鼻咽癌鸡胚绒毛尿囊膜移植瘤模型的建立及其在肿瘤生物学行为研究上的应用

发布时间：2018-06-23 19:06

本文选题：鼻咽癌 + 鸡胚绒毛尿囊膜　；参考：《广西医科大学》2010年博士论文

【摘要】： 鼻咽癌是我国高发的恶性肿瘤,而广西是全国鼻咽癌的第二高发区。在过去二十年中,广西的鼻咽癌发病率及死亡率几乎无明显改善。建立鼻咽癌体内动物实验模型是研究鼻咽癌的重要手段,也是我们急需解决的课题之一。目前常用的鼻咽癌动物模型是裸鼠移植瘤模型,其实验条件相对较高,且实验周期长,费用昂贵。而鸡胚绒毛尿囊膜(CAM)模型以简单、方便、价廉、快速等优点已日益成为肿瘤研究工作者常用的一个活体模型。我们的目的是建立基于鸡胚绒毛尿囊膜的高效、实用、经济的鼻咽癌体内动物实验模型,为将来进一步研究鼻咽癌细胞的浸润转移机制、抗肿瘤药物筛选、临床前期评价乃至肿瘤个体治疗方案等建立技术平台。经过反复摸索,我们对建立该移植瘤模型的方法和条件进行了优化：根据鸡胚孵化特点,确定实验的开始时间为鸡胚孵至第8日龄时,最迟的终止时间为鸡胚孵至第14日龄；确定以“开窗法”为我们获取CAM的方法；以硅胶圈为我们接种肿瘤细胞的载体；完善并熟练掌握了鸡胚“开窗”的技术,使鸡胚“开窗”成功率接近100%；确定鼻咽癌细胞的最佳接种细胞数。通过以上条件的优化,我们成功建立和完善了鼻咽癌鸡胚CAM移植瘤模型。同时,我们发现,鼻咽癌细胞在CAM移植瘤形成的过程中可诱导宿主新生血管形成,形成的新生血管以移植瘤为中心呈放射状的排列。我们探索了通过测算尿囊膜／血管面积比来定量研究新生血管的方法,并发现,随着接种细胞数的增多,新生血管也增多。我们构建了标记GFP的鼻咽癌细胞株,接种于CAM,通过激光共聚焦显微镜观察GFP标记的鼻咽癌细胞的侵袭行为。通过此模型我们可观察到癌细胞浸润并突破基底膜及血道转移的早期过程。我们还探讨了CAM模型用于研究鼻咽癌细胞远处转移的可行性。通过绝对定量real-time荧光定量PCR检测人β-globin基因,我们在接种鼻咽癌细胞5天的鸡胚心肺组织内检测到了微量的人癌细胞。在此基础上,我们验证了两株不同转移潜能鼻咽癌细胞株的远处转移能力差异,并证实CAM模型可以用于肿瘤转移能力的预测。总之,本研究首次建立了鼻咽癌鸡胚绒毛尿囊膜移植瘤模型,并从形态学、分子生物学等方面阐述该活体模型应用于鼻咽癌新生血管的定量研究、鼻咽癌早期浸润侵袭的生物学行为的观察以及鼻咽癌远处转移潜能的评价。本课题工作为进一步开展鼻咽癌的肿瘤生物学相关研究奠定了良好的工作基础,提供了良好的技术平台。
[Abstract]:Nasopharyngeal carcinoma is a high incidence of malignant tumor in China, and Guangxi is the second high incidence area of nasopharyngeal carcinoma in China. In the past twenty years, the incidence and mortality of nasopharyngeal carcinoma in Guangxi have almost no obvious improvement. The animal model of nasopharyngeal carcinoma (nasopharyngeal carcinoma) is a model of nude mice. The experimental conditions are relatively high, and the experimental period is long and the cost is expensive. The chicken embryo chorioallantoic membrane (CAM) model has become a living model commonly used by the cancer researchers for the advantages of simple, convenient, cheap and fast. The animal experimental model of efficient, practical and economical nasopharyngeal carcinoma in vivo is a technical platform for further study of the mechanism of invasion and metastasis of nasopharyngeal carcinoma cells, screening of antitumor drugs, pre clinical evaluation and even individual treatment of tumor.
After repeated exploration, we have optimized the method and conditions for establishing the model of the transplanted tumor. According to the characteristics of chicken embryo hatching, it is determined that the start time of the experiment is eighth days of age when the chicken embryo is hatched to the age of eighth days, the last time for the chicken embryo is hatched to fourteenth days of age. The carrier of the tumor cells was planted, and the technique of "opening the window" of the chicken embryo was perfected and mastered to make the success rate of the chicken embryo "open window" close to 100%, and to determine the optimum number of inoculation cells for nasopharyngeal carcinoma cells. Through the optimization of the above conditions, we successfully established and perfected the CAM model of the chicken embryo of nasopharyngeal carcinoma.
At the same time, we found that nasopharyngeal carcinoma cells can induce the formation of the host neovascularization in the CAM transplant tumor, and the new blood vessels form a radial arrangement in the center of the transplanted tumor. We explored the method of quantitative study of the neovascularization by measuring the allantoic membrane / vascular area ratio, and found that as the number of inoculated cells increases, The neovascularization was also increased. We constructed a nasopharyngeal carcinoma cell line marked GFP, inoculated in CAM, and observed the invasion behavior of GFP labeled nasopharyngeal carcinoma cells by laser confocal microscopy. Through this model, we can observe the infiltration of the cancer cells and break through the early process of the basement membrane and the metastasis of the blood channel. We also explored the CAM model for the study of the nose. The feasibility of distant metastasis of the pharyngeal carcinoma cells was detected by the absolute quantitative real-time fluorescence quantitative PCR detection of human beta -globin gene. We detected a small number of human cancer cells in the chicken embryo cardiopulmonary tissues of 5 days of nasopharyngeal carcinoma cells. On this basis, we verified the difference in the distant metastasis ability of two different metastatic potential nasopharyngeal carcinoma cells. It is confirmed that the CAM model can be used to predict tumor metastasis.
In conclusion, this study was the first time to establish a model of nasopharyngeal choriochorioallantoic membrane transplantation, and the quantitative study on the application of the living model to nasopharyngeal cancer neovascularization, the biological behavior of nasopharyngeal invasion and the evaluation of the distant metastasis potential of nasopharyngeal carcinoma from morphology, molecular biology and other aspects. Further research on tumor biology in nasopharyngeal carcinoma has laid a good foundation for work and provided a good technical platform.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.63

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