(xì)胞從而繼續(xù)存活，有絲分裂的忠實(shí)性也因此得到保證。放射線導(dǎo)致DNA損傷主要為DNA單鏈斷裂(Single strand break, SSB)和DNA雙鏈斷裂(Double strand break,DSB)。DSB較SSB更為嚴(yán)重，其修復(fù)能力直接關(guān)系到腫瘤細(xì)胞放射敏感性。照射后DNA鏈斷裂修復(fù)動(dòng)力學(xué)研究也表明，DSB修復(fù)存在慢修復(fù)與快修復(fù)兩種方式�？煨迯�(fù)的半修復(fù)時(shí)間多在0.08～1.2h，此時(shí)多數(shù)細(xì)胞被阻滯在G1期；而慢修復(fù)的半修復(fù)時(shí)間多在2.4～6h，此時(shí)受照射的腫瘤細(xì)胞大部分已進(jìn)入S/G2期阻滯。由于非同源末端連接修復(fù)方式（Non-Homologous End Joining, NHEJ）為G1期DSB的主要修復(fù)路徑，且具有快速有效的特點(diǎn)，一般認(rèn)為DSB的快修復(fù)方式是由NHEJ完成，而DSB的慢修復(fù)分子機(jī)制仍然存在較大爭(zhēng)議。在DSB慢修復(fù)期大部分腫瘤細(xì)胞已進(jìn)入G2期阻滯，該時(shí)相DSB的修復(fù)可能由NHEJ和同源重組修復(fù)（homologous recombination repair,HRR）共同完成。明確DSB慢修復(fù)機(jī)制中NHEJ和HRR的分工協(xié)作，有助于開發(fā)新的輔助治療手段，通過調(diào)控DSB后期修復(fù)路徑中的關(guān)鍵蛋白增強(qiáng)腫瘤細(xì)胞的放射敏感性。DNA依賴蛋白激酶催化亞基(catalytic sunbunit of the DNA-dependent protein kinase, DNA-PKcs)是NHEJ路徑的重要蛋白酶，其酶活性為非同源末端連接修復(fù)（NHEJ）所必須。 DNA-PKcs與毛細(xì)血管共濟(jì)失調(diào)突變基因(Ataxiatelangiectasia mutated, ATM)、Chk2蛋白同為細(xì)胞G2-M期轉(zhuǎn)換的關(guān)鍵調(diào)節(jié)蛋白，與ATM、ATR (ATM and Rad3-related)等同屬于磷脂酰肌醇相關(guān)蛋白激酶(phosphatidylinositol kinase related protein, PIKK)家族成員。本研究旨在通過抑制射線照射后特定時(shí)間DNA-PKcs的酶活性，明確DNA-PKcs依賴的NHEJ修復(fù)在DSB慢修復(fù)中所起的作用，為將來干預(yù)DSB修復(fù)從而提高腫瘤細(xì)胞放療敏感性提供進(jìn)一步的理論依據(jù)。方法：1、流式細(xì)胞儀檢測(cè)不同劑量照射后不同時(shí)間HNE-1細(xì)胞株細(xì)胞所處的周期時(shí)相；2、免疫熒光法測(cè)定不同劑量照射后HNE-1細(xì)胞株DNA雙鏈斷裂情況；3、克隆形成實(shí)驗(yàn)測(cè)定DNA-PKcs小分子抑制劑NU7441對(duì)不同劑量照射后HNE-1細(xì)胞株克隆存活率的影響；4、采用SAS9.1統(tǒng)計(jì)軟件，采用兩因素析因設(shè)計(jì)的方差分析進(jìn)行組間均數(shù)的比較。結(jié)果：1、流式細(xì)胞儀檢測(cè)細(xì)胞周期，2Gy照射后0.5h時(shí)處于G1、G2/M、S期的細(xì)胞分別占總數(shù)的32.67%、8.00%、59.33%；1h時(shí)處于G1、G2/M、S期的細(xì)胞分別占總數(shù)的32.05%、8.00%、59.95%；4h時(shí)處于G1、G2/M、S期的細(xì)胞分別占總數(shù)的14.62%、19.44%、65.94%。5Gy照射后0.5h時(shí)處于G1、G2/M、S期細(xì)胞分別占總數(shù)的38.94%、8.00%、53.06%；1h時(shí)處于G1、G2/M、S期細(xì)胞分別占總數(shù)的37.49%、8.00%、54.51%；4h時(shí)處于G1、G2/M、S期細(xì)胞分別占總數(shù)的14.16%、21.58%、64.26%。細(xì)胞周期檢測(cè)結(jié)果顯示2Gy和5Gy照射后4h時(shí)HNE-1細(xì)胞株發(fā)生了S期延遲及G2/M期阻滯。2、免疫熒光法測(cè)得未照射組細(xì)胞γH2AX數(shù)量為12.33±2.52個(gè)；2Gy照射后0.5h時(shí)每個(gè)細(xì)胞γH2AX數(shù)量為19.67±3.06個(gè)，1h時(shí)為22.00±3.00個(gè)，4h時(shí)為12.33±2.08個(gè)；5Gy照射后0.5h時(shí)每個(gè)細(xì)胞γH2AX數(shù)量為41.00±2.65個(gè)，1h時(shí)為48.67±3.21個(gè)，4h時(shí)為29.67±3.51個(gè)。以上結(jié)果得出，鼻咽癌HNE-1細(xì)胞株在2Gy和5Gy照射4h后有相當(dāng)數(shù)目的殘留DSB存在，并與劑量相關(guān)，此結(jié)果與其他相關(guān)文獻(xiàn)報(bào)道一致。3、克隆形成實(shí)驗(yàn)測(cè)得對(duì)照組HNE-1細(xì)胞株的克隆形成率(plating efficiency, PE)為65.50%；照射前加入10μm NU7441，2Gy照射后持續(xù)處理4h組細(xì)胞克隆存活率（survivalfraction, SF）為16.79%；2Gy照射后4h加入藥物濃度分別為0、0.1、1、10μm的NU7441持續(xù)作用4h后各組SF分別為（64.89±3.11）%、（62.03±1.91）%、（64.89±5.40）%、（63.00±1.14）%。5Gy照射后4h加入藥物濃度分別為0、0.1、1、10μm的NU7441持續(xù)作用4h后各組SF分別為（3.69±0.49）%、（4.07±1.44）%、（3.82±0.66）%、（4.09±1.14）%。初步分析顯示，在DSB修復(fù)后期不同劑量照射后加入不同藥物濃度NU7441并未對(duì)HNE-1細(xì)胞株的克隆存活率有影響。4、統(tǒng)計(jì)結(jié)果：采用SAS9.1統(tǒng)計(jì)軟件，采用兩因素析因設(shè)計(jì)的方差分析進(jìn)行組間均數(shù)的比較。2Gy照射后，四個(gè)不同藥物濃度之間細(xì)胞存活率差異無統(tǒng)計(jì)學(xué)意義（F=0.37，P=0.776）；5Gy照射后，四個(gè)不同藥物濃度之間細(xì)胞存活率差異亦無統(tǒng)計(jì)學(xué)意義（F=0.01，P=0.999）。結(jié)論：鼻咽癌細(xì)胞株HNE-1受照后4h仍然有殘留DSB存在，此時(shí)大部分腫瘤細(xì)胞已進(jìn)入G2期阻滯。我們的實(shí)驗(yàn)結(jié)果表明，在此階段抑制DNA-PKcs的酶活性，細(xì)胞克隆存活率未觀察到顯著變化，表明照射引起的DSB后期修復(fù)，即慢修復(fù)方式與DNA-PKcs的酶活性關(guān)系不大，其修復(fù)更多依賴于HRR和DNA-PKcs非依賴性的末端連接修復(fù)。我們的實(shí)驗(yàn)研究第一次明確了DNA-PKcs依賴性的NHEJ通路在照射引起的DSB后期修復(fù)中作用較為次要，與放射引起的細(xì)胞增殖性死亡無關(guān)。這一結(jié)論是對(duì)放射敏感分子基礎(chǔ)的重要補(bǔ)充，為將來干預(yù)DNA修復(fù)，增加腫瘤細(xì)胞放射敏感性提供了新的思路。
[Abstract]:Objective : Radiotherapy is one of the most important methods for the comprehensive treatment of malignant tumors . The intrinsic mechanism of radiation resistance of tumor cells is helpful to design and improve the therapeutic strategy and to improve the therapeutic effect of radiotherapy . The radiosensitivity of the cells is closely related to the regulation of cell cycle . The cell cycle arrest can be blocked in G1 and G2 phases . The cell cycle arrest will allow the cells to repair the damaged DNA at a sufficient time . The cell cycle arrest will allow the cells to continue to survive by DNA repair . The DNA damage is mainly caused by single strand break ( SSB ) and DNA double strand break . Compared with SSB , the repair ability is directly related to the radiosensitivity of tumor cells .
DNA - dependent protein kinase ( DNA - PKcs ) is an important protease in NHEJ pathway , and its enzyme activity is necessary for non - homologous end - connection repair ( NHEJ ) . DNA - PKcs and ATM and Rad3 - related protein kinase related protein kinase related protein kinase ( PIKK ) family members were identified by DNA - PKcs and ATM , ATM and Rad3 - related proteins .
2 . DNA double strand breaks of HNE - 1 cell line after different doses were measured by immunofluorescence assay .
3 . The effect of NU7441 inhibitor NU7441 on the survival rate of HNE - 1 cell line after irradiation with different doses was determined .
Results : 1 . Flow cytometry was used to detect the cell cycle , and the cells in G1 , G2 / M and S phases accounted for 32.67 % , 8.00 % and 59.33 % of total cells in G1 , G2 / M , S phase after 2Gy irradiation .
The cells in G1 , G2 / M , S phase accounted for 32.05 % , 8.00 % and 59.95 % , respectively .
Cells in G1 , G2 / M , S phase accounted for 14.62 % , 19.44 % and 65.94 % respectively at 4h . The cells in G1 , G2 / M , S phase accounted for 38.94 % , 8.00 % and 53.06 % of total cells at 0.5h after 5Gy irradiation .
At 1 h , G1 , G2 / M , S phase cells accounted for 37.49 % , 8.00 % and 54.51 % , respectively .
At 4 h , the cells in G1 , G2 / M , S phase accounted for 14.16 % , 21.58 % and 64.26 % , respectively . The results of cell cycle showed S phase delay and G2 / M arrest at 4 h after 2 Gy and 5 Gy irradiation .
緯 H2AX was 19.67 鹵 3.06 at 0.5h after 2Gy irradiation , 22.00 鹵 3.00 at 1h , 12.33 鹵 2.08 at 4h ;
The number of 緯H2AX at 0.5h after 5Gy irradiation was 41.00 鹵 2.65 , 48.67 鹵 3.21 at 1h and 29.67 鹵 3.51 at 4h . The results showed that HNE - 1 cell line of nasopharyngeal carcinoma had a significant number of residues after 2 Gy and 5 Gy irradiation .
After 10 渭m NU7441 was added before irradiation , the survival rate ( SF ) was 16.79 % after 2 Gy irradiation .
After 4 hours after irradiation with 2 Gy , the concentration of NU7441 was ( 64.89 鹵 3.11 ) % , ( 62.03 鹵 1.91 ) % , ( 64.89 鹵 5.40 ) % , ( 63.00 鹵 1.14 ) % , ( 3.82 鹵 0.66 ) % , ( 4.09 鹵 1.14 ) % .
After 5 Gy irradiation , there was no significant difference in cell viability between four different drug concentrations ( F = 0.01 , P = 0.999 ) . Conclusion : No significant changes in the activity of DNA - PKcs in nasopharyngeal carcinoma cell line HNE - 1 were observed in 4 hours after exposure . Our experimental results showed that the effect of DNA - PKcs - dependent NHEJ pathway on the repair of DNA - PKcs was not significant at this stage . The results showed that the DNA - PKcs - dependent NHEJ pathway was secondary to radiation - induced cell proliferative death . This conclusion is an important supplement to radiation - sensitive molecular basis . It provides a new idea for the future intervention of DNA repair and increasing the radiosensitivity of tumor cells .
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R730.55

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本文編號(hào)：2132090