裸鼠肌肉移植瘤模型对研究鼻咽癌亚临床灶意义

发布时间：2018-04-16 17:55

本文选题：鼻咽肿瘤 + 动物模型　；参考：《华中科技大学》2007年硕士论文

【摘要】： 目的:以人鼻咽癌CNE1细胞系为例建立的balb/c裸鼠肌肉内移植瘤模型,观察高分化细胞系CNE1在体内的生物学行为及生长侵袭能力,为下一步研究鼻咽高分化癌亚临床灶在活体内的显像建立合适的动物模型。此动物模型的移植瘤需无包膜,肿瘤细胞较易向周围组织侵袭,以便于我们对移植瘤周围正常组织中侵袭的单个肿瘤细胞的生物学特性的研究。材料:人鼻咽癌CNE1细胞株(LMP1表达阳性);胎牛血清;RPMI-1640培养基、胰蛋白酶;LMP1( CS1-4)单克隆抗体;pEGFP-C1质粒;梭华-SofastTM转染试剂;质粒抽提试剂盒(V-gene);SABC二抗试剂盒;4-6周龄balb/c裸鼠;TJ-TY-500型全自动图像分析系统;超净细胞培养实验室及SPF层流动物房。方法:体外培养LMP1阳性表达的CNE1细胞系,细胞爬片后固定,免疫荧光证实LMP1的表达清情况。pEGFP-C1质粒体外转染CNE1细胞系,筛选稳定表达绿色荧光蛋白的细胞克隆并扩大培养,做流式细胞仪和荧光显微镜检测。动物分为三组,分别将带有pEGFP基因的细胞株、不带有EGFP基因的细胞株扩增后制成浓度为1.0×107细胞悬液,将不带EGFP基因的细胞株于半小时内分别种植于两组balb/c裸鼠臀大肌或背部皮下;将带EGFP基因的细胞株于半小时内种植于第三组balb/c裸鼠臀大肌内。定期观察并测量移植瘤大小,待有裸鼠濒临死亡时处死全部裸鼠取标本,4%多聚甲醛固定后石蜡包埋,做连续石蜡切片,第三组裸鼠相邻切片分别做冰冻切片、苏木素-伊红(HE)和免疫组化染色。图像分析以及荧光显微镜观察并比较肿瘤细胞浸润及转移情况。结果:皮下移植瘤潜伏期长,生长较慢,肿瘤体积小,肿瘤周围可见明显包膜包绕;肌肉移植瘤肿瘤细胞向周围组织侵袭明显,肿瘤组织和周围正常组织界限不清,周围淋巴结和远处转移率较皮下移植瘤高。组织切片可见肌肉组织内癌巢形成,并且有癌细胞侵入肌细胞现象;不带有EGFP基因的两组细胞株进行比较,皮下移植瘤平均潜伏期为(15.40±2.46),肿瘤倍增时间为(3.01±0.32),平均瘤重(1.13±0.15),淋巴结和肝脏转移率低(41.7%)。而肌肉组移植瘤体内平均潜伏期(11.92±2.02d)和瘤体平均倍增时间(2.32±0.24d)明显缩短(P0.01),平均瘤重明显增加(1.32±0.17g,P0.05),淋巴结和肝脏转移率明显增高(41.7%/100%,P 0.05),而成瘤率无显著差异(83.3%/100%,P0.05)。 EGFP在裸鼠体内稳定表达,且不影响细胞在体内的增值与转移,移植瘤重量为(1.33±0.11g),潜伏期为(10.98±2.21),倍增时间为(2.29±0.62),与未传染EGFP组无显著统计学差异。冰冻切片在荧光显微镜下直接观察,较容易直接区分肿瘤区与非肿瘤区,并能发现向周围侵袭的肿单个肿瘤细胞,其敏感性及特异性明显优于HE染色和免疫组化方法。结论:鼻咽癌高分化细胞系CNE1的裸鼠肌肉移植瘤模型在体内的生长侵袭能力强,肿瘤周围无包膜形成,为下一步研究亚临床灶在活体内的显像建立了较为理想的动物模型。 EGFP体外转染CNE1细胞并建立裸鼠移植瘤模型,对研究鼻咽癌的体内侵袭行为具有一定的优势。
[Abstract]:Objective : To investigate the biological behavior and invasion ability of the high - differentiated cell line CNE1 in vivo and to establish a suitable animal model for the development of high - differentiated carcinoma sub - clinical range in vivo . The transplanted tumor of this animal model needs no envelope , and the tumor cells are more vulnerable to the surrounding tissues in order to study the biological characteristics of the single tumor cells that are invasive in the normal tissues around the transplanted tumor .

Materials : Human nasopharyngeal carcinoma CNE1 cell line ( LMP1 expression - positive ) ; fetal bovine serum ; RPMI - 1640 medium , trypsin ; LMP1 ( CS1 - 4 ) monoclonal antibody ; shuttle - SofastTM transfection reagent ; plasmid extraction kit ( V - gene ) ; SABC two - antibody kit ; 4 - 6 week - old balb / c nude mouse ; TJ - TY - 500 type full - automatic image analysis system ; Ultra clean cell culture laboratory and SPF laminar flow animal room .

Methods : CNE1 cell line was cultured in vitro . The expression of LMP1 was confirmed by immunofluorescence . The expression of LMP1 was confirmed by immunofluorescence . The cell line with EGFP gene was isolated and cultured in half an hour . The cell line with EGFP gene was implanted in the gluteus maximus of the third group of balb / c nude mice .

Results : The latent period of subcutaneous transplantation tumor was long , the growth was slow , the tumor volume was small , the tumor around the tumor was significantly shortened ( P0.01 ) . The average latent period of tumor was ( 15.40 卤 2.46 ) , the tumor doubling time was ( 3.01 卤 0.32 ) , the lymph node and liver metastasis rate were significantly increased ( 41.27 % / 100 % , P0.05 ) , but the rate of tumor formation was not significantly different ( 83.3 % / 100 % , P0.05 ) .

EGFP was stably expressed in nude mice without affecting the proliferation and metastasis of cells in vivo . The weight of transplanted tumor was ( 1.33 卤 0.11 g ) . The incubation period was ( 10.98 卤 2.21 ) . The doubling time was ( 2.29 卤 0.62 ) .

Conclusion : The nude mouse muscle transplantation tumor model of high differentiation cell line CNE1 in nasopharyngeal carcinoma has strong invasion ability in vivo and no envelope formation around the tumor , which provides an ideal animal model for the next study of the imaging of subclinical foci in vivo .

EGFP in vitro transfection of CNE1 cells and the establishment of nude mice transplantation tumor model has some advantages to the study of invasive behavior in patients with nasopharyngeal carcinoma .

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332;R739.63

【参考文献】