本文選題：鼻咽癌 + 放射抗拒��； 參考：《廣西醫(yī)科大學(xué)》2012年碩士論文

【摘要】：【研究背景和目的】 鼻咽癌是我國(guó)常見(jiàn)的惡性腫瘤之一，尤以華南地區(qū)發(fā)病率最高。由于鼻咽部解剖位置的特殊性以及鼻咽癌細(xì)胞對(duì)射線較敏感的特點(diǎn)，放射治療是未轉(zhuǎn)移鼻咽癌患者的首選治療方式。同步放化療提高了局部晚期鼻咽癌患者的生存時(shí)間，已經(jīng)成為局部晚期鼻咽癌患者的標(biāo)準(zhǔn)治療模式~([1-2])。臨床工作中，一些患者接受了足量的放化療也出現(xiàn)復(fù)發(fā)，這通常是由于存在對(duì)放療抗拒的細(xì)胞有關(guān)~([5])。目前放射抗拒機(jī)理研究熱點(diǎn)在于：對(duì)比親本細(xì)胞及其誘導(dǎo)出來(lái)的放射抗拒細(xì)胞，以高通量實(shí)驗(yàn)方法篩選出差異表達(dá)基因或者蛋白，經(jīng)過(guò)體外實(shí)驗(yàn)進(jìn)一步驗(yàn)證。但相應(yīng)的體內(nèi)實(shí)驗(yàn)很少報(bào)道。 目前已有報(bào)道誘導(dǎo)出人鼻咽癌放射抗拒細(xì)胞系CNE2R)，本課題組前期也已建立了人鼻咽癌放射抗拒細(xì)胞系CNE-2R)，但相應(yīng)的動(dòng)物實(shí)驗(yàn)?zāi)Ｐ蜕形唇�。腫瘤動(dòng)物模型是研究腫瘤機(jī)理和腫瘤防治研究最有用的工具之一。BALB/c-nu裸小鼠先天性胸腺缺乏，適合移植瘤生長(zhǎng)，因此裸鼠異種移植瘤模型是腫瘤研究中常用的模型。本研究將誘導(dǎo)出來(lái)的放射抗拒細(xì)胞移植到動(dòng)物體內(nèi)，建立裸鼠移植瘤模型，并用χ射線分次照射移植瘤以檢驗(yàn)放射抗拒細(xì)胞在體內(nèi)的放射抗拒性，為解決鼻咽癌放射抗拒問(wèn)題提供較好的動(dòng)物模型。本研究進(jìn)一步探討前期研究篩選出的幾個(gè)差異表達(dá)蛋白（DEPs）：NPM1蛋白、Annexin A3蛋白和nm23-H1蛋白在不同放射敏感移植瘤組織中的表達(dá)，為解決鼻咽癌放射抗拒提供新的標(biāo)志物，，這對(duì)于臨床上采取更有效的手段以提高鼻咽癌的治療效果很有必要。 【研究方法】 1.將鼻咽癌放射抗拒細(xì)胞CNE-2R和人鼻咽癌親本細(xì)胞CNE-2注射入裸鼠皮下，建立人鼻咽癌放射抗拒移植瘤模型和人鼻咽癌放射敏感移植瘤模型這一對(duì)比模型。 2.觀察移植瘤生長(zhǎng)特性，以χ射線分次照射移植瘤，觀察不同放射敏感性移植瘤受照射后生長(zhǎng)情況。 3.取出移植瘤進(jìn)行病理學(xué)檢測(cè)，光鏡下觀察移植瘤細(xì)胞形態(tài)。 4.免疫組化法檢測(cè)NPM1蛋白、Annexin A3蛋白和nm23-H1蛋白在不同放射敏感性移植瘤組織中的表達(dá)，比較其差異。 【研究結(jié)果】 1.建立了人鼻咽癌放射抗拒移植瘤模型（CNE-2R移植瘤）和人鼻咽癌放射敏感移植瘤模型（CNE-2移植瘤）。 2.未受照射CNE-2R移植瘤生長(zhǎng)速度慢于未受照射CNE-2移植瘤（P0.01），移植瘤體積倍增時(shí)間分別為4.8天、3.9天；CNE-2R移植瘤生長(zhǎng)受照射抑制不明顯(P0.05)，而CNE-2移植瘤受照射生長(zhǎng)明顯受抑（P0.05），移植瘤積倍倍增時(shí)間分別為6.2天、17.1天；CNE-2R移植瘤受照射后體積增長(zhǎng)率大于CNE-2移植瘤（P0.01）。 3.移植瘤形態(tài)大體形態(tài)及病理學(xué)檢查：肉眼見(jiàn)移植瘤呈卵圓形或不規(guī)則球形，有薄層假包膜，切面呈魚肉狀。光鏡下可見(jiàn)未受照射CNE-2R移植瘤及未受照射CNE-2移植瘤大量壞死灶，受照射CNE-2R移植瘤細(xì)胞較少壞死。未壞死的CNE-2移植瘤細(xì)胞與CNE-2R移植瘤細(xì)胞形態(tài)光鏡下無(wú)明顯差別。移植瘤細(xì)胞呈巢狀分布，呈多角形或卵圓形。核大深染，核圓形、卵圓形，胞漿豐富，可見(jiàn)病理核分裂。移植瘤細(xì)胞與人鼻咽低分化鱗癌細(xì)胞來(lái)源形態(tài)學(xué)特征一致。 4. NPM1蛋白、Annexin A3蛋白在未受照射CNE-2R移植瘤中較在未受照射CNE-2移植瘤中明顯低表達(dá)，P0.01；nm23-H1蛋白則明顯高表達(dá)，P0.05。AnnexinA3蛋白在受照射CNE-2R移植瘤中較在受照射CNE-2移植瘤中表達(dá)明顯下調(diào)，P0.01；nm23-H1蛋白表達(dá)則明顯上調(diào)，P0.01；NPM1蛋白在受照射CNE-2R中較在受照射CNE-2移植瘤中表達(dá)稍上調(diào)，差異無(wú)明顯統(tǒng)計(jì)學(xué)意義，P0.05。 【研究結(jié)論】 1.成功建立了人鼻咽癌放射抗拒移植瘤模型。 2.人鼻咽癌放射抗拒移植瘤生長(zhǎng)速度較人鼻咽癌放射敏感移植瘤慢，生長(zhǎng)受照射抑制不明顯，提示鼻咽癌放射抗拒移植瘤可能通過(guò)減慢生長(zhǎng)速度獲得放射抗拒性。 3. NPM1蛋白、Annexin A3蛋白在未受照射CNE-2R移植瘤中較在未受照射CNE-2移植瘤中明顯低表達(dá)，nm23-H1蛋白明顯高表達(dá)，與前期體外試驗(yàn)結(jié)論一致。提示NPM1蛋白、Annexin A3蛋白和nm23-H1蛋白與鼻咽癌放射抗拒有關(guān)，可能作為鼻咽癌放射抗拒新的標(biāo)志物，但需要臨床進(jìn)一步驗(yàn)證。 4. NPM1蛋白、nm23-H1蛋白在受照射CNE-2R移植瘤中較在受照射CNE-2移植瘤中表達(dá)上調(diào)，可能與照射后細(xì)胞DNA損傷修復(fù)能力增強(qiáng)有關(guān)。
[Abstract]:[research background and purpose]
Nasopharyngeal carcinoma is one of the most common malignant tumors in China, especially in Southern China. Due to the specificity of the anatomic location of the nasopharynx and the sensitivity of nasopharyngeal cancer cells to radiation, radiation therapy is the first choice for patients with non metastatic nasopharyngeal carcinoma. Synchronous radiotherapy and chemotherapy have improved the survival time of patients with locally advanced nasopharyngeal carcinoma. The standard treatment model for patients with locally advanced nasopharyngeal carcinoma ~ ([1-2]). In clinical work, some patients receive a full dose of chemotherapy and relapse, which is usually due to the presence of cells related to radiation resistance to radiation ([5]). The current research focus on the mechanism of radiation resistance is that compared to parental cells and their induced radiological resistance. The differentially expressed genes or proteins were screened by high throughput experimental methods, which were further verified by in vitro experiments. However, the corresponding in vivo experiments were rarely reported.
The radiation resistant cell line CNE2R of nasopharyngeal carcinoma (nasopharyngeal carcinoma) has been induced, and the radiation resistant cell line of human nasopharyngeal carcinoma (CNE-2R) has been established in our group, but the animal model has not been established. The tumor animal model is one of the most useful tools to study the mechanism of tumor and the prevention and control of tumor,.BALB/c-nu nude mice congenital chest. The glandular deficiency is suitable for the growth of the transplanted tumor. Therefore, the xenograft tumor model in nude mice is a common model in the study of tumor. The induced radiological resistant cells are transplanted into the animal body, and the transplanted tumor model of nude mice is established, and the radiated xenograft in the nude mice is used to test the radiological resistance of radiological resistant cells in the body, in order to solve the nasopharynx. This study further explores several differentially expressed protein (DEPs) protein (DEPs):NPM1 protein, Annexin A3 protein and nm23-H1 protein expressed in different radiosensitivity xenograft tissues, which can provide a new marker to solve the radiation resistance of nasopharyngeal carcinoma. More effective means to improve the therapeutic effect of nasopharyngeal carcinoma are necessary.
[research methods]
1. the radiation resistant cell CNE-2R of nasopharyngeal carcinoma and CNE-2 of human nasopharyngeal carcinoma were injected into the nude mice subcutaneously, and a contrast model of human nasopharyngeal carcinoma radiation resistant xenograft tumor model and human nasopharyngeal carcinoma radiosensitive xenograft model was established.
2. to observe the growth characteristics of transplanted tumors and to irradiate them with X-rays, and observe the growth of different radiosensitivity xenografts after irradiation.
3. the transplanted tumor was removed for pathological examination, and the morphology of transplanted tumor cells was observed under light microscope.
4. immunohistochemical method was used to detect the expression of NPM1 protein, Annexin A3 protein and nm23-H1 protein in different radiosensitivity xenograft tissues, and to compare their differences.
[results]
1. a radiosensitivity xenograft model of human nasopharyngeal carcinoma (CNE-2R xenograft) and a human nasopharyngeal carcinoma (NPC) radiosensitivity xenograft model (CNE-2 xenograft) were established.
2. unirradiated CNE-2R transplanted tumor was slower than unirradiated CNE-2 transplantation tumor (P0.01), and the volume doubling time of the transplanted tumor was 4.8 days, 3.9 days respectively. The growth of CNE-2R transplanted tumor was not obviously inhibited by irradiation (P0.05), and the growth of CNE-2 transplanted tumor was obviously suppressed (P0.05), and the time of multiplication of transplanted tumor was 6.2 days, 17.1 days, CNE-2R, respectively. The growth rate of transplanted tumor after irradiation was greater than that of CNE-2 transplanted tumor (P0.01).
3. the morphological and pathological examination of the morphology of the transplanted tumor: the naked eye showed an oval or irregular spheroid with a thin layer of thin layer and a fish shape. Under the light microscope, the unirradiated CNE-2R transplanted tumor and the unirradiated CNE-2 transplanted tumor had a large number of necrotic foci, and the irradiated CNE-2R transplanted tumor cells were less necrotic. The unnecrotic CNE-2 transplanted tumor cells There was no obvious difference from the morphological light microscopy of the transplanted tumor cells of CNE-2R. The transplanted tumor cells were nests and polygonal or oval. The nuclei were deeply dyed, the nucleus was round, the oval, the cytoplasm was rich, and the pathological mitosis was visible. The xenograft cells and human nasopharyngeal low differentiated squamous cell carcinoma cells were identical with the morphological characteristics.
The expression of 4. NPM1 protein and Annexin A3 protein in unirradiated CNE-2R xenografts was significantly lower than that in unirradiated CNE-2 xenografts, P0.01, nm23-H1 protein was highly expressed, P0.05.AnnexinA3 protein decreased significantly in the irradiated CNE-2R xenograft tumor than in the irradiated CNE-2 transplanted tumor, P0.01, and the expression of nm23-H1 protein was up obviously. The expression of P0.01 and NPM1 protein was slightly up-regulated in irradiated CNE-2R than in irradiated CNE-2 xenografts, and the difference was not statistically significant, P0.05.
[Conclusion]
1. a radiation rejection model of human nasopharyngeal carcinoma was successfully established.
The growth rate of 2. human nasopharyngeal carcinoma xenografts is slower than that of human nasopharyngeal carcinoma, and the inhibition of growth is not obvious. It suggests that the radiation resistance of nasopharyngeal carcinoma can be obtained by slowing down the growth rate.
3. NPM1 protein, Annexin A3 protein was significantly lower in the unirradiated CNE-2R transplanted tumor than in the unirradiated CNE-2 xenograft, and the nm23-H1 protein was highly expressed, which was consistent with the previous in vitro test. It suggests that the NPM1 protein, Annexin A3 protein and nm23-H1 protein are related to the radiation resistance of nasopharyngeal carcinoma, which may be a new standard for nasopharyngeal carcinoma. But it needs further clinical validation.
The expression of 4. NPM1 protein and nm23-H1 protein in irradiated CNE-2R xenografts was up-regulated in the irradiated CNE-2 xenografts, which may be related to the enhancement of the DNA damage repair ability of the irradiated cells.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.63

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