人鼻咽癌放射抗拒移植瘤模型的建立及放射抗拒相关蛋白的研究

发布时间：2018-06-17 00:45

本文选题：鼻咽癌 + 放射抗拒　；参考：《广西医科大学》2012年硕士论文

【摘要】：【研究背景和目的】鼻咽癌是我国常见的恶性肿瘤之一，尤以华南地区发病率最高。由于鼻咽部解剖位置的特殊性以及鼻咽癌细胞对射线较敏感的特点，放射治疗是未转移鼻咽癌患者的首选治疗方式。同步放化疗提高了局部晚期鼻咽癌患者的生存时间，已经成为局部晚期鼻咽癌患者的标准治疗模式~([1-2])。临床工作中，一些患者接受了足量的放化疗也出现复发，这通常是由于存在对放疗抗拒的细胞有关~([5])。目前放射抗拒机理研究热点在于：对比亲本细胞及其诱导出来的放射抗拒细胞，以高通量实验方法筛选出差异表达基因或者蛋白，经过体外实验进一步验证。但相应的体内实验很少报道。目前已有报道诱导出人鼻咽癌放射抗拒细胞系CNE2R)，本课题组前期也已建立了人鼻咽癌放射抗拒细胞系CNE-2R)，但相应的动物实验模型尚未建立。肿瘤动物模型是研究肿瘤机理和肿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，适合移植瘤生长，因此裸鼠异种移植瘤模型是肿瘤研究中常用的模型。本研究将诱导出来的放射抗拒细胞移植到动物体内，建立裸鼠移植瘤模型，并用χ射线分次照射移植瘤以检验放射抗拒细胞在体内的放射抗拒性，为解决鼻咽癌放射抗拒问题提供较好的动物模型。本研究进一步探讨前期研究筛选出的几个差异表达蛋白（DEPs）：NPM1蛋白、Annexin A3蛋白和nm23-H1蛋白在不同放射敏感移植瘤组织中的表达，为解决鼻咽癌放射抗拒提供新的标志物，，这对于临床上采取更有效的手段以提高鼻咽癌的治疗效果很有必要。【研究方法】 1.将鼻咽癌放射抗拒细胞CNE-2R和人鼻咽癌亲本细胞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这一对比模型。 2.观察移植瘤生长特性，以χ射线分次照射移植瘤，观察不同放射敏感性移植瘤受照射后生长情况。 3.取出移植瘤进行病理学检测，光镜下观察移植瘤细胞形态。 4.免疫组化法检测NPM1蛋白、Annexin A3蛋白和nm23-H1蛋白在不同放射敏感性移植瘤组织中的表达，比较其差异。【研究结果】 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移植瘤生长速度慢于未受照射CNE-2移植瘤（P0.01），移植瘤体积倍增时间分别为4.8天、3.9天；CNE-2R移植瘤生长受照射抑制不明显(P0.05)，而CNE-2移植瘤受照射生长明显受抑（P0.05），移植瘤积倍倍增时间分别为6.2天、17.1天；CNE-2R移植瘤受照射后体积增长率大于CNE-2移植瘤（P0.01）。 3.移植瘤形态大体形态及病理学检查：肉眼见移植瘤呈卵圆形或不规则球形，有薄层假包膜，切面呈鱼肉状。光镜下可见未受照射CNE-2R移植瘤及未受照射CNE-2移植瘤大量坏死灶，受照射CNE-2R移植瘤细胞较少坏死。未坏死的CNE-2移植瘤细胞与CNE-2R移植瘤细胞形态光镜下无明显差别。移植瘤细胞呈巢状分布，呈多角形或卵圆形。核大深染，核圆形、卵圆形，胞浆丰富，可见病理核分裂。移植瘤细胞与人鼻咽低分化鳞癌细胞来源形态学特征一致。 4. NPM1蛋白、Annexin A3蛋白在未受照射CNE-2R移植瘤中较在未受照射CNE-2移植瘤中明显低表达，P0.01；nm23-H1蛋白则明显高表达，P0.05。AnnexinA3蛋白在受照射CNE-2R移植瘤中较在受照射CNE-2移植瘤中表达明显下调，P0.01；nm23-H1蛋白表达则明显上调，P0.01；NPM1蛋白在受照射CNE-2R中较在受照射CNE-2移植瘤中表达稍上调，差异无明显统计学意义，P0.05。【研究结论】 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长速度较人鼻咽癌放射敏感移植瘤慢，生长受照射抑制不明显，提示鼻咽癌放射抗拒移植瘤可能通过减慢生长速度获得放射抗拒性。 3. NPM1蛋白、Annexin A3蛋白在未受照射CNE-2R移植瘤中较在未受照射CNE-2移植瘤中明显低表达，nm23-H1蛋白明显高表达，与前期体外试验结论一致。提示NPM1蛋白、Annexin A3蛋白和nm23-H1蛋白与鼻咽癌放射抗拒有关，可能作为鼻咽癌放射抗拒新的标志物，但需要临床进一步验证。 4. NPM1蛋白、nm23-H1蛋白在受照射CNE-2R移植瘤中较在受照射CNE-2移植瘤中表达上调，可能与照射后细胞DNA损伤修复能力增强有关。
[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.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

【参考文献】