【摘要】：大腸癌,包括結(jié)腸癌和直腸癌,是威脅人類生命健康的常見(jiàn)消化道惡性腫瘤。放射治療是大腸癌的主要治療手段之一,但放射治療大腸癌的輻射耐受現(xiàn)象嚴(yán)重影響大腸癌病人的療效,放療抵抗性成為大腸癌放療面臨嚴(yán)峻且迫切需要解決的難題。電離輻射后細(xì)胞DNA損傷的修復(fù)是腫瘤放療效果不佳的主要原因之一。X射線修復(fù)交叉互補(bǔ)(X-ray repair cross complementing, XRCC)基因家族(XRCC1～XRCC11)對(duì)電離輻射誘導(dǎo)的DNA損傷修復(fù)發(fā)揮重要作用。DNA損傷通過(guò)堿基切除修復(fù)、核苷酸切除修復(fù)、錯(cuò)配修復(fù)、同源重組修復(fù)(homologous recombination,HR)和非同源末端連接(non-homologous end joining, NHEJ)等多種方式進(jìn)行修復(fù),從而維持生物體基因組的完整性和抑制腫瘤的發(fā)生。XRCC2是重要的參與HR途徑的基因之一,其高表達(dá)與增加輻射誘導(dǎo)的DNA損傷抵抗有關(guān)。XRCC2基因修復(fù)缺陷表現(xiàn)出對(duì)電離輻射的敏感性增高,而XRCC2蛋白過(guò)表達(dá)則對(duì)放射線耐受。提示通過(guò)抑制腫瘤細(xì)胞XRCC2的表達(dá),有可能提高臨床腫瘤放射治療的敏感性。目前尚未見(jiàn)到關(guān)于大腸癌中XRCC2表達(dá)水平以及XRCC2與放射敏感性關(guān)系的研究報(bào)道。降低XRCC2的表達(dá)是否可以改變大腸癌細(xì)胞的放射敏感性,XRCC2是否可以預(yù)測(cè)大腸癌放射治療的療效,目前在國(guó)內(nèi)外未見(jiàn)相關(guān)的研究報(bào)道。 目的：本實(shí)驗(yàn)通過(guò)大腸癌體外細(xì)胞模型和體內(nèi)動(dòng)物模型,探討shRNA介導(dǎo)的XRCC2基因沉默是否影響大腸癌細(xì)胞的放療敏感性及其療效,闡明XRCC2在大腸癌放療敏感性中的關(guān)鍵作用和初步相關(guān)機(jī)制。 方法：(1)體外細(xì)胞實(shí)驗(yàn)：將shRNA-XRCC2轉(zhuǎn)染人大腸癌T84細(xì)胞以沉默XRCC2基因表達(dá),采用蛋白免疫印跡法和實(shí)時(shí)定量PCR法檢測(cè)沉默XRCC2基因的效率；采用MTT法檢測(cè)T84細(xì)胞的增殖。經(jīng)X-射線照射后,采用克隆形成法檢測(cè)T84細(xì)胞的放射敏感性；采用堿性“彗星”電泳法測(cè)定T84細(xì)胞的DNA損傷修復(fù)；流式細(xì)胞術(shù)檢測(cè)T84細(xì)胞的細(xì)胞周期；Annexin V-FITC/PI雙染法檢測(cè)T84細(xì)胞的細(xì)胞凋亡率。(2)體內(nèi)細(xì)胞實(shí)驗(yàn)：同時(shí)將shRNA-XRCC2轉(zhuǎn)染的大腸癌T84細(xì)胞接種于BALB/c裸鼠建立移植瘤模型,進(jìn)行X-射線照射,檢測(cè)腫瘤的體積和重量變化,并對(duì)腫瘤組織進(jìn)行病理分析。 結(jié)果：(1)在體外細(xì)胞實(shí)驗(yàn)中,shRNA-XRCC2轉(zhuǎn)染有效抑制了T84細(xì)胞中XRCC2蛋白和mRNA的表達(dá)。經(jīng)嘌呤酶素篩選,得到了穩(wěn)定的XRCC2基因沉默的大腸癌T84細(xì)胞系。細(xì)胞生長(zhǎng)曲線表明,沉默XRCC2表達(dá)明顯抑制了T84細(xì)胞的增殖�？寺⌒纬蓪�(shí)驗(yàn)顯示,XRCC2基因沉默的T84細(xì)胞經(jīng)X-射線照射后,克隆形成數(shù)目顯著減少,表明XRCC2基因沉默提高了T84細(xì)胞的放射敏感性。彗星實(shí)驗(yàn)表明,沉默XRCC2表達(dá)的T84細(xì)胞DNA損傷增多,DNA損傷修復(fù)能力下降。流式細(xì)胞術(shù)檢測(cè)顯示,XRCC2基因沉默顯著誘導(dǎo)了輻射導(dǎo)致的細(xì)胞凋亡和細(xì)胞阻滯在G2/M期。(2)在體內(nèi)細(xì)胞實(shí)驗(yàn)中,轉(zhuǎn)染shRNA-XRCC2的裸鼠種植瘤生長(zhǎng)緩慢,腫瘤體積和重量明顯減少。腫瘤病理組織學(xué)分析表明,轉(zhuǎn)染shRNA-XRCC2的腫瘤組織核分裂相減少,多見(jiàn)大小不等的壞死區(qū)。說(shuō)明沉默XRCC2表達(dá)提高了裸鼠大腸癌對(duì)輻射的敏感性,腫瘤生長(zhǎng)受到明顯的抑制作用。 結(jié)論：shRNA介導(dǎo)的XRCC2基因沉默有效抑制了體外大腸癌細(xì)胞和體內(nèi)裸鼠大腸癌腫瘤的生長(zhǎng),沉默XRCC2表達(dá)對(duì)體外和體內(nèi)大腸癌細(xì)胞對(duì)X射線的反應(yīng)具有一致性,即均提高了大腸癌對(duì)放射的敏感性。提示XRCC2有希望在大腸癌的臨床放射治療敏感性中作為一重要的靶向基因。
[Abstract]:Colorectal cancer, including colorectal cancer and rectal cancer, is a common malignant tumor of the digestive tract that threatens human life and health. Radiotherapy is one of the main treatments for colorectal cancer. However, the radiation tolerance of colorectal cancer seriously affects the curative effect of colorectal cancer patients. Radiotherapy resistance has become a serious and urgent problem in colorectal cancer radiotherapy. X-ray repair cross complementing (XRCC) gene family (XRCC1-XRCC11) plays an important role in the repair of DNA damage induced by ionizing radiation. DNA damage is repaired by base excision and nucleotide excision. Repair, mismatch repair, homologous recombination (HR) and non-homologous end-joining (NHEJ) repair methods to maintain the integrity of the organism genome and inhibit the occurrence of tumors. XRCC2 is one of the important genes involved in the HR pathway, its high expression and increased radiation inducement. XRCC2 gene repair deficiency shows increased sensitivity to ionizing radiation, while XRCC2 protein overexpression is radioresistant. It suggests that inhibiting the expression of XRCC2 in tumor cells may enhance the sensitivity of clinical tumor radiotherapy. Whether reducing the expression of XRCC2 can change the radiosensitivity of colorectal cancer cells and whether XRCC2 can predict the efficacy of radiotherapy for colorectal cancer have not been reported at home and abroad.
Objective: To investigate whether shRNA-mediated XRCC2 gene silencing affects the radiosensitivity of colorectal cancer cells in vitro and in vivo, and to elucidate the key role of XRCC2 in the radiosensitivity of colorectal cancer cells.
Methods: (1) In vitro cell experiment: shRNA-XRCC2 was transfected into human colorectal cancer T84 cells to silence XRCC2 gene expression, and the efficiency of XRCC2 gene silencing was detected by Western blotting and real-time quantitative PCR, and the proliferation of T84 cells was detected by MTT method. DNA damage and repair of T84 cells were detected by alkaline comet electrophoresis, cell cycle of T84 cells was detected by flow cytometry, and apoptosis rate of T84 cells was detected by Annexin V-FITC/PI double staining. (2) In vivo cell experiment: T84 cells transfected with shRNA-XRCC2 were inoculated into BALB/c nude mice to establish a transplanted tumor model. X-ray irradiation was used to detect tumor volume and weight changes and pathological analysis of tumor tissues.
Results: (1) In vitro, shRNA-XRCC2 transfection effectively inhibited the expression of XRCC2 protein and mRNA in T84 cells. A stable colon cancer T84 cell line with XRCC2 gene silencing was obtained by purinase screening. After X-ray irradiation, the number of cloned T84 cells with gene silencing decreased significantly, suggesting that XRCC2 gene silencing increased the radiosensitivity of T84 cells. Comet assay showed that the DNA damage of T84 cells expressing XRCC2 increased and DNA damage repair ability decreased. Flow cytometry showed that XRCC2 gene silencing significantly induced radiation-induced damage. Cell apoptosis and cell block were observed in G2/M phase. (2) In vivo, shRNA-XRCC2-transfected nude mice showed slow growth, significantly reduced tumor volume and weight. Tumor histopathological analysis showed that shRNA-XRCC2-transfected tumors showed less mitosis and more necrotic areas of different sizes. The sensitivity of colorectal cancer to radiation in nude mice was significantly inhibited by tumor growth.
Conclusion: XRCC2 gene silencing mediated by shRNA can effectively inhibit the growth of colorectal cancer cells in vitro and nude mice colorectal cancer cells in vivo. The silencing of XRCC2 expression is consistent with the response of colorectal cancer cells to X-ray in vitro and in vivo, that is to say, it increases the sensitivity of colorectal cancer to radiation. As an important target gene in therapeutic sensitivity.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R735.34;R730.55

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