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。
Nasopharyngeal carcinoma(NPC)is a common neoplasm in China, and the incidence of nasopharyngeal carcinoma in GuangXi province is the second high incidence in China. In the past two decades, the incidence and mortality of NPC in GuangXi show no significant change. Nasopharyngeal carcinoma specimen collection is difficult, for two reasons:on the one hand, it is due to the insidious anatomical position of NPC so that there is difficulty in obtaining clinical samples. On the other hand, NPC patients generally don't been carried out surgical treatment, resulting in a very small amount of clinical biopsy specimen. These all are bad to carry out research on the biological behavior of NPC. Therefore, the establishment of animal model of NPC in vivo is an important means of NPC investigation, and also is one of the issues we need to be resolved. The most commonly used animal model of NPC is nude mouse xenograft tumor model which requeste high experimental condition, long experimental cycle and high cost. The chick embryo chorioallantoic membrane (CAM) model with its simple, convenient, inexpensive and rapid has increasingly become a commonly used model for living by tumor researchers. Our aim is to create in vivo animal models of NPC based on the CAM which would become a technology platform for our further study on invasion and metastasis mechanism, anticancer drug screening, pre-clinical evaluation and even individual cancer treatment.
By repeating exploration, we optimized the methods and conditions of establishing the xenograft model. According to the characteristics of embryo hatching we determined that the starting time of the experiment is the eighth day after hatching and the latest termination time is the fourteenth day after hatching. We established the "window method" for CAM methods and silicone ring for the inoculating carrier in CAM. We have improved and mastered the chick embryo "window" technology, so that chick "window" success rate close to 100%. We also explored and determined the best seeded number of nasopharyngeal carcinoma cells. With Optimization of the above conditions, we successfully established and perfected the chick CAM tumor model of nasopharyngeal carcinoma.
Meanwhile we found that NPC cells also induce angiogenesis when they form xenograft in CAM, and the new blood vessels arrange radially toward to the center of tumor. By quantitative vessel area/chorioallantoic membrane ratio it was found that neovascularization increased with the increase in the number of inoculated cells. We constructed GFP labeled NPC cells and inoculated them in CAM. Then we observed invasion behavior of these GFP labeled NPC cells by using laser confocal microscope. With this model we can observed the tumor cells invasion, breaking through the basement membrane and early stage of blood metastasis. We also discussed the feasibility that CAM model is used to study distant metastasis of NPC cells. By detecting humanβ-globin gene with absolute quantitative real-time fluorescence quantitative PCR method, we detected small amounts of human tumor cells within the heart and lung tissue of inoculated 5-day chicken embryo. On this basis, we verified the distant metastasis difference between the two nasopharyngeal carcinoma cell lines with different metastatic potential, and confirmed that CAM model can be used for prediction of tumor metastasis.
In conclusion, this study established a NPC chick embryo chorioallantoic membrane transplantation tumor model for the first time, and evaluated its application on NPC angiogenesis quantitative study in vivo, observation of early invasion behavior and anticipation of the distant metastasis potential from the morphology, molecular biology and other aspects.
Our study work has laid a good working basis and provide a good platform for further work on NPC biology research.
引文
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