【作者】 刘燕；

【导师】 韩冰；

【作者基本信息】 吉林大学 ， 口腔颌面外科学（专业学位）， 2024， 硕士

【摘要】 目的:原发性颌骨鳞状细胞癌造成的大面积颌骨缺损导致严重功能障碍,甚至威胁人类生命,一直是临床治疗的难题。为深入探讨疾病发生机制,寻求治疗方法,目前主要通过动物实验模型进行研究。但现有关于颌面部骨肿瘤动物模型的研究中,均以口腔软组织肿瘤侵袭颌骨为主,不能代表颌骨内原发肿瘤的进展过程。与鼠相比,兔在生理学和解剖学上与人类更为相似,能更好模拟人体骨骼。方法:(1)首先将VX2细胞悬液注射入兔子大腿外侧肌肉内建立荷瘤种兔,以获取建模所需的VX2肿瘤组织块;(2)将20只新西兰大白兔随机分为四组(A组对照组,B、C、D实验组),每组5只。经下颌下手术入路将VX2肿瘤组织块植入兔下颌骨骨髓腔内,建立移植性兔原位下颌骨鳞状细胞癌模型;(3)造模后7天、14天、21天分别处死各组兔子,解剖并收集兔植瘤侧下颌骨、颈部淋巴结以及远隔脏器(心肝脾肺肾)样本,完全浸于4%多聚甲醛液中固定;(4)将下颌骨肿瘤样本经4%多聚甲醛液固定48小时以上行Micro-CT检查,评估下颌骨骨质破坏情况;(5)下颌骨肿瘤样本脱钙后行苏木精-伊红染色(hematoxylin-eosin staining,H&E)观察原位肿瘤增殖情况,行免疫组织化学CK5/6、P53和Ki67染色验证原位肿瘤内特异性蛋白表达情况;(6)对淋巴结样本行H&E染色及免疫组织化学细胞角蛋白(CK)染色评估下颌骨原位肿瘤模型的淋巴结转移情况;(7)对脏器组织样本(心肝脾肺肾)行H&E染色评估下颌骨原位肿瘤模型的远处转移情况。结果:(1)本研究成功建立出一种新的兔VX2下颌骨鳞状细胞癌模型;(2)本研究建立的兔VX2下颌骨鳞状细胞癌模型术后随时间进展呈原位肿瘤体积逐渐增大、全身情况逐渐衰弱及口腔内牙齿松动表现;(3)本研究建立的兔VX2下颌骨鳞状细胞癌模型在影像学上表现出由内向外进行性骨质破坏,空白组手术区段下颌骨BV/TV%分别为68.60±0.88%,7天、14天和21天骨质破坏区段下颌骨BV/TV%逐渐降低,分别为62.15±2.90%、55.89±1.01%和51.08±1.72%;(4)本研究建立的兔VX2下颌骨鳞状细胞癌模型原位下颌骨肿瘤H&E染色显示肿瘤在下颌骨髓腔内增殖并逐渐侵袭、破坏骨壁,免疫组织化学染色显示兔下颌骨在7天、14天和21天CK5/6阳性表达区域占比分别为12.78±0.75%、18.7784±2.27%和30.12±3.60%,p53阳性表达区域占比分别为15.69±0.98%、24.68±4.83%、40.64±1.97%,Ki67阳性表达区域占比分别为22.36±1.54%、31.56±1.97%和37.99±1.44%;(5)本研究建立的兔VX2下颌骨鳞状细胞癌模型术后随时间进展,淋巴结转移率和肺转移率逐渐增高,21天内心肝脾肾均未发生转移。结论:(1)本研究成功建立出一种新的兔VX2下颌骨鳞状细胞癌模型,此原位移植瘤模型在临床表现、影像学表现及组织病理学特征上与人类原发性颌骨内鳞状细胞癌高度相似,为研究原发性颌骨鳞状细胞的增殖和转移机制提供一种可靠的动物模型,为该疾病的治疗研究和新药开发提供了一种有力工具。(2)本研究建模所采用的下颌下手术入路视野清晰、简单易掌握、重复性好,未来可常规应用于建立下颌骨肿瘤动物模型。更多还原

【Abstract】 Objective:Large jawbone defects caused by primary squamous cell carcinoma of the jawbone are a serious threat to human life and have been a challenge for clinical treatment.In order to deeply explore the mechanism of disease occurrence and seek for therapeutic methods,the current research is mainly carried out through animal experimental models.However,the existing studies on animal models of maxillofacial bone tumors are dominated by oral soft tissue tumors invading the jawbone,which cannot represent the progression of primary tumor in the jawbone.Compared with rats,rabbits are more similar to humans in physiology and anatomy,and can better simulate the human skeleton.Methods:(1)Firstly,VX2 cell suspension was injected into the lateral thigh muscle of rabbits to establish a hormonal breeder to obtain the VX2 tumor tissue block required for modeling;(2)20 New Zealand Large White rabbits were randomly divided into four groups(Group A control group,Groups B,C,and D experimental group),with 5 rabbits in each group.The VX2 tumor tissue block was implanted into the bone marrow cavity of the rabbit mandible via submandibular surgical access to establish a transplanted rabbit in situ mandibular squamous cell carcinoma model;(3)the rabbits in each group were executed 7,14 and 21 days after modeling,respectively,and the samples of the mandible on the side of the rabbit implanted with the tumor,the cervical lymph nodes as well as the distal organs(heart,liver,spleen,lungs,and kidneys)were dissected and collected and fixed by completely immersing them in 4% paraformaldehyde solution;(4)the mandibular tumor The samples were fixed in 4% paraformaldehyde solution for more than 48 hours and subjected to Micro-CT to evaluate the bone destruction of the mandible;(5)After decalcification of the mandibular tumor samples,hematoxylin-eosin staining(H&E)was performed to observe the proliferation of the in situ tumors,and immunohistochemistry for CK5/6,P53,and Ki67 staining was performed to verify the specificity of the in situ tumors.H&E staining was performed to observe the proliferation of the in situ tumor,and immunohistochemical CK5/6,P53 and Ki67 staining was performed to verify the expression of specific proteins in the in situ tumor;(6)H&E staining and immunohistochemical cytokeratin(CK)staining were performed on lymph node samples to assess the metastasis of lymph nodes in the in situ tumor model of the jawbone;(7)H&E staining was performed on tissue samples of organs(heart,liver,spleen,lungs,and kidneys)to assess the distant metastasis of in situ tumor model of the jawbone.Results:(1)A new rabbit VX2 mandibular squamous cell carcinoma model was successfully established in this study;(2)The rabbit VX2 mandibular squamous cell carcinoma model established in this study showed gradual increase in the size of in situ tumor,gradual weakening of the whole body and loosening of the teeth in the oral cavity with the progression of time after surgery;(3)The rabbit VX2 mandibular squamous cell carcinoma model established in this study showed progressive bone destruction from the inside out on imaging,and the BV/TV% of the mandible in the operated section in the blank group was68.60±0.88%,respectively,and the BV/TV% of the mandible in the bone-destroying section gradually decreased at 7,14 and 21 days,with 62.15±2.90%,55.89±1.01%,and 51.08±1.01%,respectively.and 51.08±1.72%,respectively;(4)H&E staining of the in situ mandibular tumor in the rabbit VX2 mandibular squamous cell carcinoma model established in this study showed that the tumor proliferated in the bone marrow cavity of the mandible and gradually invaded and destroyed the bone wall,and immunohistochemistry staining showed that the percentage of CK5/6 positively expressing areas in the rabbit mandible at 7 days,14 days and 21 days were 12.78±0.75%,18.7784±2.27% and 30.12±3.60%,15.69±0.98%,24.68±4.83% and 40.64±1.97% for p53-positive expression regions,and 22.36±1.54%,31.56±1.97% and 37.99±1.44% for Ki67-positive expression regions,respectively;(5)In the rabbit VX2 mandibular squamous cell carcinoma model established in this study,the rate of lymph node metastasis and lung metastasis gradually increased with the progression of time after surgery,and no metastasis occurred in the heart,liver,spleen or kidney within 21 days.Conclusion:(1)In this study,a new rabbit VX2 mandibular squamous cell carcinoma model was successfully established.This in situ transplantation tumor model is highly similar to human primary intramandibular squamous cell carcinoma in terms of clinical manifestations,imaging manifestations,and histopathological features,which provides a reliable animal model for the study of the mechanism of primary mandibular squamous cell proliferation and metastasis,and a powerful tool for therapeutic research and development of new drugs for this disease.Tools.(2)The submandibular surgical approach used in this study has a clear field of view,is simple and easy to grasp,and has good reproducibility,so it can be routinely used to establish animal models of mandibular tumors in the future.更多还原